ABSTRACT
Objective To identify human leucocyte antigen (HLA)-A* 0201-restricted hepatitis C virus (HCV)-cytotoxic T lymphocyte (CTL) epitopes. Methods Based on the prediction results of RANKpep and SYFPEITHI prediction programs, six candidate CTL epitopes were selected and synthesized. The affinity of candidate CTL epitopes to HLA-A* 0201 molecules of T2 cells was explored. Subsequently, enzyme-linked immunosorbent spot (ELISPOT) assay and intracellular cytokine staining (ICS) were utilized to determine whether candidate CTL epitopes could induce the recall positive response in peripheral blood mononuclear cells (PBMC) of HLA-A* 0201 positive HCV-1b-infected patients. Results Among six candidate CTL epitopes, peptides C_181(LLSCLTTPV) and NS2_172 (VLQAGLIRV) had high affinity to HLA-A* 0201 molecules. Moreover, the affinity was proportional to the concentration of peptide. Furthermore, among ten HLA-A* 0201 positive HCV-1b-infected patients, the frequencies of C_181 and NS2_172-specific interferon (IFN)-γ-producing cells were 0-19 spots forming cells (SFC)/1 × 105 PBMC and 0-20 SFC/1 × 105 PBMC, respectively.The percentages of C_ 181 and NS2_172-specific IFN-γ+ CD8+ T lymphocytes in total CD8+ T lymphocytes were 0.006%-0.065% and 0.005%-0.080%, respectively. Conclusion Peptides C_181 (LLSCLTTPV) and NS2_172 (VLQAGLIRV) are identified as novel HLA-A* 0201-restricted HCV-CTL epitopes.
ABSTRACT
Objective To analyze human leucocyte antigen (HLA)-A0201 restricted antigen-specific cytotoxic lymphocytes (CTL), and to investigate the difference of T cell response to specific antigen epitopes between patients with acute phase of acute hepatitis B and active phase of chronic hepatitis B. Methods Peripheral blood mononuclear cells (PBMC) from 5 patients with acute phase of acute hepatitis B and 6 patients with active phase of chronic hepatitis B were isolated. The numbers and functions of CD8+ T-lymphocyte epitope peptide specific CTL were detected using enzyme-linked immunosorbent spot (ELISPOT) assay, and the 3 peptides were from HBV polymerase region (Pol575-583), envelope region (Env348-357) and core region (Core18-27), respectively. The data were analyzed using t test. Results The spot formation cell counts (SFC) of Pol575-583, Env348-357 and Core18-27 stimulations in patients with acute phase of acute hepatitis B were 110±13, 165±17 and 185±20, respectively; and those in patients with active phase of chronic hepatitis B were 22±4, 23±5 and 30±5, respectively; the differences were all significant (t=10.9, 15.2 and 8.0, respectively, all P<0.05). The CTL responses to the three peptides in patients with acute phase of acute hepatitis B were Pol575-583<Env348-357<Core18-27; and the difference between responses to Pol575-583 and Core18-27 was significant (t=4.0, P<0.05), while there was no statistical difference between CTL responses to Env348-357 and Core18-27 (P>0.05). The SFC were increased upon non-antigen specific HLA-A2404 restricted epitope (Core117-125), but the difference was not significant compared with negative control group (P>0.05). Conclusions Hepatitis B virus-specific CTL responses in patients with acute hepatitis B are significantly higher than those in patients with chronic hepatitis B. The number and function of polyclonal CTL are both impaired in patients with chronic hepatitis B.
ABSTRACT
Objective To investigate the effect of therapeutic dual-plasmid HBV DNA vaccine on specific humoral and cellular immunity in cancrivorous monkey (Macaca fascicularis). Methods The eukaryotic expression plasmids encoding human IL-2 and IFN-? fusion protein (pFP) were constructed to enhance the cellular immunity of therapeutic HBV DNA vaccine (pS2.S) encoding HBV envelope middle protein in the form of dual-plasmid (pS2.S+pFP). Thirty cancrivorous monkeys were randomly divided into 5 groups (6 each) with sex rotio of 1∶1, namely the dual-plasmid DNA vaccine group in high dosage (2000?g/kg), medium dosage (660?g/kg), low dosage (200?g/kg), and EP-mediated pFP (330?g/kg) or PBS administration groups as the controls. The monkeys were vaccinated repeatedly with the dual-plasmid HBV DNA vaccine, and then the immune responses including level of serum anti-HBs and number of IFN-? secreting T-cells induced by HBsAg were examined by means of enzyme-linked immunosobent assay (ELISA) and enzyme-linked immunosobent spot assay (ELISPOT) respectively. Results Ten weeks after immunization of HBV DNA vaccine, various levels of serum anti-HBs was detected in all the monkeys of three different dose groups. Sixteen weeks after administration of EP-mediated HBV DNA vaccine, the positive HBsAg-specific INF-? T cell responses was found in 3, 2 and 3 out of 3 monkeys, respectively in the high, medium and low losage groups, and HBsAg-specific INF-? T cell responses were positive in all the animals with the respective cell count of 30.0?13.5 SFCs/3?105 PBMCs, 30.7?26.3 SFCs/3?105 PBMCs and 17.7?6.4 SFCs/3?105 PBMCs in each corresponding group at the 29th week. However, HBsAg-specific INF-? T cell responses were negative in the pFP and PBS group at the same time. Conclusion Electroporation-mediated vaccination of the HBV DNA vaccine can effectively induce both humoral and cellular HBsAg-specific immune responses in cancrivorous monkeys.