| Hepatitis B is an important disease with great menace to people's health. Although progresses have been obtained in some aspects of the pathogenesis, many questions still to be clarified. Usually hepatitis B virus (HBV) does not directly cause hepatocyte damage. Amount intrahepatic filtration of lymphocytes indicates that cellular immune response is closely associated with pathological damage. HBV antigenic epitope-specific cytotoxic T lymphocyte (CTL) response is major mechanism for viral clearance or control. CTL epitope harbored in viral protein is the target for specific CTL to bind and attack. Since CTL epitope presentation is restricted by host HLA class I molecules and HBV genotype has significant influence on CTL epitope variation, HLA class I typing and HBV genotyping are needed for investigation of patients'CTL epitopic variation. In this study, direct sequencing was used to analyze complete HBV genome sequences. HBV-specific HLA-A2-restricted CTL epitopic variations were analyzed for patients with acute hepatitis B (CHB), chronic hepatitis B (CHB), and chronic severe hepatitis B (CSHB), respectively. Simultaneously, HBV genotype was determined based on sequence-based phylogentic analysis. The study is to seek after the variant CTL epitope(s) that may associates with the disease progress towards exacerbation. The results are summarized as follows.1. Duplex overlapping nested PCR for the amplification of complete HBV genome. The total of 516 pieces of complete HBV genomes were successfully acquired from 516 individual patients, including 131 sequences from AHB patients that have been submitted to BenBank, 239 sequences from CHB patients that have been registered in GenBank (accession number: FJ386574-FJ386689), and 146 sequences from CSHB patients that have been registered in GenBank, too (accession number: EU939536-EU939681).2. MEGA 4.0 software for phylogenetic tree analysis of HBV genotype. In AHB patients, genotype B, C, and D occupied 20.6% (27/131), 78.6% (103/131), and 0.8% (1/131), respectively. In CHB patients, genotype B, C, and D occupied 21.3 (51/239), 77.0% (184/239), and 1.7% (4/239), respectively. In CSHB patients, genotype B, C, and D occupied 22.6% (33/146), 76.0% (111/1146), and 1.4% (2/146), respectively. Proportion of each genotype had no significant difference among the three groups of patients. 3. Gene amplification and sequencing for HLA-A2 genotyping for total 516 patients. The results showed that HLA-A2 positive frequencies were 51.1% (67/131) in AHB patients, 45.6% (109/239) in CHB patients, and 48.6% (71/146) in CSHB patients. The positive frequencies had no significant difference among three groups of patients in statistics.4. Acquirement of HBV epitopic variation profile of the HLA2 positive patients by comparing HBV-specific HLA-A2-restricted epitopic sequences which had been identified in previous publications. Occurence of X92-100 and S177-185 epitopes variation were significantly different among AHB, CHB and CSHB groups despite of their genotype(p<0.01).When only HBV genotype B patients were included,Occurrence of P455-463 and P816-824 epitopes variation were significantly different among the three groups. When only HBV genotype C patients were included, occurrence of X92-100,S177-185(P<0.01), X102-110,X115-123,S131-139 and S183-191(P<0.05) epitopes variation were significantly different among the three groups,occurrence of S335-343 variation was marginal different (P=0.05). Although the occurrence of S269-278 variation didn't show significant difference (P=0.11), tendency test analysis by SAS software suggested that there might be significant difference when greater number of patients were included. For most epitope variation sites, there is only one amino acid substitution. However, multiple substitutions can be seen at some episopic variations. Such as X36-44,S201-210 and S204-212. As some of the epitopes are overlapped, e.g: S177-185 and S183-191, S201-S210 and S204-S212, S269-278 and S271-280, S335 and S338-347, one amino acid change could cause two CTL epitopes changes simultaneously. Changes of an isoleucine to a valine substitution occurred in 227 among all 247 patients.(91.9%) and the variation rate of X52-60 and S370-379 are both 100%, HLSLRGLFV to HLSLRGLPV, and SIVSPFIPLL to NIVSPFIPLL respectively.In summary, (theresults of) our study showed that variation occurrence of some HBV-specific HLA-A2 restricted CTL epitopes was associated with disease status, suggesting that the CTL epitopic variation might be a virologic factor to influence the progression of the disease and severity of chronic hepatitis B. |
