| Over the past decade, genetic (or DNA) vaccines have emerged as a viable alternative approach to traditional vaccine, such as live-attenuated, subunit, peptide or protein vaccines. Intramuscular injection of DNA vaccine induces strong, long-lasting immune response to the antigen encoded by the gene subcloned into the plasmid. Plasmid DNA immunization leads to the production of antibody, activation of both . major histocompatibility complexes (MHC) class I-restricted cytotoxic T lymphocyte (CTL) and MHC class Ⅱ-restricted CD4+ T cells secreting Th1-type cytokines. It has potential advantages compared to traditional protein vaccine due to the prolonged antigen expression in the cytoplasm and loading of peptides derived from endogenous antigen onto class I MHC molecule, which is recognized by CTL. As a consequence, DNA vaccination has potential applications in the prevention or treatment of infectious diseases and cancers. However, results from clinical trials suggest that the immunogenicity of these vaccines in humans may be limited. Therefore, there is an urgent need for the development of potent and safe adjuvants to increase the activity of this class of vaccine, we and others have been exploring the benefit of co-immunizing with various types of vaccine adjuvant.HBV is a DNA virus with a partially single-stranded 3.2 kb genome encoding several viral proteins, including the core and envelope proteins. Of these proteins, the secreted middle '"S" (surface) envelope protein is known to be highly immunogenic and can elicit both humoral and cell-mediated immune responses. So we choose the "preS2S" gene as our model antigen, then select prothymosin a (human) or staphylococcus enterotoxin A or heat shock protein 65 (tuberculosis) as an adjuvant improves antibody responses to HBV S antigen.In this study, we have constructed HBV DNA vaccine (pHBVs2s), and three plasmid containing gene of prothymosin α , staphylococcus enterotoxin A or heat shock protein 65 as gene adjuvant. We have also constructed three plasmid containinggene-fusious of prothymosin a . staphylococcus enterotoxin A or heat shock protein 65 with PS2S as DNA vaccine.When P815 (mastocytoma cell) or 293 cells were transfected with Flag- HBVs2s. Flag-pThy a/HBVS2S. Flag-pHSP65/HBVs2s, Flag- pmSEA/HBVs2s, Flag- pThy a, Flag-pmSEA. Flag-HSP65. or pcDNA3/flag vector by using lipofectamine 2000. Transfecting 24-48 hours, the cells were collected and analyzed by western blot and ELISA. Western blot assay showed that all the constructs except Flag-PS2S/PmSEA were expressed in P815 cells. The ELISA results confirmed that the cells which transfected with Flag-pHBVs2s, Flag-pThya/HBVS2S, Flag-pHSP65/HBVs2s have expressed HBsAg with corrective antigen- epitope.Anti-HBs antibody response was followed in the sera of mice injected with mixture of 100 ug of pcDNA3 , 100 u.g of pHBVS2S, a mixture of 100 ug of pThy a and 100 ug of pHBVS2S, a mixture of 100 |ag of pHSP65 and 100 ug of pHBVS2S or a mixture of 100 ug of pmSEA and 100 ug of pHBVS2S. After booster injection, mice inoculated with pHBVs2s+ pThy a generated the highest responses; a 5-fold increase in antibody titers relative to the response to the pHBVS2S alone was observed. Similarly, mice inoculated with pHBVs2s+ pmSEA or pHBVS2S+ pHSP65 generated greater responses compared to those inoculated with PreS2S alone.The ELISPOT assay was employed to determine the number of cells secreting interferon-y (IFN-y) upon in vitro re-stimulation with antigenic peptides. mice immunized with DNA encoding pHBVS2S and pThy a , pHBVS2S and pHSP65 or pHBVS2S+pmSEA generated ~3-fold more IFN-y ELISPOTs than mice immunized with the pHBVS2S alone did. Interestingly, the adjuvant also improved the readability of the ELISPOTs: both the size and intensity of spots was increased for IFN-y T cell responses to the HBV DNA vaccine.Three kinds of gene adjuvant boast the CTL activity induced by pHBVS2S. mice co-immunized with pHBVS2S and pThy a, pHBVS2S and pHSP65 or pHBVS2S+pmSEA exhibited greater cellular immune responses to HBsAg compared to those immunized with pHBVS2S alone, as determined by CTL activity of the splenocytes. Therefore, co-expression of pThy a, pHSP65 or pmSEA increased the... |
