Seasonal Influenza Exposure Elicits Functional Antibody and T-cell Responses to A(H5) Influenza Viruses in Humans

BackgroundHighly pathogenic avian influenza A(H5) viruses pose a pandemic threat, with a history of zoonotic spillovers into humans that are presumed immunologically naive. Whether the general population is currently immunologically naive to circulating A(H5) influenza viruses is unknown. MethodsTo evaluate the presence of cross-reactive immune responses to emerging A(H5) clade 2.3.4.4b influenza viruses in the general population, we conducted comprehensive immune profiling on cross-sectional samples from healthcare workers (n=107). Samples were collected in August and September 2024 in the scope of an ongoing prospective follow-up study: Surveillance of rEspiratory viruses iN healThcare and anImal workers in the NethErLands (SENTINEL). FindingsLow-level antibody responses directed against the A(H5) hemagglutinin (HA) head were detected in a limited number of individuals, but without hemagglutination inhibition activity. Nevertheless, we detected in most participants A(H5)-reactive antibodies with Fc-effector functions, likely directed at the conserved HA stalk. Additionally, we observed abundant neuraminidase (NA) inhibiting antibodies against avian N1s and T-cell responses against HAs and NAs from A(H5) influenza viruses. These responses correlated strongly with immune responses targeting an A(H1N1) seasonal influenza virus, indicating they were likely induced by prior exposures. InterpretationTogether, our findings suggest that partial cross-reactive immunity to A(H5) influenza viruses exists in humans, which may play an important role during future outbreaks, potentially by blunting disease severity. Characterizing such baseline immunity is crucial for accurate pandemic risk assessment and preparedness planning. FundingNetherlands Organization for Health Research and Development (ZonMw), European Unions EU4Health program DURABLE, Dutch Ministries of Agriculture, Fisheries, Food Security and Nature and Health, Welfare and Sport, National Institute of Health - National Institute of Allergies and Infectious Diseases (NIH-NIAID). The funding sources had no role in study design, data collection, analysis, interpretation of the data, or the decision to submit the paper for publication. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed (Jan 1997 - Jul 2025; English) using "influenza", "heterosubtypic", "immunity", "cross-immunity", "baseline", "population", "H5N1", "avian influenza", "clade 2.3.4.4b", "humoral", "antibodies", "cellular", "T-cells", and relevant combinations. Prior work to assess population immunity to A(H5) influenza viruses is fragmented: most reports measured binding antibodies, hemagglutination inhibition (HI) antibodies, or neutralizing antibodies exclusively, and few examined T-cell responses. The measurement of integrated antibody breadth, functional HI, neuraminidase inhibition (NI), and antibody-dependent cellular cytotoxicity (ADCC) activity, as well as T-cell responses, has not been described in cross-sectional population studies. Added value of this studyWe present profiling of immunity to A(H5) influenza viruses in an inferred unexposed cross-sectional sample of healthcare workers (n=107). This comprehensive analysis revealed patterns that were not detected in earlier single-endpoint studies: (i) A(H5) binding antibodies mainly targeted the conserved stalk, with higher titers in adults over 60 years, (ii) A(H5)-reactive NI-and ADCC-mediating antibodies were common, and (iii) cross-reactive T-cell responses against HAs and NAs of A(H5) influenza viruses were detected. The rational selection of antigens from both seasonal and avian influenza viruses allowed for direct comparison of multiple effector mechanisms. These design choices led to the first comprehensive map of humoral and T-cell effector functions against A(H5) influenza viruses, establishing a robust baseline for future vaccine evaluations and pandemic-risk assessments. Implications of all the available evidenceOur findings in combination with existing research shows that that immunity to A(H5) influenza viruses is widespread. It is likely that repeated exposure to seasonal influenza viruses led to the development of a cross-reactive antibody and T-cell repertoire capable of recognizing emerging A(H5) influenza viruses. While the protective value of the cross-reactive immune responses remains speculative, evidence from model systems suggests these could confer protection, potentially blunting disease severity in the case of a future outbreak.