Antigenic seniority and convergent haemagglutinin evolution shaped the immune landscape preceding influenza B/Yamagata's extinction

Background: In 2020, the B/Yamagata lineage of influenza B disappeared from circulation. Understanding the immunological conditions preceding this loss can help explain why the lineage disappeared and has implications for our understanding of viral evolution and ongoing vaccine policies. Methods: We measured neutralising antibody responses in age- and sex-matched blood donor cohorts collected in 2020, 2023, and 2025 (n=114 per cohort) against a panel of pseudotyped influenza B viruses spanning 79 years of evolution, validated against live virus neutralisation assays. We performed antibody pulldown assays using full-length and head domain HA proteins from B/Yamagata (B/Phuket/3073/2013) and B/Victoria (B/Washington/02/2019) lineages, testing purified antibodies against chronologically distinct pseudotyped viruses. We used Bayesian network analysis, and LASSO regression to identify potential molecular determinants of cross-lineage neutralisation which were then confirmed via site-directed mutagenesis, and epitope-specific peptide pulldowns. Findings: In 2020, population immunity was asymmetrically focused on B/Yamagata viruses. Despite no B/Yamagata circulation after 2020, neutralisation of recent B/Yamagata strains increased in 2023 while B/Victoria responses remained unchanged, consistent with antigenic seniority directing recall responses toward B/Yamagata. This trend inverted by 2025, with B/Yamagata immunity declining and B/Victoria immunity increasing. Antigen-specific purified antibodies showed lineage-biased potency, neutralising B/Yamagata viruses more effectively than B/Victoria viruses. We identified the 120-loop of the HA head domain as a critical cross-lineage epitope, with the charge state at a single convergently evolving residue at position 131 determining cross-reactive potency between lineages. Interpretation: B/Yamagata's acquisition of a positively charged residue at position 131, shared with B/Victoria, likely increased its cross-neutralisation by Victoria-raised immunity preceding its disappearance. Although antigenic seniority sustained cross-reactive responses to B/Yamagata after its extinction, the waning of this effect by 2025 suggests that population immunity to B/Yamagata is now declining. This has implications for influenza B vaccine policy.