| Hepatitis B is a severly human-threatened disease which will lead to liver cirrhosis and hepatocellular carcinoma (HCC) without timely treatment. Vaccine inoculation is an efficient method to prevent human from HBV infection, however, exerts little effect on HB cure. One of important reasons is that traditional vaccine induces homoral immune response mainly but very weak cell-mediated immune response which plays the central role in eliminating HBVs. Thus, therapeutic vaccines, which can trigger satisfying cellular response, are paid much attention by vaccine researchers. Amongst them, DNA vaccine is one of the most promising candidates.Though many studies illustrate that DNA vaccine could induce ideal cellular and humoral immune response, certain shortcomings cannot be ignored. It has been suggested that it primed insufficient immune response in large animals and humans and showed unsatisfactory effect. Adjuvant can enhance antigen-specific response as well as extend its time of effect, thus is regarded as an ideal way to overcome the drawbacks of DNA vaccine. APS, extracted from the Chinese Traditional Medical Herb Astragalus, possesses excellent immunopotentiatory property. Literatures reported that it could induce CD4+T cells to proliferate and secrete IL-2 and IFN-y. Meanwhile, it could stimulate splenocyte and B cell proliferation and activate macrophages. Moreover, APS could prompt DC maturation and its ability of antigen presentation. In our study, APS was selected as adjuvant of HBV DNA vaccine, combined with HBsAg-expressed plasmids, they were immunizied to mouse intramuscularly. Finally, cellular and humoral response level was determined by certain methods to evaluate the adjuvant effect of APS. Furthermore, relevant mechanism was explored to give insights into its functioning.50 6-8 week-old healthy female KM white mouse were selected and divided randomly into 5 treatment groups, each of which contained 10. Certain experimental treatments were imposed on five groups, namely, control, vehicle plasmids injection only, antigen-recombined plasmids injection only, antigen-recombined plasmids plus APS injection and APS injection only. Accordingly, five groups were designated as Naive group, pcDNA3 group, pcDS2 group, pcDS2 plus APS group and APS group. Immunization procedure was each injection on day 0 (the time of first immunization), day 14 and day 28. On day 35, namely 7 days after the second boost immunization, certain samples (serum and splenocytes) were gathered and immunological parameters were determined, including serum antibody level, T cell proliferation, expression of surface and intracellular molecules, CTL activity and expression of signaling pathway molecules. Results of this study are as following:Antibody concentrations of Naive, pcDNA3, pcDS2 groups were all negative. Group pcDS2 plus APS (8464.8ng/ml) was significantly higher than group pcDS2 (3319.6ng/ml). The IL-4-expressed CD4+T cells percentage of Naive group, pcDNA3 group, pcDS2 group, pcDS2+APS group, APS group were 0.64%,0.97%,1.06%,1.42%,0.90% respectively. Group pcDS2 plus APS also displayed the highest expression level of IL-4 of CD4+T cell, which showed that the immune response directed to Th2 type. These data demonstrated that APS as adjuvant could enhance the humoral response of mouse.Stimulation Indexes of Naive group, pcDNA3 group, pcDS2 group, pcDS2 plus APS group and APS group were 1.4,1.4,2.4,3.2 and 1.7 respectively, demonstrating that group pcDS2 plus APS induce the optimal T cell proliferation ability, which was significantly higher than the other four groups. IL-2 expression of CD4+T cell (Naive group 0.26%, pcDNA3 group 0.43%, pcDS2 group 0.58%, pcDS2 plus APS group 3.42% and APS group 0.36%) also showed the similar trend as the T cell proliferation. The level of IFN-y expressed by CD4+and CD8+T cells also showed that group pcDS2 plus APS possessed the strongest ability of inducing Thl-type immune response. Also in the in vivo CTL ability assay, group pcDS2 plus APS (56.5%) showed the highest target-cell-killing level, significantly higher than group pcDS2 (27.6%) as well as group naive (2%), pcDNA3 (5.9%) and APS (3.9%). These data in sum demonstrated that APS as adjuvant could augment the cell-mediated immune response of mouse.The result of cosimulatory signaling molecules CD40, CD80 and CD86 exhibited that APS combined with pcDS2(1.15%,1.10%,1.23%) induced the highest expression level (Naive group 0.69%,0.57%,0.49%, pcDNA3 group 0.66%,0.51%,0.55%, pcDS2 group 0.92%,0.77%, 0.88%, APS group 0.78%,0.64%,0.78%), which showed the strongest ability of inducing "second signal" for T cell activation; Expression level of MHC I, MHC II and CCR7 detected by RT-PCR also displayed that pcDS2 plus APS exerted the best effect. Signaling pathway molecules NF-κB and p38 MAPK was also detected and the results came out consistent with the aforementioned data. The data in all showed that APS as adjuvant could stimulate DC maturation effectivelyCollectively, APS combined with pcDS2 could strengthen the homoral and cellular immune response via stimulating DC maturation. The study provided a novel method for enhancing the immunological effect of HBV DNA vaccine and demonstrated that APS possess promising potential of being adjuvant and showed a good prospect of application. |
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