Multiplexed antigen panel analysis identifies B cell phenotype and receptor genetic contributions to antibody breadth

The cellular, immunogenetic, and antigenic factors affecting the breadth of viral antigen variants recognized by human antibody responses are poorly defined. We developed highly multiplexed panels of DNA-tagged SARS-CoV-2 antigens from up to 20 viral variants to label and sort 6,262 antigen-binding circulating B cells from previously naive mRNA vaccinees or infected patients, and from deceased organ donor lymphoid tissues, to enable antigen receptor and transcriptome sequencing. Atypical B cells and a subset of class-switched memory cells with evidence of recent germinal center exposure were enriched for antigen binding. In contrast to atypical B cells, post-germinal center B cells showed progressively increasing variant binding breadth and somatic hypermutation over time. Vaccination, compared with infection, preferentially stimulated B cells expressing antibodies with inherently high antigen-binding breadth. This large-scale analysis reveals key determinants of antigen-binding breadth, critical for understanding responses to viral infection and guiding vaccine development against rapidly mutating viruses.