A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Abstract

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that can cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential for cross-neutralizing epitopes on SARS-like viruses.

Cross-Neutralization of a SARS-CoV-2 Antibody to a Functionally Conserved Site Is Mediated by Avidity

“…These observations are similar to some broadly neutralizing influenza antibodies to the hemagglutinin (HA) receptor binding site, where bivalent binding can increase avidity and neutralization breadth (Ekiert et al, 2012;Lee et al, 2012). Nevertheless, we have shown recently that the CR3022 Fab and IgG have similar neutralization potency as a SARS-CoV-2 variant with enhanced binding affinity for CR3022 (Wu et al, 2020a), suggesting that an avidity effect is not universally observed for all RBD-targeting antibodies, especially to this particular epitope, which is targeted by CR3022 and COVA1-16.…”

Affinity maturation of cross-reactive CR3022 antibody against the receptor binding domain of SARS-CoV-2 via in silico site-directed mutagenesis

“…Despite the direct overlap of the epitopes with residues binding to the human ACE-2 receptor and higher binding a nity than SARS-CoV-2 S protein RBD and hACE-2, these three mutants display average interaction energies for the wild type CR3022 and the lead L-K45S. Wu et al demonstrate that the P384 residue is responsible for determining the a nity between the cross-reactive antibody and SARS-CoV-2 RBD [26]. In SARS-CoV, this position consists of alanine instead of proline and is the reason behind successful neutralization by CR3022, unlike the case with binding of the same antibody with SARS-CoV-2 RBD.…”

Synthetic protein antigens for COVID-19 diagnostics

“…The affinity of the cross reactive SARS-CoV-1 antibody, CR3022, whose epitope is located in the conserved area just upstream of the JS7 site, 28 was greatly reduced by the single amino acid change, P to A found in the SARS-CoV-2 protein. 29 Further, while all 9 COVID-19 patients in this study had antibodies that recognize the RBD of SARS-CoV-2, one patient with mild illness did not recognize the whole S protein of SARS-CoV-1 (protein 2), despite the 80% overall sequence identity (Table 1, S1). Further, some variants in this area, which also arose as escape mutants of monoclonal antibodies 40 , can render the virus insensitive to neutralization by convalescent sera 41 .…”

“…Many assays for determining prior COVID-19 infection are based on detecting antibodies in sera to recombinant versions of the surface (S) protein, which was previously identified as the binding site for neutralizing antibodies produced in response to SARS-CoV-1. At least one SARS-CoV-1 neutralizing monoclonal antibody 28,29 also binds with less affinity to the S protein SARS-CoV-2 30 , which varies by about 20% throughout its length ( Fig. S1).…”