Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection

Dysfunctional CD8+ T cells present in chronic virus infections can express programmed death 1 (PD-1) molecules, and the inhibition of the engagement of PD-1 with its ligand (PD-L1) has been reported to enhance the antiviral function of these T cells. We took advantage of the wide fluctuations in levels of viremia which are typical of chronic hepatitis B virus (HBV) infection to comprehensively analyze the impact of prolonged exposure to different virus quantities on virus-specific T-cell dysfunction and on its reversibility through the blocking of the PD-1/PD-L1 pathway. We confirm that chronic HBV infection has a profound effect on the HBV-specific T-cell repertoire. Despite the use of a comprehensive panel of peptides covering all HBV proteins, HBV-specific T cells were rarely observed directly ex vivo in samples from patients with chronic infection, in contrast to those from patients with acute HBV infection. In chronic HBV infection, virus-specific T cells were detected mainly in patients with lower levels of viremia. These HBV-specific CD8+ T cells expressed PD-1, and their function was improved by the blocking of PD-1/PD-L1 engagement. Thus, a broad spectrum of anti-HBV immunity is expressed by patients with chronic HBV infection and this spectrum is proportional to HBV replication levels and can be improved by blocking the PD-1/PD-L1 pathway. This information may be useful for the design of immunotherapeutic strategies to complement and optimize available antiviral therapies.     

Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection

The proliferative response of PBMC to hepatitis B virus (HBV) envelope, core, and e Ag was analyzed prospectively in 21 patients with acute self-limited HBV infection and compared with the response of patients with chronic HBV infection and different levels of HBV replication (i.e., hepatitis e Ag (HBeAg)- or anti-HBe-positive) and liver damage (i.e., chronic active hepatitis or chronic asymptomatic carriers). Our results indicate that: 1) HBV-infected subjects who develop a self-limited acute hepatitis show a vigorous PBMC response to hepatitis B core Ag and HBeAg, as expression of T cell activation; 2) appearance of a detectable lymphocyte response to HBV nucleocapsid Ag is temporally associated with the clearance of HBV envelope Ag; 3) in patients with chronic HBV infection the level of T cell responsiveness to hepatitis B core Ag and to HBeAg is significantly lower than that observed during acute infection; 4) T cell sensitization to HBV envelope Ag in acute and chronic HBV infection is usually undetectable and when measurable is expressed transiently and at low levels. These results may reflect immune events of pathogenetic relevance with respect to evolution of disease and viral clearance.     

Evidence of viral adaptation to HLA class I-restricted immune pressure in chronic hepatitis C virus infection

Cellular immune responses are an important correlate of hepatitis C virus (HCV) infection outcome. These responses are governed by the host's human leukocyte antigen (HLA) type, and HLA-restricted viral escape mutants are a critical aspect of this host-virus interaction. We examined the driving forces of HCV evolution by characterizing the in vivo selective pressure(s) exerted on single amino acid residues within nonstructural protein 3 (NS3) by the HLA types present in two host populations. Associations between polymorphisms within NS3 and HLA class I alleles were assessed in 118 individuals from Western Australia and Switzerland with chronic hepatitis C infection, of whom 82 (69%) were coinfected with human immunodeficiency virus. The levels and locations of amino acid polymorphisms exhibited within NS3 were remarkably similar between the two cohorts and revealed regions under functional constraint and selective pressures. We identified specific HCV mutations within and flanking published epitopes with the correct HLA restriction and predicted escaped amino acid. Additional HLA-restricted mutations were identified that mark putative epitopes targeted by cell-mediated immune responses. This analysis of host-virus interaction reveals evidence of HCV adaptation to HLA class I-restricted immune pressure and identifies in vivo targets of cellular immune responses at the population level.     

Cellular immune responses to the hepatitis B virus polymerase

CD4 T cells play an important role in hepatitis B virus (HBV) infection by secretion of Th1 cytokines that down-regulate HBV replication, and by promoting CD8 T cell and B cell responses. We have identified and characterized 10 CD4 T cell epitopes within polymerase and used them to analyze the immunological effects of long-term antiviral therapy as compared with spontaneous recovery from HBV infection. Candidate epitopes were tested for binding to 14 HLA-DR molecules and in IFN-gamma ELISPOT and cytotoxicity assays using peripheral blood lymphocytes from 66 HBV-infected patients and 16 uninfected controls. All 10 epitopes bound with high affinity to the most prevalent HLA-DR Ags, were conserved among HBV genomes, and induced IFN-gamma responses from HBV-specific CD4+ T cells. Several epitopes contained nested MHC class I motifs and stimulated HBV-specific IFN-gamma production and cytotoxicity of CD8+ T cells. HBV polymerase-specific responses were more frequent during acute, self-limited hepatitis and after recovery (12 of 18; 67%) than during chronic hepatitis (16 of 48 (33%); p=0.02). Antiviral therapy of chronic patients restored HBV polymerase and core-specific T cell responses during the first year of treatment, but thereafter, responses decreased and, after 3 years, were no more frequent than in untreated patients. Decreased T cell responsiveness during prolonged therapy was associated with increased prevalence of lamivudine-resistant HBV mutants and increased HBV titers. The data provide a rationale for the combination of antiviral and immunostimulatory therapy. These newly described HBV polymerase epitopes could be a valuable component of a therapeutic vaccine for a large and ethnically diverse patient population.     

