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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) in cell culture. This cross-neutralizing antibody targets a communal epitope on these viruses and may offer potential for prevention and treatment of COVID-19. Vaccines and targeted therapeutics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are currently lacking. Here, the authors report a human monoclonal antibody capable of neutralizing both authentic SARS-CoV and SARS-CoV-2 by targeting a common epitope.

Coronaviruses cause respiratory tract infections in humans and outbreaks of deadly pneumonia worldwide. Infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host receptors and fuses the viral and cellular membranes. To understand the molecular basis of coronavirus attachment to oligosaccharide receptors, we determined cryo-EM structures of coronavirus OC43 S glycoprotein trimer in isolation and in complex with a 9-O-acetylated sialic acid. We show that the ligand binds with fast kinetics to a surface-exposed groove and that interactions at the identified site are essential for S-mediated viral entry into host cells, but free monosaccharide does not trigger fusogenic conformational changes. The receptor-interacting site is conserved in all coronavirus S glycoproteins that engage 9-O-acetyl-sialogycans, with an architecture similar to those of the ligand-binding pockets of coronavirus hemagglutinin esterases and influenza virus C/D hemagglutinin-esterase fusion glycoproteins. Our results demonstrate these viruses evolved similar strategies to engage sialoglycans at the surface of target cells.Structural and functional analyses reveal how 9-O-acetyl sialic acid is recognized by the human coronavirus OC43 S glycoprotein and how this interaction promotes viral entry.

The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. The coronavirus spike (S) glycoprotein initiates infection by promoting fusion of the viral and cellular membranes through conformational changes that remain largely uncharacterized. Here we report the cryoEM structure of a coronavirus S glycoprotein in the postfusion state, showing large-scale secondary, tertiary, and quaternary rearrangements compared with the prefusion trimer and rationalizing the free-energy landscape of this conformational machine. We also biochemically characterized the molecular events associated with refolding of the metastable prefusion S glycoprotein to the postfusion conformation using limited proteolysis, mass spectrometry, and single-particle EM. The observed similarity between postfusion coronavirus S and paramyxovirus F structures demonstrates that a conserved refolding trajectory mediates entry of these viruses and supports the evolutionary relatedness of their fusion subunits. Finally, our data provide a structural framework for understanding the mode of neutralization of antibodies targeting the fusion machinery and for engineering next-generation subunit vaccines or inhibitors against this medically important virus family.

Three highly pathogenic β-coronaviruses have crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. To evaluate the possibility of identifying antibodies with broad neutralizing activity, we isolated a monoclonal antibody, termed B6, that cross-reacts with eight β-coronavirus spike glycoproteins, including all five human-infecting β-coronaviruses. B6 broadly neutralizes entry of pseudotyped viruses from lineages A and C, but not from lineage B, and the latter includes SARS-CoV and SARS-CoV-2. Cryo-EM, X-ray crystallography and membrane fusion assays reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery. The data indicate that antibody binding sterically interferes with the spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with β-coronaviruses from three lineages, along with a 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-β-coronavirus vaccine. Structural characterization of B6, a monoclonal antibody that cross-reacts with eight β-coronavirus spike proteins from three viral lineages, reveals a conserved cryptic epitope that could serve as a target for structure-guided design of a pan-β-coronavirus vaccine.

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth. Here, the authors report the isolation and characterization of two human monoclonal antibodies (mAbs) from immunized mice with trimeric spike ectodomains of three human betacoronaviruses HCoV-OC43, SARS-CoV and MERSCoV, and show that while exhibiting cross-reactivity, the mAbs only neutralize MERS-CoV but not SARS-CoV nor SARS-CoV-2, likely due to the subtle epitope differences in the spike S2 fusion subunit.

Cryo-EM and mass spectrometry analyses of the spike glycoprotein trimer from coronavirus HcoV-NL63 reveal an extensive glycan shield that covers the protein surface, including an epitope targeted by neutralizing antibodies against several coronaviruses. The threat of a major coronavirus pandemic urges the development of strategies to combat these pathogens. Human coronavirus NL63 (HCoV-NL63) is an α-coronavirus that can cause severe lower-respiratory-tract infections requiring hospitalization. We report here the 3.4-Å-resolution cryo-EM reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which mediates entry into host cells and is the main target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass spectrometry, provides a structural framework to understand the accessibility to antibodies. The structure reveals the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on epitope masking with glycans and activating conformational changes, to evade the immune system of infected hosts.

