Pre-existing hemagglutinin-specific antibody levels are associated with B-cell repertoire maturation and antibody breadth after influenza vaccination.

Repeated influenza exposures generate complex antibody landscapes, yet how pre-existing antibodies are associated with subsequent vaccine responses remains unclear. We longitudinally tracked 21 adults stratified by pre-vaccination neutralizing titers to examine B-cell dynamics using flow cytometry, B cell receptor (BCR) sequencing, and monoclonal antibody characterization. After vaccination, participants with high baseline titers showed a longitudinal increase in the frequency of HA+CD27-IgD+ naive-phenotype B cells. BCRs recovered from hemagglutinin (HA)-binding (HA+) B cells in the high-titer group displayed lower somatic hypermutation and shorter CDR3s, whereas those recovered from the lower-titer groups showed higher mutation levels and more extensive class switching. Correspondingly, monoclonal antibodies from the high-titer group generally showed narrow reactivity, while those from lower-titer groups showed broader neutralizing activity against historical strains. Clonal tracing further suggested that mAbs from lower-titer groups were more often linked to pre-existing sequences. Together, these findings suggest that pre-existing antibody levels are associated with differences in B-cell repertoire maturation and antibody breadth after influenza vaccination.