ABSTRACT
Objective:To clarify the differences of host immune responses at different stages of HBV infection. Methods:We constructed three HLA-A*0201/HBV tetramers with immunodominant epitopes of core18-27,polymerase 575-583 and envelope 335-343,and analyzed antigen specific CD8+ T cells and the expression of CD127 in peripheral blood mononuclear cells ( PBMCs) from patients infected with HBV using these HLA-A*0201/HBV tetramers. Results: The frequencies and expansion ability of antigen specific CD8+ T cells in most self-limited HBV infected individuals were higher than that in chronically HBV infected patients. In low copy period the frequencies of antigen specific CD8+ T cells were similar to those in immune clearance phase at a high viral load and liver damage and in immune clearance phase, which had no significant correlation with virus quantitation and ALT level. In chronic infection the ability of antigen specific CD8+ T cells proliferation was inversely proportional to the viral titer. In most self-limited HBV infected individuals the IFN-γsecretion functions of antigen specific CD8+ T cells were higher than in chronic infection,but in immune tolerance phase these cells lost the ability. HBsAg level was different at different stages after HBV infection:it was highest in immune tolerance phase,but in immune clearance phase,activity period and low copy period the correlation with HBV DNA replication gradually declined. The frequency of CD8+ CD127+ T cells in chronic HBV infection was lower than the control group and self-limited infection group,especially in immune tolerance with HBeAg+ and immune clearance phase. Conclusion: The frequencies of antigen specific CD8+ T cells are not the main determinant of immune-mediated protection in chronic HBV infection,memory antigen specific CD8+ T cells are not clear or missing,which provides the possibility for therapeutic vaccines and immunization therapy.
ABSTRACT
Objective:To prepare and test tetrameric sH-2K~d-HBc complex for the further measurement of the specific CTL response.Methods:PE labled streptavidin with 4 biotinylated binding sites can bind to 4 biotinylated monomer to form the corresponding tetramer.Mice were immunized via different methods of genetic immunization by use of the construted pcDNA3-C plasmid to get the specific CTLs.Then our prepared tetramer was applied to stain the specific CTLs by the analysis of flow cytometry.Results:We applied our prepared tetramer to stain the cells from the experimental groups and control group.The results showed the tetramer was able to discriminate the frequencies of specific CTL induced by the three immunol methods(0.24%,0.26%,0.36% vs 0.07%,P≤0.05).This demonstrated that the prepared tetramer could bind its targets specifically and efficiently.The three immunol methods induced different levels of immune responses.Compared with the traditional muscle injection,gene gun induced weaker humoral immune response and stronger cellular immune response,and hydrodynamic injection induced the strongest humoral and cellular immune responses.Conclusion:Have successfully constructed the sH-2K~d-HBc tetramer.The techniques and methods can be used for preparation of tetramers of other types of MHCⅠ molecules.
ABSTRACT
Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are believed to play a major role in viral clearance and disease pathogenesis during HBV infection. To clarify the differences in host immune responses between self-limited and chronic HBV infections, we constructed three HLA-A*0201/HBV tetramers with immunodominant epitopes of core18-27, polymerase 575-583 and envelope 335-343, and analyzed the HBV-specific CTLs in peripheral blood mononuclear cells (PBMCs) from patients infected with HBV. The frequencies and expansion ability of HBV-specific CD8(+) T cells in most self-limited HBV infected individuals were higher than those in chronic HBV-infected patients. HBV-specific CD8(+) T cells could be induced by in vitro peptide stimulation from chronic patients with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. In chronic infection, no significant correlation was found either between the frequencies of HBV-specific CD8(+) T cells and the viral load, or between the frequencies and the levels of alanine transaminase. Our results suggested that the frequencies of HBV-specific CTLs are not the main determinant of immune-mediated protection in chronic HBV infection and immunotherapeutic approaches should be aimed at not only boosting a HBV-specific CD8(+) T response but also improving its function.
ABSTRACT
Previous studies have shown that expression of the interferon-sensitive gene (ISG)I5 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by Hi (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by HI (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.