The Study Of The Immune Regulation Effector Of HBEAg In HBV Mouse Model

Objective 1. To investigate how HBe Ag regulate the immune response in HBV persistence replication mouse model and what is the mechanism..Methods1. HI of wild type plasmid PAAV/HBV1.2 and mutant plasmid HBe Ag(-) into C57BL/6 mice to construct chronic replication mouse model. HI the mutant plasmid E(-) and HBe Ag expression plasmid p CDNA3.1/HBe Ag into C57BL/6 mouse, at the same time, to explore the influence of HBe Ag on immune response.2. According to the designed plan, we bleed the mice every week and stored liver tissues from the killed mice at the indicated time points. The serum antigen, antibody and antibody isotype, Ig G1 and Ig G2 a, were monitored by ELISA. Serum HBV DNA was detected by realtime PCR.3. Liver tissue and NPC total RNA was extracted and tested relevant cytokines by RT-PCR. Intrahepic HBc Ag level were visualized by immunohistochemical staining. Lymphocytic infiltration of spleen were tested by HE staining.4. The separated spleen lymphocytes were cultured and assayed for the number of antigen-specific IFN-γ secreting cells by ELISPOT. The frequencies of CD4, CD8, mccrophage, DC and other cells and the HBs/HBc petide-specific IFNγ secreting CD8+ cells from from NPC and spleen were detected by FACS. The figures were made and datas were analysed by Graph Pad Prime 5.Results1. In the chronic replication mouse model, serum HBV DNA were persisted replication in wild type mice; however the serum HBV DNA were cleared faster in mutant group.2. Serum HBsAg and HBV DNA level were higher in HI HBe Ag expressed vector mice than E(-) group. It is hints that HBe Ag may play the role of immune tolerence.3. The proportion of HBcAb appear earlier and longer in H1.2 group than E(-) group. For the HBV DNA cleared, HBs Ab produced in part of E(-) group. According to antibody isotype we find that Ig G2 a plays a role in HBcAb in H1.2 and E(-) group. While it has Ig G1 and Ig G2 a in HBs Ab. It is hints that virus eliminated in E(-) group by the balance of Th1/Th2 immune activation.4. According to the analysis of PDL1 RNA level, we find that it is higher in E(-)+E group, medium in H1.2 group and lower in E(-) group. It is hints that the PDL1 level may up-regulation by HBe Ag expressed and PDL1-PD1 signal pathway activated.5. After analysis the FACS result, we find that the proportion of CD8 Th cell higher in E(-) group than H1.2 group at the time of HI D21 in spleen and HI D28 in NPC. It is further indicate that HBe Ag negative control the CD8 T cells recruitment in liver tissues, and take a tolerance role in immune response.Conclusions1. HBe Ag take a tolerance role in immune response in the chronic replication mouse model. HBe Ag could activate PD-L1-PD1 signal pathway by up-regulation PD-L1 of Kupffer and DC in NPC, or suppress the T cell response by activate Treg to take the tolerance effect.2. For lack of the effect of tolerance, E(-) mutant strain could stimulate a stronger Th1-Th2 immune response. It can recruitment more CD8+T led to the mutant strain cleared and severe inflammation.