In Vivo Evolution of Antibody CR3022 Expands Cross-Neutralization of SARS-CoV-2 Variants and Informs Pan-sarbecovirus Immunity

Monoclonal antibody CR3022, isolated from a patient infected during the 2003 SARS-CoV outbreak, shows cross-reactive binding and, with targeted mutagenesis, neutralization of SARS-CoV-2. These findings suggest that targeting the CR3022 epitope may be a promising candidate for broadly protecting against a wide range of coronavirus variants and zoonotic strains that may pose future pandemic threats. However, it is unclear how readily and broadly this epitope can be adapted against variant coronavirus strains in vivo. To address this issue, we generated immunoglobulin (Ig)-humanized mice expressing the predicted germline heavy chain of CR3022 for a direct evaluation of the epitopes' in vivo adaptation potential. Following a SARS-CoV/SARS-CoV-2 sequential immunization regimen, we observed the convergent evolution of the germline CR3022 variable gene through somatic hypermutation to resemble that of the mutated WT counterpart. Most CR3022-like clones acquired cross-neutralizing activity against SARS-CoV-2, plus adaptation against key variants and more divergent Sarbecovirus strains. Notably, while simple prime-boost strategies drove CR3022-epitope targeting, an intensive vaccination protocol drove dominant responses to other epitopes even with a predominance of CR3022-like precursors. X-ray crystallography revealed that SARS-CoV-2-neutralizing CR3022 antibodies readily exhibited enhanced affinity due to varied adaptive changes, resulting in significantly increased electrostatic interactions with the receptor-binding domain (RBD). Overall, these findings show ready adaptation of CR3022-like clones by minor shifts in affinity with appropriate vaccination strategies. Overall design: This study includes B cells sorted from CR3022 knock-in mice following immunization with SARS-CoV or SARS-CoV-2 spike proteins under different regimens. Samples represent distinct time points post-immunization and include biological replicates.