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Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe., Competing Interests: Declaration of interests J.S.M. is an inventor on the following U.S. patent applications: no. 62/412,703 (“Prefusion Coronavirus Spike Proteins and Their Use”); no. 62/972,886 (“2019-nCoV Vaccine”); no. 63/032,502 (“Engineered Coronavirus Spike (S) Protein and Methods of Use Thereof”)., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 healthcare workers were recruited by open invitation via UHBFT trust email and social media between 27 April 2020 and the 8 June 2020., 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 in self-isolating individuals a time when Wuhan strain SARS-CoV-2 was predominant., 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., Competing Interests: Competing interests: MTD reports personal fees from Abingdon Health, outside the submitted work., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

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 relatively 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. We systematically developed an ELISA, optimizing different antigens and amplification steps, in serum and saliva from non-hospitalized SARS-CoV-2-infected subjects. 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 IgG, IgA and IgM antibody responses were readily detectable in saliva from a minority of RT-PCR confirmed, non-hospitalized symptomatic individuals, and these were mostly subjects who had the highest levels of anti-spike serum antibodies. Therefore, detecting antibody responses in both saliva and serum can contribute to determining virus exposure and understanding immune responses after SARS-CoV-2 infection., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

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 among 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 S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support 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.

The severity of SARS-CoV-2 infection is highly variable and yet the molecular basis for this effect remains elusive. One potential contribution are differences in the glycosylation of target human cells, particularly as SARS-CoV-2 has the capacity to bind sialic acid which is a common, and highly variable, terminal modification of glycans. The viral spike glycoprotein (S) of SARS-CoV-2 and the human cellular receptor, angiotensin-converting enzyme 2 (ACE2) are both densely glycosylated. We therefore sought to investigate whether the glycosylation state of ACE2 impacts the interaction with SARS-CoV-2 viral spike. We generated a panel of engineered ACE2 glycoforms which were analyzed by mass spectrometry to reveal the site-specific glycan modifications. We then probed the impact of ACE2 glycosylation on S binding and revealed a subtle sensitivity with hypersialylated or oligomannose-type glycans slightly impeding the interaction. In contrast, deglycosylation of ACE2 did not influence SARS-CoV-2 binding. Overall, ACE2 glycosylation does not significantly influence viral spike binding. We suggest that any role of glycosylation in the pathobiology of SARS-CoV-2 will lie beyond its immediate impact of receptor glycosylation on virus binding., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Dried blood spot (DBS) samples can be used for the detection of severe acute respiratory syndrome coronavirus 2 spike antibodies. DBS sampling is comparable to matched serum samples with a relative 98.1% sensitivity and 100% specificity. Thus, DBS sampling offers an alternative for population-wide serologic testing in the coronavirus pandemic.

Objective: To determine the rates of asymptomatic viral carriage and seroprevalence of SARS-CoV-2 antibodies in healthcare workers., Design: A cross-sectional study of asymptomatic healthcare workers undertaken on 24/25 April 2020., Setting: University Hospitals Birmingham NHS Foundation Trust (UHBFT), UK., Participants: 545 asymptomatic healthcare workers were recruited while at work. Participants were invited to participate via the UHBFT social media. Exclusion criteria included current symptoms consistent with COVID-19. No potential participants were excluded., Intervention: Participants volunteered a nasopharyngeal swab and a venous blood sample that were tested for SARS-CoV-2 RNA and anti-SARS-CoV-2 spike glycoprotein antibodies, respectively. Results were interpreted in the context of prior illnesses and the hospital departments in which participants worked., Main Outcome Measure: Proportion of participants demonstrating infection and positive SARS-CoV-2 serology., Results: The point prevalence of SARS-CoV-2 viral carriage was 2.4% (n=13/545). The overall seroprevalence of SARS-CoV-2 antibodies was 24.4% (n=126/516). Participants who reported prior symptomatic illness had higher seroprevalence (37.5% vs 17.1%, χ 2 =21.1034, p<0.0001) and quantitatively greater antibody responses than those who had remained asymptomatic. Seroprevalence was greatest among 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%). BAME (Black, Asian and minority ethnic) ethnicity was associated with a significantly increased risk of seropositivity (OR: 1.92, 95% CI 1.14 to 3.23, p=0.01). Working on the intensive care unit was associated with a significantly lower risk of seropositivity compared with working in other areas of the hospital (OR: 0.28, 95% CI 0.09 to 0.78, p=0.02)., Conclusions and Relevance: We identify differences in the occupational risk of exposure to SARS-CoV-2 between hospital departments and confirm asymptomatic seroconversion occurs in healthcare workers. Further investigation of these observations is required to inform future infection control and occupational health practices., Competing Interests: Competing interests: MTD reports personal fees from Abingdon Health, outside the submitted work. All other authors declare no competing interests., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The severe acute respiratory syndrome coronavirus 2 (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 protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication in the upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic. • Two-component protein nanoparticles display multiple copies of the SARS-CoV-2 spike • Spike protein nanoparticles enhance cognate B cell activation in vitro • Vaccination induces potent neutralization in mice, rabbits, and cynomolgus macaques • Vaccination protects macaques against a high-dose SARS-CoV-2 challenge Brouwer et al. present preclinical evidence in support of a COVID-19 vaccine candidate, designed as a self-assembling two-component protein nanoparticle displaying multiple copies of the SARS-CoV-2 spike protein, which induces strong neutralizing antibody responses and protects from high-dose SARS-CoV-2 challenge. [ABSTRACT FROM AUTHOR]