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Background: Administration of potent anti-receptor-binding domain (RBD) monoclonal antibodies has been shown to curtail viral shedding and reduce hospitalization in patients with SARS-CoV-2 infection. However, the structure-function analysis of potent human anti-RBD monoclonal antibodies and its links to the formulation of antibody cocktails remains largely elusive. Methods: Previously, we isolated a panel of neutralizing anti-RBD monoclonal antibodies from convalescent patients and showed their neutralization efficacy in vitro. Here, we elucidate the mechanism of action of antibodies and dissect antibodies at the epitope level, which leads to a formation of a potent antibody cocktail. Results: We found that representative antibodies which target non-overlapping epitopes are effective against wild type virus and recently emerging variants of concern, whilst being encoded by antibody genes with few somatic mutations. Neutralization is associated with the inhibition of binding of viral RBD to ACE2 and possibly of the subsequent fusion process. Structural analysis of representative antibodies, by cryo-electron microscopy and crystallography, reveals that they have some unique aspects that are of potential value while sharing some features in common with previously reported neutralizing monoclonal antibodies. For instance, one has a common VH 3-53 public variable region yet is unusually resilient to mutation at residue 501 of the RBD. We evaluate the in vivo efficacy of an antibody cocktail consisting of two potent non-competing anti-RBD antibodies in a Syrian hamster model. We demonstrate that the cocktail prevents weight loss, reduces lung viral load and attenuates pulmonary inflammation in hamsters in both prophylactic and therapeutic settings. Although neutralization of one of these antibodies is abrogated by the mutations of variant B.1.351, it is also possible to produce a bi-valent cocktail of antibodies both of which are resilient to variants B.1.1.7, B.1.351 and B.1.617.2. Conclusions: These findings support the up-to-date and rational design of an anti-RBD antibody cocktail as a therapeutic candidate against COVID-19.

Background Previous infection with SARS-CoV-2 affects the immune response to the first dose of the SARS-CoV-2 vaccine. We aimed to compare SARS-CoV-2-specific T-cell and antibody responses in health-care workers with and without previous SARS-CoV-2 infection following a single dose of the BNT162b2 (tozinameran; Pfizer–BioNTech) mRNA vaccine. Methods We sampled health-care workers enrolled in the PITCH study across four hospital sites in the UK (Oxford, Liverpool, Newcastle, and Sheffield). All health-care workers aged 18 years or older consenting to participate in this prospective cohort study were included, with no exclusion criteria applied. Blood samples were collected where possible before vaccination and 28 (±7) days following one or two doses (given 3–4 weeks apart) of the BNT162b2 vaccine. Previous infection was determined by a documented SARS-CoV-2-positive RT-PCR result or the presence of positive anti-SARS-CoV-2 nucleocapsid antibodies. We measured spike-specific IgG antibodies and quantified T-cell responses by interferon-γ enzyme-linked immunospot assay in all participants where samples were available at the time of analysis, comparing SARS-CoV-2-naive individuals to those with previous infection. Findings Between Dec 9, 2020, and Feb 9, 2021, 119 SARS-CoV-2-naive and 145 previously infected health-care workers received one dose, and 25 SARS-CoV-2-naive health-care workers received two doses, of the BNT162b2 vaccine. In previously infected health-care workers, the median time from previous infection to vaccination was 268 days (IQR 232–285). At 28 days (IQR 27–33) after a single dose, the spike-specific T-cell response measured in fresh peripheral blood mononuclear cells (PBMCs) was higher in previously infected (n=76) than in infection-naive (n=45) health-care workers (median 284 [IQR 150–461] vs 55 [IQR 24–132] spot-forming units [SFUs] per 106 PBMCs; pInterpretation A single dose of the BNT162b2 vaccine is likely to provide greater protection against SARS-CoV-2 infection in individuals with previous SARS-CoV-2 infection, than in SARS-CoV-2-naive individuals, including against variants of concern. Future studies should determine the additional benefit of a second dose on the magnitude and durability of immune responses in individuals vaccinated following infection, alongside evaluation of the impact of extending the interval between vaccine doses. Funding UK Department of Health and Social Care, and UK Coronavirus Immunology Consortium.

The SARS-CoV-2 pandemic continues despite the presence of effective vaccines, and novel vaccine approaches may help to reduce viral spread and associated COVID-19 disease. Current vaccine administration modalities are based on systemic needle-administered immunisation which may be suboptimal for mucosal pathogens. Here we demonstrate in a mouse model that small-volume intranasal administration of purified spike (S) protein in the adjuvant polyethylenemine (PEI) elicits robust antibody responses with modest systemic neutralisation activity. Further, we test a heterologous intranasal immunisation regimen, priming with S and boosting with RBD-Fc. Our data identify small volume PEI adjuvantation as a novel platform with potential for protective mucosal vaccine development.

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 severe acute respiratory syndrome coronavirus 2 (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, a combinatorial human antibody library constructed 20 years before the coronavirus disease 2019 (COVID‐19) pandemic is used 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 three antibodies contain over 13–22 SHMs, many of which are involved in specific interactions in their crystal structures with SARS‐CoV‐2 spike receptor binding domain. The identification of these somatically mutated antibodies in a pre‐pandemic library raises intriguing questions about the origin and evolution of these antibodies with respect to their reactivity with SARS‐CoV‐2., Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the causative agent of the coronavirus disease 2019 pandemic. From a 20‐year‐old, combinatorial library, the authors isolate three neutralizing antibodies with high somatic hypermutations that are involved in antigen recognition, suggesting cross‐reactivity with a related antigen present in the pre‐pandemic‐era.

