The Role Of JAK/STAT Signal Pathway In Immune Activation Of Hepatitis B Mediated By Tapasin Modified Fusion Protein

Objective:To further investigate the mechanism of the immune response induced by a fusion protein containing HBcAg_18-27 CTL epitope and molecular chaperone Tapasin coupled with CTP in mice by inhibiting JAK/STAT signal pathway.Methods:1.HLA-A2 mice were randomly divided into 6 groups:CTP-HBcAg_18-27-Tapasin?50?g?,CTP-HBcAg_18-27?50?g?,HBcAg_18-27-Tapasin?50?g?,CTP-HBcAg_18-27-Tapasin+AG490?50?g?,AG490 and vehicle group?PBS?.HBV transgenic mice were randomly divided into 7 groups:CTP-HBcAg_18-27-Tapasin?50?g?,CTP-HBcAg_18-27?50?g?,HBcAg_18-27-Tapasin?50?g?,IFN-??2000 IU?CTP-HBcAg_18-27-Tapasin+AG490?50?g?,AG490 and vehicle group?PBS?.The fusion protein was intramuscularly injected to mice at 1-week interval three times.AG490?5mg/kg?was intraperitoneally injected to mice in total volume of 100?l 1times a day.The levels of interferon?IFN?-?and interleukin?IL?-2 in the supernatant were determined by Enzyme linked immunosorbent assay?ELISA?.T lymphocyte proliferation activity was detected by CCK-8 Kit.The number of IFN-?-secreting specific T lymphocytes was analyzed by Enzyme linked immunosorbent spot?ELISPOT?.The percentages of IFN-?-secreting CD4⁺and CD8⁺cells in spleen lymphocytes were detected by flow cytometry?FCM?.2.HBV transgenic mice were randomly divided into 7 groups:CTP-HBcAg_18-27-Tapasin?50?g?,CTP-HBcAg_18-27?50?g?,HBcAg_18-27-Tapasin?50?g?,IFN-??2000 IU?,CTP-HBcAg_18-27-Tapasin+AG490?50?g?,AG490 and vehicle group?PBS?.HBV transgenic mice were intramuscularly immunized with the recombinant protein at1-week interval three times.AG490?5mg/kg?was intraperitoneally injected to HLA-A2 mice in total volume of 100?l 1 times a day.Serum samples were respectively collected at day 7 after the first and third immunization.All HBV transgenic mice were sacrificed by cervical dislocation under general anesthesia at day 7 after the last immunization.The levels of serum HBV DNA were analyzed by fluorescence quantitative polymerase chain reaction?PCR?.The serum level of HBsAg was determined by microparticle enzyme immunoassay?MEIA?.The expression of hepatitis B surface antigen?HBsAg?and HBcAg in liver samples were detected by immunohistochemical detection.The expression level of JAK/STAT signal pathway quantitative PCR and Western blotting.The serum levels of Alanine transaminase?ALT?and aspartate aminotransferase?AST?were detected with ARCHITECT Automatic Biochemistry Analyzer.Results:1.Mice were immunized with fusion protein with or without blocking JAK/STAT signaling pathway.The concentrations of TH1 cytokines?IL-2 and IFN-??secreted by T lymphocytes in CTP-HBcAg_18-27-Tapasin with AG490 group were significantly decreased compared with CTP-HBcAg_18-27-Tapasin group.The percentages of CD8⁺and CD4⁺cells-secreting IFN-?and CD4⁺cells-secreting IL-2 in the spleen lymphocytes of mice from fusion protein with AG490 group were remarkably lower than those in CTP-HBcAg_18-27-Tapasin group.Also the proliferation activity of T lymphocytes was reduced.2.HBV transgenic mice were immunized with fusion protein with or without blocking JAK/STAT signaling pathway.The serum levels of HBV DNA and HBsAg were reduced and expression levels of HBsAg and HBcAg in liver tissue were decreased.Minimal infiltrations of lymphocytes in the liver of HBV transgenic mice were observed from CTP-HBcAg_18-27-Tapasin group with AG490 group.The expression levels of JAK2,TYK2,STAT1 and STAT4expression were significantly decreased.Conclusion:Tapasin-modified CTL epitope peptide coupled with cytoplasmic transduction peptide could elicit Th1 immune response with robust HBV-specific CTL activity and inhibit HBV replication via activation of Jak2,Tyk2,STAT1 and STAT4molecules in the JAK/STAT signaling pathway.