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

Vaccines capable of eliciting broadly neutralizing antibodies (bnAbs) are a major goal for pandemic preparedness. A challenge across vaccine fields is how to deliberately recruit the rare B cell clones that recognize conserved epitopes shared across diverse viral variants. BnAbs have been known to frequently emerge through extensive somatic hypermutation during affinity maturation, here we describe an alternative mechanism for bnAb selection. We designed an mRNA vaccine in which two antigenically distinct (severe acute respiratory syndrome coronavirus 2) SARS-CoV-2 variant's (Omicron and Delta; O-Δ) receptor binding domains (RBDs) are physically fused on a single polypeptide. This design is predicted to favor B cell antigen receptors capable of engaging conserved epitopes on both RBDs with enhanced avidity. A matched nondivergent tandem RBD (Delta-Delta; Δ-Δ) served as a control. In mice, immunization elicited potent antibody responses and increased the frequency of cross-reactive B cells, recognizing Delta, Omicron, and the 2002 pandemic strain SARS-CoV RBDs. Using multicolor RBD tetramers and single-cell B cell receptor sequencing, we show that breadth arises via two distinct pathways. The divergent vaccine preferentially enriches clonally distinct cross-reactive B cells (not present within non-cross-reactive B cell pools) with low levels of somatic hypermutation, consistent with selection of germline-biased precursors. In contrast, the matched control vaccine yields 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. This work establishes a simple, modular antigen-design principle in which juxtaposing appropriately divergent antigens on a single scaffold promotes the enrichment of bnAb-prone B cells.