| The ideal adjuvant for hepatitis B vaccines can elicit both strong humoral and cellular immune responses. So far, Alum is used as adjuvant in the hepatitis B vaccines currently commercialized, but it does work only by intramuscular injection route and it can’t elicit effective cellular immune responses. We isolate the WHlfungin peptide from the culture of Bacillus, the preliminary study demonstrated that it has been explored for its potential as an alternative adjuvant. In this study, a formulation containing hepatitis B surface antigen (HBsAg) adjuvanted with WHlfungin was used to immune KM mice by different routes, and we evaluate the adjuvant effect of the WH1fungin peptide on the immune responses to the hepatitis B surface antigen (HBsAg) by ELISA method. We also studied on the immune dose and mechanism underlying of HBsAg with WHlfungin by different immune routes, the results were as follows:(1)The immune dose study of WHlfungin and HBsAg. The immune response was positively correlated with antigen dose between 10μg and 1μg with subcutaneous injection route. By intramuscular injection, the the group with 2μg HBsAg was detected the highest specific IgG antibody titer, which was significantly higher than the group with 1μg or 0.5μg HBsAg. The specific antibody titers of anti-HBsAg IgG was highest with 50μug WH1fungin by intranasal drug delivery, and the levels of specific IgG antibody were increasd by antigen dose-dependent means.(2) Adjuvant effect of WHlfungin peptide on specific IgG antibody titer. The specific antibody titers of anti-HBsAg IgG in mice serum showed WHlfungin peptide could significantly increase the specific antibody titers with subcutaneous injection, intramuscular injection or intranasal drug delivery route. The specific IgG antibody provoked by Hz-Au peptide could last at a high level in mice for more than four months.(3)The anaylsis of the specific IgG subclasse titers. IgG subclasse anaylsis showed that the group with WH1fungin peptide by subcutaneous injection and intramuscular injection was detected high specific antibody titers of IgGl and IgG2a, which was significantly higher than the group with Alum. And WHlfungin only slightly increased titer of specific IgG2a but pronounced abundance of IgGl. By intranasal drug delivery, only the specific IgGl antibody titer of the group with WHlfungin was higher than the group with CpG. The results suggested that WH1fungin peptide preferentially induces the Th2-dependent response.(4)FACS analysis of TNF-a/IFN-y level in T lymphocyte. The data indicated that WHlfungin peptide could promot the shift of immune balance to Th2 type.(5)The anaylsis of CTL activity. When the mice immuned by intramuscular injection or intranasal drug delivery, WHlfungin peptide could inhance CTL activity, and the specific CTL activity with intramuscular injection was more significant. However, the group with WH1fungin peptide by subcutaneous injection could not be detected CTL activity.(6)The antigen specific lymphocyte proliferation assay. When treated with HBsAg, splenocytes from subcutaneous and intramuscular immunized mice with HBsAg plus WHlfungin showed slightly higher proliferative response than those from immunized with HBsAg only, and lower than the group with Alum. By intranasal drug delivery, WHlfungin peptide could markedly enhance the specific lymphocyte proliferative response, and higher than the group with CpG.(7)The specific IgA antibody titers in alveolus and small intestine. IgA anaylsis showed that the group with WH1fungin peptide by intranasal drug delivery was detected specific antibody titers of IgA in alveolus, which was significantly higher than the group with HBsAg only. And the specific IgA antibody titer in small intestine of the group with WH1fungin was higher than the group with CpG. It suggests that WH1fungin could induce mucosal immune response.In summary, WH1fungin peptide is a novel adjuvant in enhancing the immune response to recombinant HBsAg with subcutaneous injection, intramuscular injection or intranasal drug delivery. |
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