Single‐Dose Intranasal Chimeric Influenza A–B Nucleoprotein Vaccine Confers Dual Protection in a Mouse Model

The influenza nucleoprotein (NP) is conserved and type‑specific, making it a key target for universal vaccine development. NP sequences mutate slowly and share > 90% amino‑acid homology within a virus type; NP is recognized by cytotoxic T lymphocytes (CTLs), which clear infected cells and contribute to broad heterosubtypic immunity. Intranasal NP‑based vaccines have previously protected mice against diverse influenza A subtypes (H1N1, H3N2, H5N2, H7N9 and H9N2) and two lineages of influenza B (B/Yamagata and B/Victoria lineages), yet NP is type‑specific, and vaccines often target only one type. To achieve cross‑type protection, we engineered recombinant adenoviruses expressing NP from influenza A (A‑NP), influenza B (B‑NP) or chimeric constructs linking the two sequences (AB‑NP and BA‑NP). Four replication‑deficient adenoviruses encoding A‑NP, B‑NP, AB‑NP, or BA‑NP were produced. BALB/c mice were anaesthetized and immunized intranasally once with either a mixture of A‑NP plus B‑NP (A + B), or a single chimeric vaccine (AB‑NP or BA‑NP). All regimens induced robust systemic specific IgG, mucosal IgA responses, and CTLs, reactive to both A and B NP. Each group showed complete protection from both influenza A and B challenges compared with controls. Of note, the AB‑NP fusion vaccine conferred the highest survival rate and the least morbidity. Our data demonstrate that fusing influenza A and B NP sequences into a single recombinant antigen induces cross‑type immunity after a single intranasal dose. This extends previous findings that NP‑based vaccines protect against multiple influenza A subtypes and both B lineages and suggests that chimeric NP vaccines could form the basis of a real universal influenza vaccine strategy.