A new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently emerged to cause a human pandemic. Although molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. We developed serologic assays for detection of SARS-CoV-2 neutralizing, spike protein-specific, and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed SARS-CoV-2 infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrated that most PCR-confirmed SARS-CoV-2-infected persons seroconverted by 2 weeks after disease onset. We found that commercial S1 IgG or IgA ELISAs were of lower specificity, and sensitivity varied between the 2 assays; the IgA ELISA showed higher sensitivity. Overall, the validated assays described can be instrumental for detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies.

Porcine deltacoronavirus (PDCoV), identified in 2012, is a common enteropathogen of swine with worldwide distribution. The source and evolutionary history of this virus is, however, unknown. PDCoV belongs to the Deltacoronavirus genus that comprises predominantly avian CoV. Phylogenetic analysis suggests that PDCoV originated relatively recently from a host-switching event between birds and mammals. Insight into receptor engagement by PDCoV may shed light into such an exceptional phenomenon. Here we report that PDCoV employs host aminopeptidase N (APN) as an entry receptor and interacts with APN via domain B of its spike (S) protein. Infection of porcine cells with PDCoV was drastically reduced by APN knockout and rescued after reconstitution of APN expression. In addition, we observed that PDCoV efficiently infects cells of unusual broad species range, including human and chicken. Accordingly, PDCoV S was found to target the phylogenetically conserved catalytic domain of APN. Moreover, transient expression of porcine, feline, human, and chicken APN renders cells susceptible to PDCoV infection. Binding of PDCoV to an interspecies conserved site on APN may facilitate direct transmission of PDCoV to nonreservoir species, including humans, potentially reflecting the mechanism that enabled a virus, ancestral to PDCoV, to breach the species barrier between birds and mammals. The APN cell surface protein is also used by several members of the Alphacoronavirus genus. Hence, our data constitute the second identification of CoVs from different genera that use the same receptor, implying that CoV receptor selection is subjected to specific restrictions that are still poorly understood.

Human betacoronaviruses OC43 and HKU1 are endemic respiratory pathogens and, while related, originated from independent zoonotic introductions. OC43 is in fact a host-range variant of the species Betacoronavirus-1, and more closely related to bovine coronavirus (BCoV)—its presumptive ancestor—and porcine hemagglutinating encephalomyelitis virus (PHEV). The β1-coronaviruses (β1CoVs) and HKU1 employ glycan-based receptors carrying 9-O-acetylated sialic acid (9-O-Ac-Sia). Receptor binding is mediated by spike protein S, the main determinant of coronavirus host specificity. For BCoV, a crystal structure for the receptor-binding domain S1A is available and for HKU1 a cryoelectron microscopy structure of the complete S ectodomain. However, the location of the receptor-binding site (RBS), arguably the single-most important piece of information, is unknown. Here we solved the 3.0-Å crystal structure of PHEV S1A. We then took a comparative structural analysis approach to map the β1CoV S RBS, using the general design of 9-O-Ac-Sia-binding sites as blueprint, backed-up by automated ligand docking, structure-guided mutagenesis of OC43, BCoV, and PHEV S1A, and infectivity assays with BCoV-S–pseudotyped vesicular stomatitis viruses. The RBS is not exclusive to OC43 and related animal viruses, but is apparently conserved and functional also in HKU1 S1A. The binding affinity of the HKU1 S RBS toward short sialoglycans is significantly lower than that of OC43, which we attribute to differences in local architecture and accessibility, and which may be indicative for differences between the two viruses in receptor fine-specificity. Our findings challenge reports that would map the OC43 RBS elsewhere in S1A and that of HKU1 in domain S1B.

Although serological studies have shown that antibodies against SARS-CoV-2 play an important role in protection against (re)infection, the dynamics of mucosal antibodies during primary infection and their potential impact on viral load and the resolution of disease symptoms remain unclear. During the first pandemic wave, we assessed the longitudinal nasal antibody response in index cases with mild COVID-19 and their household contacts. Nasal and serum antibody responses were analysed for up to nine months. Higher nasal receptor binding domain and spike protein-specific antibody levels at study inclusion were associated with lower viral load. Older age was correlated with more frequent COVID-19 related symptoms. Receptor binding domain and spike protein-specific mucosal antibodies were associated with the resolution of systemic, but not respiratory symptoms. Finally, receptor binding domain and spike protein-specific mucosal antibodies remained elevated up to nine months after symptom onset. There has been limited research on the role of the mucosal immune response to SARS-CoV-2 infection. Here, the authors perform a prospective observational household study and find that mucosal antibody responses are associated with decreased viral load and faster resolution of systemic symptoms.