Background: The emergence of highly pathogenic avian influenza A (H5N1) clade 2.3.4.4b in dairy cattle and human cases raises urgent pandemic concerns. A critical question is whether seasonal influenza vaccines elicit cross-reactive immunity against this novel zoonotic strain, potentially contributing to pre-existing population immunity.

Methods: We combined structural bioinformatics and serological analysis. Hemagglutinin (HA) protein sequence and structural conservation were assessed between World Health Organization WHO-recommended 2024-2025 vaccine strains (H1N1 A/Victoria/4897/2022, H3N2 A/Thailand/8/2022) and bovine H5N1 (A/Texas/37/2024). Cross-reactive antibodies were measured in serum from 46 vaccinated individuals using ELISA against A/Texas/37/2024 HA. Endpoint titers were the highest reciprocal dilution with absorbance >2.1-fold background. Statistical analyses included Pearson correlation (age, dose, time) and Wilcoxon rank-sum/Chi-squared tests (group comparisons).

Results: Structural analysis revealed 79.3% amino acid identity in the HA2 subunit between H1N1 and H5N1, with conserved epitopes in the stalk domain. Serologically, 41.3% (19/46) of vaccinated individuals had cross-reactive HA-binding antibodies with titers ≥1,280. No significant associations were found with sex, vaccine type, brand, or number of doses. A significant positive correlation existed between age and antibody titer (Pearson´s R = 0.51, p < 0.001); individuals over 60 years had higher titers than younger groups.

Conclusions: Seasonal influenza vaccination is associated with cross-reactive HA-binding antibodies against bovine H5N1 clade 2.3.4.4b in a substantial proportion of individuals, with responses increasing with age. This suggests pre-existing immunity from vaccination or prior exposures may influence responses to this zoonotic threat. However, the functional neutralizing capacity and protective efficacy of these antibodies are unproven. These findings highlight the potential immunological footprint of current vaccines against emerging strains and support further investigation into cross-protection. They also reinforce the importance of conserved HA epitopes as targets for next-generation universal influenza vaccines.