Functional and antigenic characterization of SARS-CoV-2 spike fusion peptide by deep mutational scanning (preprint)

The fusion peptide of SARS-CoV-2 spike protein is functionally important for membrane fusion during virus entry and is part of a broadly neutralizing epitope. However, sequence determinants at the fusion peptide and its adjacent regions for pathogenicity and antigenicity remain elusive. In this study, we performed a series of deep mutational scanning (DMS) experiments on an S2 region spanning the fusion peptide of authentic SARS-CoV-2 in different cell lines and in the presence of broadly neutralizing antibodies. We identified mutations at residue 813 of the spike protein that reduced TMPRSS2-mediated entry with decreased virulence. In addition, we showed that an F823Y mutation, present in bat betacoronavirus HKU9 spike protein, confers resistance to broadly neutralizing antibodies. Our findings provide mechanistic insights into SARS-CoV-2 pathogenicity and also highlight a potential challenge in developing broadly protective S2-based coronavirus vaccines.

A single C-terminal residue controls SARS-CoV-2 spike trafficking and virion assembly (preprint)

The spike (S) protein of SARS-CoV-2 is delivered to the virion assembly site in the ER-Golgi Intermediate Compartment (ERGIC) from both the ER and cis-Golgi in infected cells1-3. However, the relevance and modulatory mechanism of this bidirectional trafficking are unclear. Here, using structure-function analyses, we show that S incorporation into virions and viral fusogenicity are determined by coatomer-dependent S delivery from the cis-Golgi and restricted by S-coatomer dissociation. Although S mimicry of the host coatomer-binding dibasic motif ensures retrograde trafficking to the ERGIC, avoidance of the host-like C-terminal acidic residue is critical for S-coatomer dissociation and therefore incorporation into virions or export for cell-cell fusion. Because this C-terminal residue is the key determinant of SARS-CoV-2 assembly and fusogenicity, our work provides a framework for the export of S protein encoded in genetic vaccines for surface display and immune activation.

Limited variation between SARS-CoV-2-infected individuals in domain specificity and relative potency of the antibody response against the spike glycoprotein (preprint)

The spike protein of SARS-CoV-2 is arranged as a trimer on the virus surface, composed of three S1 and three S2 subunits. Infected and vaccinated individuals generate antibodies against spike, which can neutralize the virus. Most antibodies target the receptor-binding domain (RBD) and N-terminal domain (NTD) of S1; however, antibodies against other regions of spike have also been isolated. The variation between infected individuals in domain specificity of the antibodies and in their relative neutralization efficacy is still poorly characterized. To this end, we tested serum and plasma samples from 85 COVID-19 convalescent subjects using 7 immunoassays that employ different domains, subunits and oligomeric forms of spike to capture the antibodies. Samples were also tested for their neutralization of pseudovirus containing SARS-CoV-2 spike and of replication-competent SARS-CoV-2. We observed strong correlations between the levels of NTD- and RBD-specific antibodies, with a fixed ratio of each type to all anti-spike antibodies. The relative potency of the response (defined as the measured neutralization efficacy relative to the total level of spike-targeting antibodies) also exhibited limited variation between subjects, and was not associated with the overall amount of anti-spike antibodies produced. Accordingly, the ability of immunoassays that use RBD, NTD and different forms of S1 or S1/S2 as capture antigens to estimate the neutralizing efficacy of convalescent samples was largely similar. These studies suggest that host-to-host variation in the polyclonal response elicited against SARS-CoV-2 spike is primarily limited to the quantity of antibodies generated rather than their domain specificity or relative neutralization potency. IMPORTANCEInfection by SARS-CoV-2 elicits antibodies against various domains of the spike protein, including the RBD, NTD and S2. Different infected individuals generate vastly different amounts of anti-spike antibodies. By contrast, as we show here, there is a remarkable similarity in the properties of the antibodies produced. Different individuals generate the same proportions of antibodies against each domain of the spike protein. Furthermore, the relationship between the amount of anti-spike antibodies produced and their neutralization efficacy of SARS-CoV-2 is highly conserved. Therefore, the observed variation in the neutralizing activity of the antibody response in COVID-19 convalescent subjects is caused by differences in the amounts of antibodies rather than their recognition properties or relative antiviral activity. These findings suggest that COVID-19 vaccine strategies that focus on enhancing the overall level of the antibodies will likely elicit a more uniformly efficacious protective response.