Different vaccine platforms result in distinct antibody responses to the same antigen in haemodialysis patients (preprint)

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.

Mechanisms of SARS-CoV-2 Inactivation using UVC Laser Radiation (preprint)

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 the ability of the SARS-CoV- 2 spike protein 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.

The diversity of the glycan shield of sarbecoviruses closely related to SARS-CoV-2 (preprint)

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.

Natural variations within the glycan shield of SARS-CoV-2 impact viral spike dynamics (preprint)

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.

Broad SARS-CoV-2 spike-specific CD4+ T-cell response preserves recognition of variants of concern (preprint)

CD4 + T-cells are essential for protection against viruses including SARS-CoV-2. Mutations in SARS-CoV-2 variants of concern (VOC) can enhance infectivity and reduce antibody recognition. CD4 + T-cell sensitivity to mutations is less well understood because few epitopes have been mapped. Characterising > 100 SARS-CoV-2-specific CD4 + T-cell clones from convalescent healthcare workers, we mapped and HLAII restricted 21 epitopes across three viral proteins. Responses to the same spike epitopes were also present after vaccination of uninfected individuals. Lack of CD4 + T-cell cross-reactivity with endemic beta-coronaviruses suggests these responses arose from naïve T-cells rather than pre-existing cross-reactive coronavirus-specific T-cell responses. 10/17 spike epitopes were mutated in VOCs and CD4 + T-cell recognition of 7 was impaired, including 3 of 4 epitopes mutated in Omicron. Broad CD4 + T-cell targeting of epitopes likely limits evasion by current VOCs. However, continued genomic surveillance is vital to identify new emerging mutations able to evade CD4 + T-cell immunity.

In vitro, classical complement activation differs by disease severity and between SARS-CoV-2 antigens (preprint)

Antibodies specific for the spike glycoprotein (S) and nucleocapsid (N) SARS-CoV-2 proteins are typically present during severe COVID-19, and induced to S after vaccination. The binding of viral antigens by antibody can initiate the classical complement pathway. Since complement could play pathological or protective roles at distinct times during SARS-CoV-2 infection we determined levels of antibody-dependent complement activation along the complement cascade. Here, we used an ELISA assay to assess complement protein binding (C1q) and the deposition of C4b, C3b, and C5b to S and N antigens in the presence of anti-SARS-CoV-2 antibodies from different test groups: non-infected, single and double vaccinees, non-hospitalised convalescent (NHC) COVID-19 patients and convalescent hospitalised (ITU-CONV) COVID-19 patients. C1q binding correlates strongly with antibody responses, especially IgG1 levels. However, detection of downstream complement components, C4b, C3b and C5b shows some variability associated with the antigen and subjects studied. In the ITU-CONV, detection of C3b-C5b to S was observed consistently, but this was not the case in the NHC group. This is in contrast to responses to N, where median levels of complement deposition did not differ between the NHC and ITU-CONV groups. Moreover, for S but not N, downstream complement components were only detected in sera with higher IgG1 levels. Therefore, the classical pathway is activated by antibodies to multiple SARS-CoV-2 antigens, but the downstream effects of this activation may differ depending on the specific antigen targeted and the disease status of the subject. O_LISpike- and nucleocapsid-specific antibodies activate complement in vitro C_LIO_LIC1q binding correlates with IgG1 antibody levels C_LIO_LIGeneration of C4b, C3b and C5b relates to the antigen targeted and the patient group tested C_LI

Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein (preprint)

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.

Stimulation of vascular organoids with SARS-CoV-2 antigens increases endothelial permeability and regulates vasculopathy (preprint)

Objective Thrombotic complications and vasculopathy have been extensively associated with severe COVID-19 infection, however the mechanisms by which endotheliitis is induced remain poorly understood. Here we investigate vascular permeability in the context of SARS-CoV-2-mediated endotheliitis in patient samples and a vascular organoid model. Methods and Results We report the presence of the Spike glycoprotein in pericytes associated with pericyte activation and increased endothelial permeability in post-mortem COVID-19 lung autopsies. A pronounced decrease in the expression of the adhesion molecule VE-cadherin is observed in patients with thrombotic complications. Interestingly, fibrin-rich thrombi did not contain platelets, did not colocalize with tissue factor and have heterogenous levels of Von Willebrand factor, suggesting a biomarker-guided therapy might be required to target thrombosis in severe patients. Using a 3D vascular organoid model, we observe that ACE2 is primarily expressed in pericytes adjacent to vascular networks, consistent with patient data, indicating a preferential uptake of the S glycoprotein by these cells. Exposure of vascular organoids to SARS-CoV-2 or its antigens, recombinant trimeric Spike glycoprotein and Nucleocapsid protein, reduced endothelial cell and pericyte viability as well as CD144 expression with no additive effect upon endothelial activation via IL-1β. Conclusions Our data suggest that pericyte uptake of SARS-CoV-2 or Spike glycoprotein contributes to vasculopathy by altering endothelial permeability increasing the risk of thrombotic complications.

