Retooling a novel influenza B hemagglutinin to redirect neutralizing antibodies against B/Victoria strains

Since the COVID-19 pandemic, only influenza B viruses (IBVs) from the Victoria (B/VIC) lineage have continued to circulate in humans. The hemagglutinin (HA) protein of these viruses has undergone substantial antigenic evolution, rendering antibodies elicited by older IBVs less effective at neutralization and protection. The computationally optimized broadly reactive antigen (COBRA) influenza B HA protein, BC2, elicits cross-lineage neutralizing antibodies against pre-COVID-19 IBVs. However, following the disappearance of the Yamagata (B/YAM) lineage, BC2 HA required updating to improve protection against contemporary B/VIC strains. To address this, a series of point mutations were introduced into BC2 HA to redirect antibody responses toward modern B/VIC-like viruses. This modified HA, named BC17, was evaluated in mice for immunogenicity and protective efficacy. Immune responses were compared with those elicited by BC2 HA and a representative wild-type B/VIC HA, B/CO/17. Mice vaccinated with BC17 HA exhibited significantly higher hemagglutination-inhibition (HAI) and neutralization titers against B/VIC viruses compared to mice vaccinated with BC2 HA or B/CO/17 HA. Consistent with these findings, BC17-vaccinated mice experienced significantly less weight loss than BC2-vaccinated mice following viral challenge. Overall, BC17 HA elicited broadly protective antibody responses against recently circulating B/VIC strains. The enhanced neutralization elicited by BC17 HA relative to BC2 HA was hypothesized to result primarily from amino acid changes within the 160-loop, a key antigenic region located at the membrane-distal tip of HA. Modern B/VIC viruses in the V1a.3 subclade contain a characteristic three-amino acid deletion in this loop that is absent in older strains. The design of the BC17 HA incorporated these deletions along with additional mutations. Restoration of these deleted residues in a BC17 mutant HA modestly reduced HAI titers but did not abrogate protection against viral challenge, indicating that the 160-loop contributes to, but does not solely determine, the enhanced antigenicity of BC17 HA.