Prior immunity to seasonal influenza A(H3N2) virus confers varying levels of cross-protection against challenge with clade 2.3.4.4b A(H5N1), A(H7N9), or A(H9N2) virus in a ferret model

Evaluating how prior immunity to seasonal influenza viruses influences subsequent zoonotic influenza A virus (IAV) infection in animal models is critical for pandemic preparedness. In this study, we investigated the cross-protective effect of pre-existing A(H3N2) immunity in ferrets challenged with three distinct subtypes of zoonotic IAVs: low pathogenic A(H7N9) and A(H9N2) viruses, and highly pathogenic clade 2.3.4.4b A(H5N1) virus. Our results show that A(H3N2) preimmunity conferred some protection against A(H5N1) and A(H9N2) virus infection, as evidenced by more rapid viral clearance in the upper respiratory tract, reduced virus shedding in the nasal wash on select days post-inoculation, and a lowered frequency of viral detection in specific tissues compared with naive animals. In contrast, A(H3N2) preimmunity provided minimal cross-protection against A(H7N9) infection, as weight loss and viral dissemination in tissues were not significantly reduced in A(H3N2) preimmune ferrets relative to naive animals. These findings highlight the variable breadth and magnitude of cross-protection elicited by prior seasonal IAV immunity against zoonotic influenza virus challenges in the ferret model. Seasonal influenza A(H3N2) preimmunity provided differing levels of cross-protection against zoonotic influenza A virus infections in ferrets. IMPORTANCE: Understanding how pre-existing influenza immunity shapes susceptibility to zoonotic IAV infections is essential for risk assessment and pandemic preparedness. By evaluating the extent and magnitude of cross-protection conferred by A(H3N2) preimmunity against three distinct zoonotic subtypes-A(H5N1), A(H7N9), and A(H9N2)-in the ferret model, we show that HA or NA relatedness to the priming A(H3N2) virus is not sufficient to predict cross-protection outcomes. Notably, A(H3N2) preimmunity provided some protection against clade 2.3.4.4b A(H5N1) virus infection despite sharing neither an HA within the same phylogenetic group nor an NA within the same subtype, whereas cross-protection against A(H7N9), which shares a group 2 HA, was limited. These findings underscore the need to evaluate both humoral and cell-mediated immunity against multiple viral antigens when assessing cross-protection elicited by seasonal influenza viruses.