Site-specific steric control of SARS-CoV-2 spike glycosylation (preprint)

A central tenet in the design of recombinant vaccines is the display of native-like antigens in the elicitation of protective immunity. However, the diversity of global vaccine strategies against Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses challenges to benchmark antigens across global vaccine programs. Here, we investigate the glycosylation of a variety of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against the glycan shield of an infectious virus. The site-specific stalling of glycan maturation is a highly sensitive reporter of local protein structure and we find there is remarkable conservation of this feature across all samples. Analysis of molecular dynamics simulations of a fully glycosylated spike supports a model of steric restrictions that shape enzymatic processing of the glycans. Furthermore, we show that there is a conserved glycosylation pattern across the monomeric receptor binding domain (RBD) protein and the complete trimeric spike (S) protein. This is in contrast to RBD glycosylation in Middle East respiratory syndrome coronavirus (MERS-CoV) where quaternary architecture limits glycan processing when in the context of full-length MERS-CoV S protein. These results suggest that spike-based immunogen glycosylation reproducibly recapitulates viral glycosylation.

Interferon-regulated genetic programs and JAK/STAT pathway activate the intronic promoter of the short ACE2 isoform in renal proximal tubules (preprint)

Summary: 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 and IFN{beta}, 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.

An all-solid-state heterojunction oxide transistor for the rapid detection of biomolecules and SARS-CoV-2 spike S1 protein (preprint)

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.

The impact of Spike mutations on SARS-CoV-2 neutralization (preprint)

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 5-10-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.

A trans-complementation system for SARS-CoV-2 (preprint)

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.

Azithromycin Plus Zinc Sulfate Rapidly and Synergistically Suppresses IκBα-Mediated In Vitro Human Airway Cell ACE2 Expression for SARS-CoV-2 Entry (preprint)

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.

Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine (preprint)

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.

Comprehensive mapping of SARS-CoV-2 interactions in vivo reveals functional virus-host interactions (preprint)

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 ({Delta}382) 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 {Delta}382 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'-O-methylated. 2'-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'-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.

Validation of a combined ELISA to detect IgG, IgA and IgM antibody responses to SARS-CoV-2 in mild or moderate non-hospitalised patients (preprint)

BackgroundFrequently SARS-CoV-2 results in mild or moderate disease with potentially lower concentrations of antibodies compared to those that are hospitalised. Here, we validated an ELISA using SARS-CoV-2 trimeric spike glycoprotein, with targeted detection of IgG, IgA and IgM (IgGAM) using serum and dried blood spots (DBS) from adults with mild or moderate disease. MethodsTargeting the SARS-CoV-2 trimeric spike, a combined anti-IgG, IgA and IgM serology ELISA assay was developed using 62 PCR-confirmed non-hospitalised, mild or moderate COVID-19 samples, [≥]14 days post symptom onset and 624 COVID-19 negative samples. The assay was validated using 73 PCR-confirmed non-hospitalised COVID-19 and 359 COVID-19 negative serum samples with an additional 81 DBSs, and further validated in 226 PCR-confirmed non-hospitalised COVID-19 and 426 COVID-19 negative clinical samples. ResultsA sensitivity and specificity of 98.6% (95% CI, 92.6-100.0), 98.3% (95% CI, 96.4-99.4), respectively, was observed following validation of the SARS-CoV-2 ELISA. No cross-reactivities with endemic coronaviruses or other human viruses were observed, and no change in results were recorded for interfering substances. The assay was stable at temperature extremes and components were stable for 15 days once opened. A matrix comparison showed DBS to correlate with serum results. Clinical validation of the assay reported a sensitivity of 94.7% (95% CI, 90.9-97.2%) and a specificity of 98.4% (95% CI, 96.6-99.3%). ConclusionsThe human anti-IgGAM SARS-CoV-2 ELISA provides accurate and sensitive detection of SARS-CoV-2 antibodies in non-hospitalised adults with mild or moderate disease. The use of dried blood spots makes the assay accessible to the wider community. Supplementary MaterialNo

Two-Component Spike Nanoparticle Vaccine Protects Macaques from SARS-CoV-2 Infection (preprint)

