Multi-compartmental analysis of SARS-CoV-2 spike-immunized macaques highlights extensive B cell lineage diversification underpinning neutralizing breadth

The continued evolution of SARS-CoV-2 underscores the need to understand qualitative aspects of the humoral immune response elicited by spike immunization. Here, we combined monoclonal antibody (mAb) isolation with deep B cell receptor (BCR) repertoire sequencing of rhesus macaques immunized with prefusion-stabilized ancestral spike glycoprotein administered in adjuvant. By tracing spike-specific B cell lineages at different time points and in multiple immune compartments, we demonstrate gradual accumulation of somatic hypermutation (SHM) and extensive dissemination of vaccine-elicited B cells in draining and non-draining lymphoid compartments, including the bone marrow (BM), spleen and, most notably, the periaortic lymph node (perLN). Phylogenetic analysis of spike-specific antibody lineages identified through deep repertoire sequencing delineated extensive intra-clonal diversification that broadened neutralizing antibody activity. A cryo-EM structure of the spike in complex with a broadly neutralizing mAb pinpointed interactions with a set of semi-conserved residues on the RBD, in part mediated by amino acids acquired by SHM, providing a molecular basis for the observed breadth. Our findings highlight that immunization leads to extensive intra-clonal B cell evolution where an individual lineage can both retain the original epitope specificity and evolve to recognize previously not encountered spike variants, providing a deeper understanding of Ab repertoire development following vaccination.