| The prevalence and infection hepatitis B virus (HBV) is a serious public health problem in the world and totally the people more than 300 million were infected worldwide. It can lead to several severe or end-stage liver diseases, including uncompensated liver cirrhosis and hepatocellular carcinoma. Chronic hepatitis B patients are usually associated with abnormal immune function, especially the dysfunction of anti-HBV-specific cellular immune, which are presented by a lower anti-viral cytokine secretion and lead to the persistence of the virus in the body. Recent studies showed that the PD-1/PD-L pathway contributed directly to T cell dysfunction and lack of viral control in established chronic infection, and the function of HBV-specific CD8-T cells can be improved by blocking this pathway. In the present study, we try to improve the immune effect of HBsAg vaccination in HBV transgenic mice by blocking PD-1/PD-L pathway in vivo and to seek new methods for the treatment of chronic hepatitis B. Objective:To study the effects of B7-H1 protein vaccine on the immune responses to HBsAg in HBV transgenic(Tg)mice.Methods:①Fifteen HBV Tg mice were randomized into three groups with 5 mice each, and designated as HBsAg vaccine group, vaccine and B7-H1 group and control group, respectively.②The Tg mouse in vaccine group was immunized with 10μg HBsAg protein. The mouse in vaccine adding B7-H1 group was immunized with 10μg HBsAg protein and 40μg B7-H1 protein. The mouse in control group was immunized with the same volume of saline. The vaccination schedule was 0,2nd,4th,6th week,and the splenocytes were harvested on the 8th week.③At various times before and after immunization(0,2nd,4th,6th,8thweek), the blood/sera of mice were collected and stored.④The weigh and the expression of HBsAg in sera of the mice were recorded before immunization.⑤The B7-H1 antibody were detected by ELISA after immunization(2nd,4th,6th,8thweek).⑥The anti-HBs of the three groups were detected by ELISA.⑦The splenocytes of the mice were stimulated with HBsAg ( 10μg/ ml ) .The concentrations of IL-2 and IFN-γwere detected by ELISA.⑧The number of IFN-γsecreting cells was measured by ELISPOT.⑨The proliferation of splenocytes was measured by MTT.⑩All data were analyzed using SPSS version 13.0 for windows. A P values of less than 0.05 were considered as significant.Results:①Each of mice were weighted at the beginning of the experiment,and the average weight of the three groups was 27.2±0.96g, 26.6±0.55g, 27.1±0.75g. There was no difference among the three groups(P>0.05). In addition, serum HBsAg level of the three groups before injection was 5307.9±437.8 IU/ml, 5335.4±523.5 IU/ml, 5554.8±439.5 IU/ml, and there was no difference among the three groups either (P>0.05).②The anti-B7-H1 in serum was detected in the mice immunized with B7-H1 at 2nd,4th,6th,8th week,and the antibody titer increased with the immune times.③There was no difference of anti-HBs positive rate between HBsAg group and HBsAg+B7-H1 group at 6th week and 8th week after immunization (P<0.05). Anti-HBs was negative in control group.④Concentrations of IL-2 and IFN-γwere elevated in both HBsAg group and HBsAg+B7-H1 group than that in control group(P<0.05),results of IL-2 were 306.7±94.8pg/ml , 327.9±109.6pg/ml, 124.5±55.7pg/ml, and IFN-γwere 569.0±132.1pg/ml, 647.8±161.8pg/ml, 99.2±50.2pg/ml, but there was no difference between the two experimental groups (P>0.05).⑤Moreover, The numbers of HBs-specifc IFN-γsecreting T cells is also increased in both the HBsAg group and HBsAg+B7-H1 group when compared with the control group(P<0.05), and the outcomes were 57.8±5.4, 52.4±4.5, 23.6±3.6 respectively that showed no difference between the two experimental groups (P>0.05).⑥Strong lymphocyte proliferation reaction was induced in the both HBsAg group and HBsAg+B7-H1 group based on the MTT results, but there was no significant difference between the two experimental groups (P>0.05).Conclusion:①HBsAg vaccine can increase the cellular and humoral immune response in HBV Tg mice and reconstruct immune response to HBsAg.②B7-H1 vaccine can not enhance the humoral and cell immune responses in HBV Tg mice immunized with HBsAg. |
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