NP_mut- and M2e-decorated T4 phage nanoparticles induce mucosal immunity and cross-group protection against influenza...

Inducing cross-group protection against diverse influenza A virus (IAV) subtypes remains a major goal in next-generation influenza vaccine development. Nucleoprotein (NP) and the extracellular domain of matrix protein 2 (M2e) are attractive conserved targets, but face distinct hurdles: NP co-purified from Escherichia coli often contains pro-inflammatory bacterial RNA, raising safety concerns, while its nucleic-acid-binding-deficient version (NP_mut) improves safety but suffers from reduced immunogenicity. In addition, M2e alone is weakly immunogenic. To address these limitations, we developed a dual-antigen T4 phage-based nanoparticle vaccine by assembling an NP_mut-3M2e fusion protein, containing three tandem M2e segments (3M2e), onto T4 phage capsids. Adjuvant-free intranasal immunization in mice elicited robust mucosal and systemic immunity, including high sIgA and IgG titers and potent CD8⁺ T-cell responses. It also conferred significant cross-group protection against lethal challenge with IAV strains from distinct hemagglutinin (HA) groups, as evidenced by improved survival, reduced body weight loss, and attenuated lung pathology. These findings support intranasal NP_mut-3M2e-T4 VLP as a promising vaccine candidate that combines conserved cross-group antigens with a T4-based delivery platform to achieve cross-group protection against IAV.