Worldwide appearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) with increased transmissibility and resistance to therapeutic antibodies necessitates the discovery of broadly reactive antibodies. We isolated receptor binding domain (RBD) targeting antibodies that potently neutralize 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Structural and functional studies revealed the molecular basis for antibody binding and showed that antibody combinations reduce the generation of escape mutants, suggesting a potential means to mitigate development of therapeutic resistance.

Investigation of antibody responses from convalescent subjects infected with the Washington-1 (WA-1) strain for reactivity against WA-1 and VOCs can inform improvements to vaccine design and therapeutics.

Blood from 22 convalescent subjects who recovered from SARS-CoV-2 WA-1 infection was screened for neutralizing and binding activity, and four subjects with high reactivity against the WA-1 variant were selected for antibody isolation. SARS-CoV-2 spike (S)–reactive antibodies were identified through B cell sorting with S protein–based probes. WA-1 live-virus neutralization assays identified four RBD-targeting antibodies with high potency [half-maximal inhibitory concentration (IC₅₀) 2.1 to 4.8 ng/ml], two of which were derived from the same IGHV1-58 germline but from different donors. Antigen-binding fragments (Fabs) of these antibodies exhibited nanomolar affinity to S (2.3 to 7.3 nM). Competition assays and electron microscopy indicated that two of the most potent antibodies blocked angiotensin-converting enzyme 2 (ACE2) and bound open conformation RBD, whereas the other two bound both up and down conformations of RBD and blocked ACE2 binding. Binding and lentivirus neutralization assays against 13 circulating VOCs or variants of interest—including B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, P.1, P.2, B.1.617.1, and B.1.617.2—indicated that these antibodies were highly potent against VOCs despite being isolated from subjects infected with early ancestral SARS-CoV-2 viruses. Cryo-EM studies of the two most potent antibodies in complex with S revealed that these antibodies target a site of vulnerability on RBD but have minimal contacts with mutational hotspots, defining the structural basis for their high effectiveness against the emerging VOCs and further delineating an IGHV1-58 antibody supersite. To investigate potential mechanisms of escape, we applied antibody selection pressure to replication-competent vesicular stomatitis virus (rcVSV) expressing the WA-1 SARS-CoV-2 S (rcVSV-SARS2) and identified S mutations that conferred in vitro resistance. We evaluated these antibodies individually or in combinations for their capacity to prevent rcVSV-SARS2 escape and discovered that antibody combinations with complementary modes of recognition to the RBD lowered the risk of resistance.

Our study demonstrates that convalescent subjects previously infected with ancestral variant SARS-CoV-2 produce antibodies that cross-neutralize emerging VOCs with high potency. Structural and functional analyses reveal that antibody breadth is mediated by targeting a site of vulnerability at the RBD tip offset from major mutational hotspots in VOCs. Selective boosting of immune responses targeting specific RBD epitopes, such as the sites defined by these antibodies, may induce breadth against current and future VOCs.