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Hepatitis B, a liver disease, is caused by hepatitis B virus (HBV) infection. It is a worldwide health problem. Commercial recombinant hepatitis B surface antigen (rHBsAg) vaccine has been widely used and provided an effective prophylaxis against HBV infections. However, such vaccine, consisted of alum and HBsAg, primarily induces humoral immune response, but it is unable to promote effective cellular responses.The adjuvant is believed to activate innate immunities, and improve immune responses by increasing the interaction between antigen and APC cells, improving antigen presentation to T cells and prolonging the immune memory responses. Astragalus polysaccharides(APS) extracted from roots of Astragalus membranaceus, a kind of traditional Chinese medicinal herb, has extensive pharmacological effects and strong immunity adjustment actions. APS can inflence substance metabolism in immune cells, induce related immune-regulating factors, enhance specific immune response and non-specific immune response, and regulate immunologic function of abnormal body.In this study, Astragalus polysaccharides was used as an adjuvant for the hepatitis B vaccine in enhancing humoral and cellular responses. The female Kunming Species mice aged 6-8 weeks were randomly divided into five groups (n=7 each), and immunized intramuscularly on days 0 and 14 with either rHBsAg alone, rHBsAg combined with APS, APS, or with rHBsAg puls aulm, and naive mice were as negative control. Immunological tests were carried out on day 7 after boost and results were as follow:After detection of the concentration of the total IgG by ELISA, T-cell proliferation by MTT method and CTL activity by in vivo CTL assay, we found that compared to the immunization with APS alone, rHBsAg alone and rHBsAg puls aulm, combinations of rHBsAg and APS significantly increased levels of IgG, T cell proliferation and CTL activity in vivo. It demonstrated that APS can enhance immune responses to hepatitis B vaccine.Furthermore, the levels of PFP, GraB, Fas L, Fas and several types of cytokines were detected by RT-PCR and by the flow cytometry respectively. It showed that compared to other groups, the mice immunized with rHBsAg plus APS could induce higher levels of PFP, GraB, Fas L, Fas and IFN-γin CD8+ cells, which was in accordance with CTL activity in vivo, and also induce higher levels of IFN-γ、IL-2 and IL-4 in CD4+ cells, which indicating that HBV vaccine plus APS could improve both Th1 and Th2 immune responses.In order to find out the mechanism of enhancing the immune responses to HBV vaccine by APS, we detected the levels of TLR2, TLR4, TGF-β1 and Foxp3 by RT-PCR. We found that rHBsAg plus APS could up-regulate the expression of TLR2 and TLR4, which meant that costimulatory molecules and cytokines were induced and involved in the adaptive immune responses via improvement of TLR2 and TLR4 expression. On the other hand, rHBsAg plus APS could decrease the expression of TGF-β1 and Foxp3, thereby promoting the immune response.In summary, Astragalus polysaccharides as an adjuvant of hepatitis B subunit vaccine can enhance immune responses, especially cellular responses.This study demonstrated that APS is an effective immunologic adjuvant, and can improve both humoral and cellular responses to HBV vaccine. It may provide basic research data for research and development of hepatitis B vaccine.
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