| BackgroundHepatitis B virus infections are global chanllenge with estimated worldwide distribution of 350 million and 170 million chronic carriers, respectively. All of these patients are at risk of developing adverse sequalae, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. For a long time, the pathogenese of chronic hepatitis B is not completely understood, which lack of ex vivo infection cell cultured system and narrow spectrum of infectious host were the major problems. HBV is a noncytopathic DNA virus with a small, circular DNA genome that causes acute and chronic liver disease and hepatocellular carcinoma. A mount of lymphocytes residual in liver indicated T lymphocyte immune response may play a critical role in the inflammary. Specific CTL response plays a crucial role in viral clearance and clinical recovery during acute hepatitis, however, the mechanisms of viral clearance are involved in many factors, such as genetic background, virological background and the activity of specific CTL. The current studys were found that HBV specific lymphocyte epitope is acting as initiator and stimulator of specific cellular immune response, in other words, as the central role of immune recognition and stimulation. HLA-A*0201 restricted CTL specific epitope (FLPSDFFPSV) was considered as a classical sequence used to epitope research, which mostly cited for positive contral of chronic infection in the study of HBV cellular immunity. However, in the process of citation, analysis of virological background was usually ignored. Therefore, maybe some bias could be induced by this ignorance. Thus, to demonstrated the distribution of the major HLA-A alleles in chronic hepatitis B virus (CHB) patients in China, explored the frequence and influence of HBcAg18-27V/I mutant on CD8+T lymphocytes with HLA-A2-peptide complexes in patients with chronic hepatitis B infection could be importance for identification of HLA-All-restricted and HBV-specific new CTL epitopes no less than that of HLA-A2-restricted HBV-specific CTL epitopes for the research of basic and clinical immunology of HBV infection.Objective1. To investigate the distribution of HLA-A11, A2, A24, and A33 alleles in chronic hepatitis B virus (CHB) patients in Southern China, and to provide essential background information for the screening of new HBV CTL epitopes.2. To investigate the distribution of HBcAg18-27V/I mutant in different HBV genotypes in China, and compared to the European and American, therefore we could make sure the virological background for the selection of CTL specific epitope.3. To analyze the difference of quantity between HBcAg18-27V-tetramer special CTL and HBcAg18-27I-tetramer special CTL in the peripheral blood of patients with chronic hepatitis B infection.Methods1. Chromosomal genomic DNA specimen extracted from peripheral blood of 267 patients and 300 healthy donors were obtained for HLA-A genotyping with PCR-SSP assays, and the data were analysed with SPSS10.0 software.2. Investigate the HBcAg18-27V/I mutant on HBVDNA specimen extracted from population which is HBV infectious in different areas and HBV genotypes by using polymerase chain reaction-RFLP and analysed the influence on the affinity of different HBcAg18-27 specific CTL epitope mutant combined with HLA-A*0201.3. Peripheral blood mononuclear cells (PBMC) were collected from patients with chronic hepatitis B infection (ALT≥2×ULN); the backgrounds of HBV virology (genotype of HBV, genotype of HLA-A and mutation of HBcAg18-27) were analyzed through polymerase chain reaction; the counting of HBcAg18-27V/I special and CD8+CTL was performed through FCM.Results1. The study showes that the frequency of HLA-A11, A2, A24 and A33 alleles was 57%, 41%, 21% and 7% respectively in CHB patients in Southern China, and the most common kind of allele is HLA-A11 which is about 16% more than that of HLA-A2. The frequency of these four alleles in healthy donors was 57%, 46%, 24% and 14% respectively, and HLA-A11 is about 11% more than that of HLA-A2. Only the distribution of HLA-A33 between the two groups had signifiacant difference. The combination of HLA-A2/A11, A2/A24, A2/A33, A11/A24, A11/A33 or A24/A33 alleles was 17%, 4%, 1%, 16%, 4%, 0.4% in CHB patients, and 18%, 8%, 4%, 11%, 5%, 1% in healthy donors respectively; there was no significant difference between the two groups. The distribution of HLA-A11, A2, A24 and A33 alleles was similar between HBeAg-positive and HBeAg-negative CHB patients.2. We chose the HBV infectious serum from 8 different areas in China, which B and C genotypes were the most popular HBV genotype. By PCR-RFLP analysed, we found that 98.12 % of all specimen determined were HBcAg18-27I, and 3/160 of all samples were detected as HBcAg18-27V. It is no significance between genotype B and C in the frequence of HBcAg18-27V or HBcAg18-27I. One humdred and eight of investigators were diagnosed as chronic actived hepatitis B, which genotype B and C was equal in this cluster, and one of them was determined as HBcAg18-27V. In different HBe antigen serological status, we found that in e antigen positive population, there was 57 cases of genotype B and 55 cases of genotype C, respectively detected as 2 cases and 1 case of HBcAg18-27V. about the affinity prediction trial, we could find the affinity of HBcAg18-27V or HBcAg18-27I combined with HLA-A*0201 anchor was different, and the affinity of HBcAg18-27V was higher than the HBcAg18-27I.3. In patients with CHB of Guangdong area, both HLA-A2 genotype (22/44) and HBV genotype B (35/44) were dominant genotypes, respectively; and HBcAg18-27I was also the dominant variant (43/44). In the HLA-A2 (+) group, HBcAg18-27I-tetramer special CTL was more than HBcAg18-27V-tetramer special CTL (0.52±0.31 VS 0.13±0.08, P=0.000)Conclusions1. HLA-A11, A2, A24 and A33 were the major HLA-A alleles in CHB patients of Southern China, of them the most common one is HLA-A11. The importance for identification of HLA-A11-restricted and HBV-specific new CTL epitopes is no less than that of HLA-A2-restricted HBV-specific CTL epitopes for the research of basic and clinical immunology of HBV infection. The distribution of these four major HLA-A alleles and their combination patterns have no significant differences between HBeAg-positive and HBeAg-negative CHB patients, but their influence on other clinical characteristics and the response of antiviral therapies in CHB patients should be further investigated.2. HBcAg18-27I was the major HBcAg18-27 phenotype in HBV infectious of China, and it was said that the distribution of HBcAg18-27V/I mutant in China was predominantly different to the distribution in European and American. The virological background may induce different outcome of antiviral therapy and mechanism of cellular immunity in different HBV genotype infectious.3. In HBV genotype B/C area, HBcAg18-27V being used to research the patients with CHB can induce the consequence deviation, when HBV virology background was not considered.
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