Development And Immune Responses Of H7N9 Influenza Subunit Vaccine Based On Salmonella Typhimurium Flagellin

An influenza virus, subtype H7N9, has circulated through China since March 2013. Its higher rates of infection in a wide range of locations within China and the associated increased likelihood of human-to-human transmission have caused global concern. Recombinant subunit vaccines provide safe and targeted protection against viral infections. However, the protective efficacy of recombinant subunit vaccines tends to be less potent than vaccines made from whole viruses. Studies have shown that bacterial flagellin has strong adjuvant activity and induces protective immune responses. Consecutive cases of human infection with H7N9 influenza viruses in China promoted efforts to develop a safe and effective vaccine. Subunit vaccines introduced by intranasal administration are highly desired because it can not only prevent disease but can also block an infection at its primary site.1. The expression and characterization of the fusion protein of the H7N9 influenza hemagglutinin HA1-2 gene and Salmonella Typhimuriumy fliC geneIn this study, we used overlap-PCR to generate an H7N9 influenza recombinant subunit vaccine that fused the globular head domain (HA1-2, aa 62-284) of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, Salmonella Typhimurium flagellin (fliC), resulting fusion gene HA1-2-fliC with 2187 bp. The HA1-2 gene fragment (669 bp) was also amplified by PCR. Both HA1-2 and HA1-2-fliC PCR products were cloned into the pCold vectors, and efficiently expressed in an Escherichia coli prokaryotic expression system, and the immunoreactivity of the purified HA1-2-fliC and HA 1-2 were confirmed by Western blotting using anti-fliC or anti-H7N9 virus polyclonal antibodies. Moreover, a significantly stronger IL-8 secreted by HEK293-mTLR5 cells was induced by HA1-2-fliC in the TLR5-stimulating activity assay. These fingdings confirmed that the HA1-2-fliC moiety could be faithfully refolded to take on the native HA and fliC conformations.2. Immune responses elicited by the fusion protein HA1-2-fliC following intraperitoneal immunizationGroups of C3H/HeJ mice were vaccinated intraperitoneally with three doses of HA1-2, HA1-2-fliC, or PBS on days 0,14, and 28. The animals were bled 12 days following the second and third immunization. Antibody titers were measured by ELISA or HAI assay. The fusion protein elicited significant and robust HA1-2-specific serum IgG titers, maintaining high levels for at least 3 months in the vaccinated animals, and induced similar levels of HA1-2-specific IgG1 and IgG2a that were significantly higher than HA1-2 group. HA1-2-fliC was also found to be capable of triggering the production of neutralizing antibodies, as assessed by measuring hemagglutination inhibition titers. We concluded that immunization with HA1-2-fliC induces potent HA1-2-specific responses, offering significant promise for the development of a successful recombinant subunit vaccine for avian influenza A (H7N9).3. Immune responses elicited by the fusion protein HAl-2-fliC following intranasal immunizationTo further evaluated the mucosal adjuvant activity of flagellin for the H7N9 influenza subunit vaccine. Groups of C3H/HeJ mice were vaccinated intranasally with three doses of HA 1-2, HA1-2-fliC, or PBS on days 0,14, and 28, using CTB adjuvant as a positive control. The humoral, cellular, and mucosal immune responses were measured. Mice immunized with the HA1-2-fliC showed significantly higher levels of HA1-2 specific IgG and IgA titers in serum, nasal wash and bronchial alveolar lavage fluid. Hemagglutination inhibition antibody titers were enhanced in HA1-2-fliC groups. Moreover, we observed that the activation and proliferation of splenocytes and HA1-2 specific IFN-γ and IL-4-producing splenocytes were markedly increased in the HA1-2-fliC groups, which suggested that HA1-2-fliC induced both Thl and Th2 immune responses, supporting the pattern of antibody isotypes (IgG1/IgG2a ratios) in serum. These findings provide the basis of further study toward the development of H7N9 influenza HA1-2 mucosal subunit vaccines.