Serological imprinting and vaccination history shape adult influenza antibody landscapes and H5N1 cross‑reactivity

Influenza vaccines are central to mitigating the ~1 billion annual influenza cases, yet their effectiveness remains modest, and how immune history shapes protection against emerging strains is unclear. The rapid spread of highly pathogenic avian influenza H5N1 clade 2.3.4.4b in birds, poultry, and cattle has intensified concerns about pandemic risk, but how pre‑existing seasonal immunity and lifelong imprinting influence responses to this lineage is unknown. Here, we use a customized 26‑plex bead‑based immunoassay to longitudinally profile hemagglutinin (HA)‑ and neuraminidase (NA)‑specific antibodies in 30 adults in New York City over three consecutive influenza seasons (2021–2024). Seasonal vaccination induced broad, predominantly IgG1 responses that rose significantly one month post‑vaccination, waned between seasons, and left stable, antigen‑specific serological imprints. We observe low‑to‑intermediate but consistent antibody levels to contemporary H5N1 clade 2.3.4.4b HAs following routine seasonal vaccination, revealing partial H5 cross‑reactivity without known H5 exposure. For six antigens, baseline levels or vaccine‑induced boosts tracked with birth year in patterns that mirror historical subtype circulation, demonstrating subtype‑ and antigen‑specific imprinting, including for H5N1. These findings define how imprinting and vaccination history jointly structure adult influenza antibody landscapes and suggest that multiplex, personalized serology could inform tailored vaccination strategies and pre-pandemic H5N1 preparedness.