The SARS-CoV-2 S2'-helix compared to stem helix confers higher genetic barrier to antibody resistance.

Understanding the potential for resistance to antibodies targeting conserved epitopes on the coronavirus spike protein is essential for developing broad-spectrum antivirals. The S2'-helix and stem helix represent two key conserved epitopes across multiple coronaviruses, with the S2'-helix being broadly conserved throughout the coronavirus subfamily and the stem helix primarily conserved among betacoronaviruses. Here, we demonstrate that the S2'-helix in SARS-CoV-2 possesses a higher genetic barrier to antibody resistance than the stem helix. Potent escape mutations in the stem helix, including D1153G, Y1155S, F1156L and F1156V, were selected under antibody pressure, and variants such as S1147L, E1151D, D1153G, D1153H, D1153Y and Y1155H were frequently identified in naturally circulating strains. These mutations completely abolished neutralization by some stem helix-targeting antibodies. In contrast, under pressure from S2'-helix-targeting antibodies, we did not detect clear viral escape. Only one naturally occurring mutation in the S2'-helix, L822F, was observed at considerable frequency; it weakly or mildly reduced but did not abolish neutralizing activity. Furthermore, combination therapy with S2'-helix-targeting antibodies synergistically suppressed the emergence of escape mutants selected by stem helix-directed antibodies. Our findings underscore the S2'-helix as a promising target for the design of broadly protective coronavirus therapeutics with a reduced risk of viral escape.