Bacterial flagellins are potent inducers of innate immune responses in the mouse lung because they bind to TLR5 expressed on the apical surfaces of airway epithelial cells. TLR engagement leads to the initiation of a signaling cascade that results in a pro-inflammatory response with subsequent up-regulation of several cytokines and leads to adaptive immune responses. We examined the ability of two soluble flagellins, a monomeric flagellin expressed in E. coli and a highly purified polymeric flagellin directly isolated from Salmonella, to enhance the efficacy of influenza vaccines in mice. Here we demonstrate that both flagellins co-administered intranasally with inactivated A/PR/8/34 (PR8) virus induced robust increases of systemic influenza-specific IgA and IgG titers and resulted in a more comprehensive humoral response as indicated by the increase of IgG2a and IgG2b subclass responses. Groups immunized with the adjuvanted vaccines were fully protected against high dose lethal challenge by homologous virus whereas inactivated PR8 alone conferred only partial protection. Finally we show that shortly after immunization the adjuvanted vaccines induced a dramatic increase in pro-inflammatory cytokines in the lung, resulting in extensive lung infiltration by granulocytes and monocytes/macrophages. Our results reveal a promising perspective for the use of both soluble monomeric and polymeric flagellin as mucosal vaccine adjuvants to improve protection against influenza epidemics as well as a range of other infectious diseases.

Influenza vaccines with broad cross-protection are urgently needed to prevent an emerging influenza pandemic. A fusion protein of the TLR5-agonist domains from flagellin and multiple repeats of the conserved extracellular domain of the influenza matrix protein 2 (M2e) was constructed, purified and evaluated as such a vaccine. A painless vaccination method suitable for possible self-administration using coated microneedle arrays was investigated for skin-targeted delivery of the fusion protein in a mouse model. The results demonstrate that microneedle immunization induced strong humoral as well as mucosal antibody responses and conferred complete protection against homo- and heterosubtypic lethal virus challenges. Protective efficacy with microneedles was found to be significantly better than that seen with conventional intramuscular injection, and comparable to that observed with intranasal immunization. Because of its advantages for administration, safety and storage, microneedle delivery of M2e-flagellin fusion protein is a promising approach for an easy-to-administer universal influenza vaccine.

Recurring influenza viruses pose an annual threat to public health. A time-saving, cost-effective and egg-independent influenza vaccine approach is important particularly when responding to an emerging pandemic. We fabricated coated, two-layer protein nanoclusters from recombinant trimeric hemagglutinin from an avian-origin H7N9 influenza A virus as an approach for vaccine development in response to an emerging pandemic. Assessment of the virus-specific immune responses and protective efficacy in mice immunized with the nanoclusters demonstrated that the vaccine candidates were highly immunogenic, able to induce protective immunity and long-lasting humoral antibody responses to this virus without the use of adjuvants. Because the advantages of the highly immunogenic coated nanoclusters also include rapid productions in an egg-independent system, this approach has great potential for influenza vaccine production not only in response to an emerging pandemic, but also as a replacement for conventional seasonal influenza vaccines.

The development of a universal influenza vaccine is an ideal strategy to eliminate public health threats from influenza epidemics and pandemics. This ultimate goal is restricted by the low immunogenicity of conserved influenza epitopes. Layered protein nanoparticles composed of well‐designed conserved influenza structures have shown improved immunogenicity with new physical and biochemical features. Herein, structure‐stabilized influenza matrix protein 2 ectodomain (M2e) and M2e‐neuraminidase fusion (M2e‐NA) recombinant proteins are generated and M2e protein nanoparticles and double‐layered M2e‐NA protein nanoparticles are produced by ethanol desolvation and chemical crosslinking. Immunizations with these protein nanoparticles induce immune protection against different viruses of homologous and heterosubtypic NA in mice. Double‐layered M2e‐NA protein nanoparticles induce higher levels of humoral and cellular responses compared with their comprising protein mixture or M2e nanoparticles. Strong cytotoxic T cell responses are induced in the layered M2e‐NA protein nanoparticle groups. Antibody responses contribute to the heterosubtypic NA immune protection. The protective immunity is long lasting. These results demonstrate that double‐layered protein nanoparticles containing structure‐stabilized M2e and NA can be developed into a universal influenza vaccine or a synergistic component of such vaccines. Layered protein nanoparticles can be a general vaccine platform for different pathogens.