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

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 is able to 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 cross-neutralizing epitopes on SARS-like viruses. Author summary The ongoing COVID-19 pandemic is caused by SARS-CoV-2. Due to the genetic similarity of SARS-CoV-2 and SARS-CoV, which caused an epidemic in 2003, a few of the SARS-CoV antibodies have now been found to also cross-react with SARS-CoV-2. One such antibody is CR3022, which was isolated from a convalescent SARS patient 14 years ago. However, the 100-fold lower binding to SARS-CoV-2 does not enable neutralization of SARS-CoV-2 compared to SARS-CoV. This study shows that one (P384A) of the four mutational differences in the CR3022 epitope between SARS-CoV and SARS-COV-2 fully accounts for the differences in CR3022 binding affinity and neutralization. These findings advance our understanding of antibody cross-reactivity among SARS-like CoVs with implications for vaccine and therapeutic design.