Memory B Cell Repertoire of a Repeatedly Vaccinated Individual Includes Influenza Hemagglutinin-Specific Cross-Group Binders with Affinity Beyond A(H1N1)pdm09 and A(H3N2) Seasonal Strains.

Background/Objectives: Humans are primarily infected by two distinct seasonal influenza A virus (IAV) subtypes, A(H1N1)pdm09 and A(H3N2). Because IAV mutates rapidly, it continuously escapes existing protective antibodies. However, repeated exposure to these evolving strains increases the diversity of virus-specific memory B cell (MBC) population. To investigate the extent of this diversity and cross-reactivity of IAV- specific immunity in adults, and its possible impact on emerging strains, we analyzed the hemagglutinin (HA)-specific MBC repertoire of an adult frequently immunized with seasonal vaccines. Methods: High-throughput single-cell sequencing of HA probe-positive MBCs collected from an individual with repeated seasonal influenza vaccination was performed. Monoclonal antibodies (mAbs) from multicell clonotypes were tested for HA binding and influenza virus neutralization. Results: Two clonotypes were identified that bound to both A(H1) and A(H3) HAs from strains circulating before and after the season of blood collection. Both clonotypes neutralized A(H3N2) strains, while one also neutralized A(H1N1)pdm09 strains. Notably, these cross-group binders showed affinity for A(H5), A(H7) and A(H9) avian influenza HAs and neutralized bovine A(H5N1) IAV. Conclusions: Our findings suggest that seasonal vaccination can support pre-existing cross-reactive B-cell immunity in some individuals. This underscores the importance of studying influenza-specific MBC repertoires across diverse populations to better understand immune dynamics and improve vaccine design.