The development of a universal influenza vaccine is an ideal strategy to eliminate public health threats from influenza epidemics and pandemics. Influenza conserved structures are outstanding immunogens for such a vaccine. Layered protein nanoparticles composed of well-designed conserved influenza structures have improved immunogenicity with new physical and biochemical features. We constructed and purified structure-stabilized influenza matrix protein 2 ectodomain (M2e) and M2e-neuraminidase fusion proteins (M2e-NAs). We produced double-layered M2e-NA protein nanoparticles by chemical crosslinking M2e-NA onto M2e nanoparticle surfaces. Immunizations with these protein nanoparticles induced immune protection against influenza viruses with homologous and heterosubtypic NA in mice. Double-layered M2e-NA protein nanoparticles induced higher levels of humoral and cellular responses compared with their comprising protein mixture or M2e nanoparticles. In addition, the layered M2e-NA protein nanoparticle group demonstrated strong cytotoxic T cell responses. We also found that antibody responses contributed to heterosubtypic influenza viral immune protection. The protective immunity was long-lasting.

To favorite rapid distribution and convenient administration of a universal influenza vaccine, we incorporated the FliC-adjuvanted M2e-NA nanoparticles into logistically simple microneedle patches (MNPs) for skin vaccination in mice. The nanoparticle MNPs significantly increased M2e and NA-specific antibody levels, the numbers of germinal center B cells, and IL-4 secreting splenocytes. MNPs incorporating the double-layered nanoparticles protected mice against homologous and heterosubtypic influenza viruses. Our results demonstrated that M2e-NA nanoparticles could be developed into a standalone or synergistic component of a universal influenza vaccine strategy. MNP skin vaccination can logistically simplify the vaccines for extensive distribution and administration.