GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE316nnn/GSE316421/primaryOK200TranscriptomicsMus musculusExpression profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE316421GEOGSEfalseEngineering antigenic breadth against SARS-CoV-2 by pairing divergent RBDs within a single mRNA immunogenVaccines capable of eliciting broadly neutralising antibodies (bnAbs)are a major goal for pandemic preparedness. A persistent challengeacross vaccine fields is how to deliberately recruit the rare B cellclones that recognise conserved epitopes shared across diverse viralvariants. BnAbs have been known to frequently emerge throughextensive somatic hypermutation during affinity maturation, here wedescribe an alternative, structure-driven mechanism for bnAbselection. We designed an mRNA vaccine in which two antigenicallydistinct SARS-CoV-2 variant’s (Omicron and Delta; O-Δ) receptorbinding domains (RBDs) are physically fused on a singlepolypeptide. This design is predicted to favour B cell antigenreceptors capable of engaging conserved epitopes on both RBDs withenhanced avidity. A matched non-divergent tandem RBD (Delta-Delta;Δ-Δ) served as a control. 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.2026/08/13GSE316421GSM9453026GSM945302532159316421Mus musculus