Cyclic GMP‐AMP (cGAMP) is an immunostimulatory molecule produced by cGAS that activates STING. cGAMP is an adjuvant when administered alongside antigens. cGAMP is also incorporated into enveloped virus particles during budding. Here, we investigate whether inclusion of cGAMP within viral vaccine vectors enhances their immunogenicity. We immunise mice with virus‐like particles (VLPs) containing HIV‐1 Gag and the vesicular stomatitis virus envelope glycoprotein G (VSV‐G). cGAMP loading of VLPs augments CD4 and CD8 T‐cell responses. It also increases VLP‐ and VSV‐G‐specific antibody titres in a STING‐dependent manner and enhances virus neutralisation, accompanied by increased numbers of T follicular helper cells. Vaccination with cGAMP‐loaded VLPs containing haemagglutinin induces high titres of influenza A virus neutralising antibodies and confers protection upon virus challenge. This requires cGAMP inclusion within VLPs and is achieved at markedly reduced cGAMP doses. Similarly, cGAMP loading of VLPs containing the SARS‐CoV‐2 Spike protein enhances Spike‐specific antibody titres. cGAMP‐loaded VLPs are thus an attractive platform for vaccination., cGAMP, a cyclic di‐nucleotide, is an adjuvant and activates STING. This study shows that cGAMP‐loaded virus‐like particles, designed to deliver antigen and cGAMP to the same antigen‐presenting cell, are an attractive platform for vaccination.

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike, and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50, Dejnirattisai et al. present an in-depth study of the human antibody response to SARS-CoV-2 infection. By characterizing 377 human mAbs from recovered COVID-19 patients, and determining 19 protein structures, they construct a map of antibody footprints on the RBD describe in great detail its antigenic anatomy.

ISG products are believed to directly block viral replication. We propose an alternative mechanism based on emerging evidence. We discuss that the metabolic products of two ISG proteins, namely RSAD2 and NOS, inhibit activity of GAPDH to regulate central carbon metabolism and support a broad‐spectrum antiviral immune response via at least four mechanisms: eicosanoids storm, antigen cross‐presentation via MHC‐I, modulating NFAT and NF‐κB pathways, and S‐nitrosylation of viral proteins., In response to viral infections, the innate immune system rapidly activates expression of several interferon‐stimulated genes (ISGs), whose protein and metabolic products are believed to directly interfere with the viral life cycle. Here, we argue that biochemical reactions performed by two specific protein products of ISGs modulate central carbon metabolism to support a broad‐spectrum antiviral response. We demonstrate that the metabolites generated by metalloenzymes nitric oxide synthase and the radical S‐adenosylmethionine (SAM) enzyme RSAD2 inhibit the activity of the housekeeping and glycolytic enzyme glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). We discuss that this inhibition is likely to stimulate a range of metabolic and signalling processes to support a broad‐spectrum immune response. Based on these analyses, we propose that inhibiting GAPDH in individuals with deteriorated cellular innate immune response like elderly might help in treating viral diseases such as COVID‐19.

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 2.6Å 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.

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.

Background: Laboratory diagnosis of SARS-CoV-2 infection (the cause of COVID-19) uses PCR to detect viral RNA (vRNA) in respiratory samples. SARS-CoV-2 RNA has also been detected in other sample types, but there is limited understanding of the clinical or laboratory significance of its detection in blood.Methods: We undertook a systematic literature review to assimilate the evidence for the frequency of vRNA in blood, and to identify associated clinical characteristics. We performed RT-PCR in serum samples from a UK clinical cohort of acute and convalescent COVID-19 cases (n=212), together with convalescent plasma samples collected by NHS Blood and Transplant (NHSBT) (n=462 additional samples). To determine whether PCR-positive blood samples could pose an infection risk, we attempted virus isolation from a subset of RNA-positive samples.Results: We identified 28 relevant studies, reporting SARS-CoV-2 RNA in 0-76% of blood samples; pooled estimate 10% (95%CI 5-18%). Among serum samples from our clinical cohort, 27/212 (12.7%) had SARS-CoV-2 RNA detected by RT-PCR. RNA detection occurred in samples up to day 20 post symptom onset, and was associated with more severe disease (multivariable odds ratio 7.5). Across all samples collected ≥28 days post symptom onset, 0/494 (0%, 95%CI 0-0.7%) had vRNA detected. Among our PCR-positive samples, cycle threshold (ct) values were high (range 33.5-44.8), suggesting low vRNA copy numbers. PCR-positive sera inoculated into cell culture did not produce any cytopathic effect or yield an increase in detectable SARS-CoV-2 RNA. There was a relationship between RT-PCR negativity and the presence of total SARS-CoV-2 antibody (p=0.02).Conclusions: vRNA was detectable at low viral loads in a minority of serum samples collected in acute infection, but was not associated with infectious SARS-CoV-2 (within the limitations of the assays used). This work helps to inform biosafety precautions for handling blood products from patients with current or previous COVID-19.