免疫選擇和B型肝炎病毒聚合酶基因序列變異對長期病毒量及肝臟疾病發展之影響

Abstract

Background During chronic hepatitis B virus (HBV) infection, evolution of viral genome continues under immune selection pressure, and the occurrence of genetic diversity, including those specific mutations that facilitate viral adaptation, can impact viral replication and thereafter the host's susceptibility to hepatic disease progression. However, studies of HBV sequence divergence with relevance to evolution, viral replication activity, and disease pathogenesis have been limited. Specific Aims There are four distinct but complementary aims to understand HBV sequence variation in relation to viral evolutionary change during the natural course of chronic infection and subsequent risk for disease progression in persons chronically infected with HBV: (i) To conduct phylogenetic analysis of HBV based on sequencing of the polymerase gene across a region of 2403 bp in a population-based study; (ii) To describe change in HBV sequence diversity across the four phases (i.e., immune-tolerant, immune-clearance, non/low replicative, and HBeAg-negative hepatitis phases) of the natural history of HBV infection at the population level; (iii) To determine the associations with levels of viral sequence divergence and specific viral polymorphisms (or mutations) for long-term dynamics of viral load and the risk for transition from healthy HBV carrier state to hepatocellular carcinoma and/or liver cirrhosis; as well as (iv) To study the potential role of sequence variation in the T-cell epitopes of the HBV-polymerase region in HBeAg seroconversion and subsequent development of hepatocellular carcinoma and/or liver cirrhosis. Subjects and Methods We performed direct sequencing of the HBV-polymerase region in baseline plasma samples from 867 treatment-naïve individuals presenting plasma HBV-DNA levels at least 1000 copies/mL in a case-cohort study (n=1143) on the longitudinal analysis of HBV viral load embedded in a cohort study of 2903 male HBsAg-positive government employees, followed up from study entry in 1989-1992 through 2006. We used linear mix models to assess the influences of viral evolutionary parameters and single nucleotide polymorphisms (SNPs) on the longitudinal levels of viral load. Multiple logistic regression was used to evaluate the associations between viral SNPs and clinical outcomes. Results: Phylogenetic tree analysis identified 5 distinct subgenotypes: Ba (80.0%), Ce (16.5%), Cs (1.2%), B3 (0.2%), and D (0.2%). Patterns of viral sequence diversity across the four phases of the natural history of HBV infection were similar between HBV subgenotypes Ba and Ce, despite of greater diversity observed for subgenotype Ce vs. Ba. There was a greater than 1.5-fold increase in the HBV-polymerase sequence diversity associated with HBeAg seroconversion, regardless of HBV subgenotype. Furthermore, levels of viral genetic divergence apart from the population consensus sequence, estimated by three viral evolutionary parameters (Kimura two-parameter distance, the number of synonymous substitutions per synonymous site, and the number of nonsynonymous substitutions per nonsynonymous site), were inversely associated with longitudinal levels of viral load in a dose-dependent manner even in HBeAg-negative subjects. Longitudinal analysis of each SNP in the entire sequence region identified 153 viral load-associated SNPs in overall and 136 in HBeAg-negative subjects, showing distinct profiles between HBV subgenotype Ba and Ce. These viral SNPs appeared to be most evident at sites within or flanking T-cell epitopes. Seven viral SNPs revealed associations with both enhanced viral load and a more than 4-fold increase in subsequent risks for hepatocellular carcinoma and/or liver cirrhosis. There is significant sequence variation in the T-cell epitopes between HBV genotypes. Polymorphisms in 96 (37 for genotype B alone, 16 for genotype C alone, and 43 for both genotype B and C) T-cell epitopes were significantly associated with HBeAg status, with higher frequencies of variant epitopes seen among HBeAg-negative relative to HBeAg-positive subjects. These associations remained even after adjustment for age. Conclusions: Our study highlights the importance of change in virus-host interaction in the natural history during chronic HBV infection. Sequence variation occurring naturally in the HBV-polymerase region is associated with long-term dynamics of viral load and/or disease progression. Increased sequence diversity within certain T-cell eptopes may be accompanied by immune pressure during HBeAg seroconversion, thereby affecting the outcome of liver disease.