Engineering antigenic breadth against SARS-CoV-2 by pairing divergent RBDs within a single mRNA immunogen

The divergent (O-Δ) immunogen was robustlyexpressed and retained high-affinity ACE2 binding. In mice,immunisation elicited potent antibody responses and increased thefrequency of antigen-specific cross-reactive B cells, recognisingDelta, Omicron, and the 2002 pandemic strain SARS-CoV RBDs. Usingmulticolour RBD tetramers and single-cell B cell receptor sequencing,we show that breadth arises via two distinct pathways.

The divergentvaccine preferentially enriches clonally distinct cross-reactive Bcells (not present within non-cross-reactive B cell pools) with lowlevels of somatic hypermutation (SHM), consistent with selection ofgermline-biased precursors. In contrast, the matched control vaccineyields cross-reactivity primarily within existing clonal lineages(clonal overlap between cross- reactive and non-cross-reactive cells)and at higher mutational burdens, consistent with affinity-maturation-driven acquisition of breadth.

Together, these findingsdemonstrate that antigen structure can bias B cell selection towardscross-reactive specificities without requiring extensive SHM. Thiswork establishes a simple, modular antigen-design principle in whichjuxtaposing appropriately divergent antigens on a single scaffoldpromotes the enrichment of bnAb-prone B cells, providing a scalablestrategy for vaccine development against rapidly evolving pathogens.