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.

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.

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.

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.

Sensitive detection of SARS-CoV-2-specific-antibodies in dried blood spot samples (preprint)

ImportancePopulation-wide serological testing is an essential component in understanding the COVID-19 pandemic. The logistical challenges of undertaking widespread serological testing could be eased through use of a reliable dried blood spot (DBS) sampling method. ObjectiveTo validate the use of dried blood spot sampling for the detection of SARS-CoV-2-specific antibodies. Design, setting and participantsEighty-seven matched DBS and serum samples were obtained from eighty individuals, including thirty-one who were previously PCR-positive for SARS-CoV-2. DBS eluates and sera were used in an ELISA to detect antibodies to the viral spike protein. ResultsSpecific anti-SARS-Cov-2 spike glycoprotein antibodies were detectable in both serum and DBS eluate and there was a significant correlation between the antibody levels detected in matched samples (r = 0.96, p<0.0001). Using serum as the gold standard in the assay, matched DBS samples achieved a Cohens kappa coefficient of 0.975 (near-perfect agreement), a sensitivity of 98.1% and specificity of 100%, for detecting anti-spike glycoprotein antibodies. Conclusions and relevanceEluates from DBS samples are a reliable and reproducible source of antibodies to be used for the detection of SARS-CoV-2-specific antibodies. The use of DBS sampling could complement the use of venepuncture in the immunosurveillance of COVID-19 in both low and high income settings.

Detection of antibodies to the SARS-CoV-2 spike glycoprotein in both serum and saliva enhances detection of infection (preprint)

Background: Detecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. Methods: We systemically developed an ELISA assay, optimising different antigens and amplification steps, in serum and saliva from symptomatic and asymptomatic SARS-CoV-2-infected subjects. Results: Using trimeric spike glycoprotein, rather than nucleocapsid enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike, but not nucleocapsid, IgG, IgA and IgM antibody responses were readily detectable in saliva from non-hospitalized symptomatic and asymptomatic individuals. Antibody responses in saliva and serum were largely independent of each other and symptom reporting. Conclusions. Detecting antibody responses in both saliva and serum is optimal for determining virus exposure and understanding immune responses after SARS-CoV-2 infection. Funding. This work was funded by the University of Birmingham, the National Institute for Health Research (UK), the NIH National Institute for Allergy and Infectious Diseases, the Bill and Melinda Gates Foundation and the University of Southampton.

Serology confirms SARS-CoV-2 infection in PCR-negative children presenting with Paediatric Inflammatory Multi-System Syndrome (preprint)

Background. During the COVID-19 outbreak, reports have surfaced of children who present with features of a multisystem inflammatory syndrome with overlapping features of Kawasaki disease and toxic shock syndrome - Paediatric Inflammatory Multisystem Syndrome- temporally associated with SARS-CoV-2 pandemic (PIMS-TS). Initial reports find that many of the children are PCR-negative for SARS-CoV-2, so it is difficult to confirm whether this syndrome is a late complication of viral infection in an age group largely spared the worst consequences of this infection, or if this syndrome reflects enhanced surveillance. Methods. Children hospitalised for symptoms consistent with PIMS-TS between 28 April and 8 May 2020, and who were PCR-negative for SARS-CoV-2, were tested for antibodies to viral spike glycoprotein using an ELISA test. Results. Eight patients (age range 7-14 years, 63% male) fulfilled case-definition for PIMS-TS during the study period. Six of the eight patients required admission to intensive care. All patients exhibited significant IgG and IgA responses to viral spike glycoprotein. Further assessment showed that the IgG isotypes detected in children with PIMS-TS were of the IgG1 and IgG3 subclasses, a distribution similar to that observed in samples from hospitalised adult COVID-19 patients. In contrast, IgG2 and IgG4 were not detected in children or adults. IgM was not detected in children, which contrasts with adult hospitalised adult COVID-19 patients of whom all had positive IgM responses. Conclusions. Strong IgG antibody responses can be detected in PCR-negative children with PIMS-TS. The low detection rate of IgM in these patients is consistent with infection having occurred weeks previously and that the syndrome onset occurs well after the control of SARS-CoV-2 viral load. This implies that the disease is largely immune-mediated. Lastly, this indicates that serology can be an appropriate diagnostic tool in select patient groups.

SARS-CoV-2 seroconversion in health care workers (preprint)

Background The correlates of protection against SARS-CoV-2 and their longevity remain unclear. Studies in severely ill individuals have identified robust cellular and humoral immune responses against the virus. Asymptomatic infection with SARS-CoV-2 has also been described, but it is unknown whether this is sufficient to produce antibody responses. Methods We performed a cross-sectional study recruiting 554 health care workers from University Hospitals Birmingham NHS Foundation Trust who were at work and asymptomatic. Participants were tested for current infection with SARS-CoV-2 by nasopharyngeal swab for real-time polymerase chain reaction and for seroconversion by the measurement of anti-SARS-CoV-2 spike glycoprotein antibodies by enzyme linked immunosorbent assay. Results were interpreted in the context of previous, self-reported symptoms of illness consistent with COVID-19. Results The point prevalence of infection with SARS-CoV-2, determined by the detection of SARS-CoV-2 RNA on nasopharnygeal swab was 2.39% (n=13/544). Serum was available on 516 participants. The overall rate of seroconversion in the cohort was 24.4% (n=126/516). Individuals who had previously experienced a symptomatic illness consistent with COVID-19 had significantly greater seroconversion rates than those who had remained asymptomatic (37.5% vs 17.1%, {chi}2 =21.1034, p<0.0001). In the week preceding peak COVID-19-related mortality at UHBFT, seroconversion rates amongst those who were suffering from symptomatic illnesses peaked at 77.8%. Prior symptomatic illness generated quantitatively higher antibody responses than asymptomatic seroconversion. Seroconversion rates were highest amongst those working in housekeeping (34.5%), acute medicine (33.3%) and general internal medicine (30.3%) with lower rates observed in participants working in intensive care (14.8%) and emergency medicine (13.3%). Conclusions In a large cross-sectional seroprevalence study of health-care workers, we demonstrate that asymptomatic seroconversion occurs, however prior symptomatic illness is associated with quantitatively higher antibody responses. The identification that the potential for seroconversion in health-care workers can associate differentially with certain hospital departments may inform future infection control and occupational health practices.

Site-specific analysis of the SARS-CoV-2 glycan shield (preprint)

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.

Vulnerabilities in coronavirus glycan shields despite extensive glycosylation (preprint)

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.