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.Funding: This work was supported by a Netherlands Organization for Scientific Research (NWO) Vici grant (to R.W.S.); by the Bill & Melinda Gates Foundation through the Collaboration for AIDS Vaccine Discovery (CAVD) grants OPP1111923, OPP1132237, and INV-002022 (to R.W.S. and/or N.P.K.), INV-008352/OPP1153692 and OPP1196345/INV-008813 (to M.C.), and grant OPP1170236 (to A.B.W.); by the Fondation Dormeur, Vaduz (to R.W.S. and to M.J.v.G.) and Health Holland PPS-allowance LSHM20040 (to M.J.v.G.); the University of Southampton Coronavirus Response Fund (to M.C.); and by the Netherlands Organisation for Health Research and Development ZONMW (to B.L.H). M.J.v.G. is a recipient of an AMC Fellowship from Amsterdam UMC and a COVID-19 grant from the Amsterdam Institute for Infection and Immunity. R.W.S and M.J.v.G. are recipients of support from the University of Amsterdam Proof of Concept fund (contract no. 200421) as managed by Innovation Exchange Amsterdam (IXA). The Infectious Disease Models and Innovative Therapies (IDMIT) research infrastructure is supported by the ‘Programme Investissements d’Avenir, managed by the ANR under reference ANR-11-INBS-0008. The Fondation Bettencourt Schueller and the Region Ile-de-France contributed to the implementation of IDMIT’s facilities and imaging technologies. The NHP study received financial support from REACTing, the National Research Agency (ANR; AM-CoV-Path) and the European Infrastructure TRANSVAC2 (730964). Conflict of Interest: N.P.K. is a co-founder, shareholder, and chair of the scientific advisory board of Icosavax, Inc. All other authors declare no competing interests.Ethical Approval: The protocols were approved by the institutional ethical committee “Comité d’Ethique en Expérimentation Animale du Commissariat à l’Energie Atomique et aux Energies Alternatives” (CEtEA #44) under statement number A20-011. The study was authorized by the “Research, Innovation and Education Ministry” under registration number APAFIS#24434-2020030216532863v1.

Serological responses to SARS-CoV-2 following non-hospitalised infection: clinical and ethnodemographic features associated with the magnitude of the antibody response (preprint)

Objective To determine clinical and ethnodemographic correlates of serological responses against the SARS-CoV-2 spike glycoprotein following mild-to-moderate COVID-19. Design A retrospective cohort study of healthcare workers who had self-isolated due to COVID-19. Setting University Hospitals Birmingham NHS Foundation Trust, UK (UHBFT). Participants 956 health care workers were recruited by open invitation via UHBFT trust email and social media. Intervention Participants volunteered a venous blood sample that was tested for the presence of anti-SARS-CoV-2 spike glycoprotein antibodies. Results were interpreted in the context of the symptoms of their original illness and ethnodemographic variables. Results Using an assay that simultaneously measures the combined IgG, IgA and IgM response against the spike glycoprotein (IgGAM), the overall seroprevalence within this cohort was 46.2% (n=442/956). The seroprevalence of immunoglobulin isotypes was 36.3%, 18.7% and 8.1% for IgG, IgA and IgM respectively. IgGAM identified serological responses in 40.6% (n=52/128) of symptomatic individuals who reported a negative SARS-CoV-2 PCR test. Increasing age, non-white ethnicity and obesity were independently associated with greater IgG antibody response against the spike glycoprotein. Self-reported fever and fatigue were associated with greater IgG and IgA responses against the spike glycoprotein. The combination of fever and/or cough and/or anosmia had a positive predictive value of 92.3% for seropositivity. Conclusions and relevance Assays employing combined antibody detection demonstrate enhanced seroepidemiological sensitivity and can detect prior viral exposure even when PCR swabs have been negative. We demonstrate an association between known ethnodemographic risk factors associated with mortality from COVID-19 and the magnitude of serological responses in mild-to-moderate disease. The combination of cough, and/or fever and/or anosmia identifies the majority of individuals who should self-isolate for COVID-19.

Landscape analysis of escape variants identifies SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization (preprint)

Although neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of most COVID-19 vaccines and being developed as therapeutics, escape mutations could compromise such countermeasures. To define the immune-mediated 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 48 escape mutants. These variants were mapped onto the RBD structure and evaluated for cross-resistance by convalescent human plasma. Although each mAb had unique resistance profiles, many shared residues within an epitope, as several variants were resistant to multiple mAbs. Remarkably, we identified mutants that escaped neutralization by convalescent human sera, suggesting that some humans induce a narrow repertoire of neutralizing antibodies. By comparing the antibody-mediated mutational landscape in S protein with sequence variation in circulating SARS-CoV-2 strains, we identified single amino acid substitutions that could attenuate neutralizing immune responses in some humans.