C48/80 And Mastoparan Influenza A (h1n1) Protein Subunit Vaccine Adjuvant Effect Of Mucosal Immunity In Mice

Increasing evidences suggest that mucosal immune responses, in particular secretary IgA (SIgA), play an important role in protection against pathogenic infections. Many pathogens initiate infection through mucosae surfaces such as respiratory, gastrointestinal or urogenital mucosae. Influenza virus is a typical kind of pathogens spreading through respiratory tract. In 2009, the global pandemic of H1N1 influenza reminds us of the importance of influenza vaccine research and production. Currently, the influenza vaccine used in China is a kind of injectable split-virus vaccine. However, there is an urgent need to develop a kind of protein based subunit vaccine administered through mucosal route (such as nasal spray), which is not only able to offer the needle-free convenience compared with traditional influenza vaccines, but also to get rid of dependence on embryonated eggs for vaccine production. Obviously, these mucosal influenza vaccines meet the current conditions in China, namely, a large population and unbalanced economic development. Mucosal immune responses are most efficiently induced by the administration of antigens onto mucosal surfaces, but such immunization strategies rarely induce effective systemic and mucosal protective immune responses. The development of mucosal vaccines requires new antigen delivery and adjuvant systems that can efficiently make up these drawbacks of mucosal immunity and promote the application of mucosal vaccines.Compound 48/80 (Compound 48/80, C48/80) is a new kind of mucosal adjuvant developed recently. In this study, H1N1 influenza virus (A/California/04/2009) hemoglutinin (HA) protein was produced and purified in insect cells, then it was combined with C48/80 adjuvant, which made a protein based influenza subunit vaccine. HA-specific IgG antibodies were detected in mice serum after three times of intranasal immunization, HA-specific IgA antibodies were also detected in mice lung, nasal, vaginal washings and fecal extracts. Further, in vitro micro-neutralization assay confirmed that the specific antibodies in serum can neutralize H1N1 influenza viruses (A/California/04/2009). Subsequent live virus challenge results showed that the subunit vaccine had a good protective effect in mice, and 90% mice survived from the lethal dose (1×106TCID50) attack of influenza virus of the same type. Since the adjuvant effect of C48/80 correlates with mast cell degranulation, we hypothesize that another mast cell activator Mastoparan (MP), which has the same ability as C48/80, may also have some mucosal adjuvant effect. We mixed different doses of MP with influenza HA protein and immunized mice intranasally. The experimental results showed that, with increasing doses of MP, HA-specific IgG antibodies in mice serum also increased correspondingly. When the MP dose reached 10μg, HA-specific IgA could be detected in mice mucosal washings and fecal extracts, while the mice survival rate reached 100% in live virus challenge experiments. Detection of total IgE concentration in mice serum indicated that these two adjuvants had no significant effect on total IgE levels in serum. However, no HA-specific IgE could be detected in mice serum by ELISA. Both Thl and Th2 cellular responses could be detected in immunized mice, and mast cell degranulation could be observed in mice nasal mucosae. These results suggest that the C48/80 and MP adjuvanted protein based H1N1 influenza subunit vaccine can protect mice from lethal challenge of influenza virus (1×106TCID50) of the same type. Serving as mucosal adjuvants, C48/80 and MP play an effective role in these subunit vaccines. No increasing serum total IgE concentration can be detected in immunized mice. MP adjuvant mechanism may also correlate with mast cells as C48/80.Innovations of this study mainly exhibit in three aspects. Firstly, we confirm that mast cell activator C48/80 has mucosal adjuvant effect in protein based influenza subunit vaccine, and we explore the MP mucosal adjuvant activity for the first time. Secondly, we indicate that the novel vaccine approach combining recombinant HA and mucosal adjuvant C48/80 or MP is effective in eliciting protective immunity in mice. Lastly, this protein based subunit vaccine immunize by intranasal approach, free from needle injections and embryonated eggs, meets the current situations of China, and has potential values for further research and development.