Skin Vaccination With Dissolvable Microneedle Patches Incorporating Influenza Neuraminidase And Flagellin Protein Nanoparticles Induces Broad Immune Protection Against Multiple Influenza Viruses

We generated self-adjuvanted protein nanopartides of conserved influenza antigens and immunized mice via skin vaccination with dissolvable microneedle patches (MNPs) to increase the strength and breadth of immune responses. We produced influenza matrix protein 2 (M2) ectodomain (M2e) nanopartides via ethanol desolvation and double-layered protein nanoparticles NA1/M2e (shell/core), NA1-FliC/M2e, NA2/M2e, and NA2-FIiC/M2e by chemically cross-linking influenza neuraminidase (NA) and flagellin (FliC) onto the surfaces of the M2e nanoparticles. The resulting nanopartides retained the FliC toll-like receptor 5 (TLRS) innate signaling activity and significantly increased antigen uptake and dendritic cell maturation in vitro. We incorporated the nanopartides into MNPs for skin vaccination in mice. The nanoparticle MNPs significantly increased the M2e- and NA-specific antibody levels, the numbers of germinal center B cells, and IL-4 positive splenocytes. Double-layered nanoparticle MNP skin vaccination protected mice against homologous and heterosubtypic influenza viruses. Our results demonstrated that the MNP skin vaccination of NA-FliC/M2e nanoparticles could be developed into a standalone or synergistic component of a universal influenza vaccine strategy.