The optimized fusion protein HA1-2-FliC△D2D3 promotes mixed Th1/Th2 immune responses to influenza H7N9 with low induction of systemic proinflammatory cytokines in mice

H7N9 influenza virus has an unusually high fatality rate of approximately 40%, and a safe and effective vaccine against this subtype is urgently needed. Flagellin, a Toll-like receptor (TLR) 5 agonist, has been deemed as a potent adjuvant candidate. However, its high antigenicity and potential for causing inflammatory injury might restrict its clinical application. Previously, we demonstrated that a fusion protein, HA1-2-FliC, comprising the hemagglutinin globular head protein (HA1-2) of H7N9 influenza virus and the full-length Salmonella typhimurium flagellin protein (FliC), had high efficiency against H7N9 in mouse and chicken models. Here, we constructed an improved fusion protein, HA1-2-FliC△D2D3, with HA1-2 fused to the FliC△D2D3 (lacking the hypervariable-region domains D2 and D3 of FliC). HA1-2-FliC△D2D3 exhibited efficient immunoreactivity and TLR5 agonist efficacy, and promoted innate immune-response activation in mouse macrophages, peripheral blood mononuclear cells, and splenocytes, based on cytokine- and chemokine-expression profiles. Mice immunized with HA1-2-FliCΔD2D3 showed significantly lower systemic inflammatory responses (compared with HA1-2-FliC) and highly reduced flagellin-specific antibody production, without affecting HA1-2-specific antibody production and cellular immune responses. Enhanced IFN-γ/IL-4 generation suggested that HA1-2-FliCΔD2D3 maintained balanced Th1/Th2 immune responses. Furthermore, virus challenge was performed in a chicken model. The results showed that chickens receiving FliCΔD2D3 adjuvant vaccine induced high levels of serum neutralizing antibodies, and exhibited a significant reduction of viral loads in throat and cloaca compared to chickens receiving only HA1-2. In conclusion, we constructed the H7N9 influenza subunit vaccine candidate HA1-2-FliCΔD2D3, with reduced immunogenicity against FliC and lower adverse events. The improved adjuvant FliCΔD2D3 can potentially help in developing safe and effective universal protein-based influenza vaccines for humans.