The Role Of The Immune Regulation In The Chronic HBV Infection And The Research Of Its Immunopathogensis

Objective1. To study the role of the immnunouppressive drug CsA in the process of HBVreplication, we treated the mice with CsA and hydrodynamic injected withpAAV/HBV1.2, detected the antigenemia, viremia and antibody response.2. Treated the BALB/c mouse with NS continuously, CsA continuously and CsAshort-term respectively, and established the HBV replication model. We measuredthe innate and adaptive immune response to illuminate the immunopathogenesisduring chronic HBV infection induced by the CsA treated.3. Established the chronic HBV infection mice by CsA treated. After thediscontinued CsA treated, we active or adoptive immunized the mice, and studythe role of immunoregulation in the treatment of HBV infection.Methods1. The female BALB/c and mice,6-8weeks old, were obtained from Center ofDisease Control in Hubei province.2. Hydrodynamic injected the plasmid pAAV/HBV1.2into the tail veins of BALB/cmice treated NS or CsA respectively. Monitor the titer of HBsAg, HBsAb andHBcAb in the serum using ECLIA. In addition, extraction the HBV DNA fromthe serum and measured the viremia by Real-Time PCR. Analyze the role of theCsA treatment in the process of HBV replication. 3. Treated the BALB/c mice with NS continuously, CsA continuously and CsAshort-term respectively, and killed the mice at the indicated time points. Thefrequencies of IFN-γ secreting splenocytes were assayed by ELISPOT after HBcpeptide stimulation. Extraction the RNA from spleen and liver tissue, and themRNA levels of CD molecules and cytokines were measured by Real-Time PCR.The HBV replication in the liver was analyzed by Southern Blot. The expressionof the HBcAg in the liver was detected by immunohistochemistry. Analyzed theimmune mechanism of prolong HBV replication in the CsA treated mice.4. Hydrodynamic injection pAAV/HBV1.2into the tail veins of the BALB/c micetreated with CsA to establish the chronic HBV replication mice model. After theCsA treatment discontinued, active immunized the mice with PBS or yeastrecombinant HBsAg vaccine, or adoptive immunized with splenocytes fromHBc-immunized mice or na ve mice, respectively. Analyze the role of the immuneregulation in the clearance of HBV infection.Results1. The BALB/c mice developed high antigenemia and viremia after thehydrodynamic injection pAAV/HBV1.2into the tail veins. In NS treated group,the HBsAg disappeared in7weeks after hydrodynamic injection. In the CsAtreated group, the antigenemia and viremia maintained on a high level during thetreatment. After the withdrawal treatment at10weeks, the HBsAg in the serumpersistent for more than6month in75%animals.2. The expression of HBsAg is prolonged corresponding with the prolonged CsAtreatment course. And the HBsAg can persistent for more than6months in mostof the mice treated with CsA for6weeks.3. The CsA treatment can not inhibit the boosting of specific T cell response. Incontast, the specific T cell response was enhanced in the CsA treated miceobviously.4. Treatment with CsA can inhibit the expression of some CD molecules andcytokines in the spleen and liver tissue, including CD3, CD4, CD8, Fas-L,Perforin and IL-2. But there are no influences for the expression of IFN-γ, TNF-αand IFN-β. After the discontinued of the short-term CsA treatment, the expression of some CD molecules and cytokines rebound accompanied with the clearance ofHBV infection.5. In the chronic HBV infection mice discontinued CsA treatment, immunizationwith yeast recombination HBsAg vaccine can induce the HBV clearance, andinduce the production of HBsAb and HBcAb, enhance the specific T cellresponse.6. In the chronic HBV infection mice discontinued CsA treatment, adoptive immunewith spenocytes from HBc-immunized mice or na ve mice can control the HBVreplication, and it’s more vigorous for the spenocytes from HBc-immunized mice toinhibit the HBV replication, to induce the production of HBcAb and to enhancethe specific T cell response.Conclusions1. In the hydrodynamic injection mice model, treated the mice with CsA canmodulate the course of the HBV infection. Treated the mice with CsA for morethan6weeks can prolong the HBV replication obviously and induce the infectionto be chronic.2. Treated with CsA cann’t inhibit the production of specific T cell, and cann’tsuppress the expression and secreting of IFN-γ and TNF-α in the liver and spleen.But it can partially suppressed the function of the T cell response, including thepathways of perforin and Fas-L, and induce the persistence of HBV replication. Itindicated that an intact immune system is required for HBV clearance and theimpaired specific T cell cann’t clear the HBV infection.3. After the discontinued of short term CsA treatment, the expression of many CDmolecules and cytokines rebound and the ability of secreting IFN-γ for thesplenocytes enhanced accompanied with HBV clearance. It indicated that theimmune response can be restored after the discontinued of short term CsAtreatment and control the HBV infection.4. In the chronic HBV infection mice discontinued CsA treatment, immunizationwith recombination HBsAg vaccine or transferred with specific splenocytes canclear or control the HBV replication, restore the humoral immunity and enhancethe specific T cell response.