Hepatitis C virus (HCV) and hepatitis B virus (HBV) can coinfect the same hepatocyte in the liver of patients with chronic HCV and occult HBV infection

In this work, we have shown that hepatitis C virus (HCV) and hepatitis B virus (HBV) can coexist in the same hepatocyte using double fluorescent in situ hybridization in liver biopsy samples from patients with chronic HCV infection with occult HBV infection. Digital image analysis of hybridization signals showed that the HBV DNA levels in coinfected hepatocytes were lower than those in cells infected only with HBV. This finding supports the hypothesis of inhibition of HBV replication by HCV. Furthermore, HCV RNA levels were lower in coinfected cells than in cells infected only with HCV, suggesting that HBV may also inhibit HCV replication.     

Genotype and subtype analyses of hepatitis B virus (HBV) and possible co-infection of HBV and hepatitis C virus (HCV) or hepatitis D virus (HDV) in blood donors, patients with chronic liver disease and patients on hemodialysis in Surabaya, Indonesia

Four subtypes (adw, adr, ayw, and ayr ) and eight genotypes (A to H) of the hepatitis B virus (HBV) have been identified. They appear to be associated with particular geographic distribution, ethnicity, and possibly clinical outcomes. In this study, hepatitis B surface antigen (HBsAg) subtyping and HBV genotyping were carried out on sera obtained from HBsAg-positive HBV carriers, including healthy blood donors; patients with acute hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; and patients on hemodialysis all located in Surabaya, Indonesia. We report here that all HBV isolates tested in Surabaya belonged to genotype B, with more than 90% of them being classified into subtype adw. Our results also revealed that prevalence of hepatitis C virus (HCV) co-infection among HBV carriers in Surabaya was approximately 10% for healthy blood donors and patients with chronic liver disease, and approximately 60% for patients on maintenance hemodialysis. Interestingly, HBsAg titers were lower in HBV carriers with HCV co-infection than in those without HCV co-infection. We also found that prevalence of hepatitis D virus (HDV) co-infection was < 0.5% among HBV carriers in Surabaya.     

The ecology of host immune responses to chronic avian haemosporidian infection

Host responses to parasitism in the wild are often studied in the context of single host–parasite systems, which provide little insight into the ecological dynamics of host–parasite interactions within a community. Here we characterized immune system responses to mostly low-intensity, chronic infection by haemosporidian parasites in a sample of 424 individuals of 22 avian host species from the same local assemblage in the Missouri Ozarks. Two types of white blood cells (heterophils and lymphocytes) were elevated in infected individuals across species, as was the acute-phase protein haptoglobin, which is associated with inflammatory immune responses. Linear discriminant analysis indicated that individuals infected by haemosporidians occupied a subset of the overall white blood cell multivariate space that was also occupied by uninfected individuals, suggesting that these latter individuals might have harbored other pathogens or that parasites more readily infect individuals with a specific white blood cell profile. DNA sequence-defined lineages of haemosporidian parasites were sparsely distributed across the assemblage of hosts. In one well-sampled host species, the red-eyed vireo (Vireo olivaceus), heterophils were significantly elevated in individuals infected with one but not another of two common parasite lineages. Another well-sampled host, the yellow-breasted chat (Icteria virens), exhibited no differences in immune response to different haemosporidian lineages. Our results indicate that while immune responses to infection may be generalized across host species, parasite-specific immune responses may also occur.     

Recognition of hepatitis B virus envelope proteins by liver-infiltrating T lymphocytes in chronic HBV infection

The Ag specificity and cytotoxic function of human T cell clones, generated from lymphocytes infiltrating the liver of a chronic hepatitis B patient, were studied. Both class I- and class II-restricted T clones specifically proliferated to hepatitis B virus envelope proteins, but not to hepatitis B core Ag. The fine specificity of T cells was studied by using rAg having different composition in relation to HBV-envelope proteins or synthetic peptides of preS regions. The antigenic determinant recognized by T cell clones mapped to the preS2 region based on the response to r(preS1+preS2+S) and to r(preS2+S) and the failure to respond to S or preS1 alone. More precise epitope mapping was based on synthetic preS2 peptides 120-150 or 120-134, which stimulated both class I- and class II-restricted T clones, whereas preS2 153-171 or preS1 1-110 peptides did not; thus, the preS2 120-134 appears to contain both the residues binding to class I molecules and the residues binding to class II molecules. Moreover, strong and specific cytotoxic responses of these clones were observed only when HLA-matched EBV-lines, used as target cells, were previously sensitized with r(preS1+preS2+S) or preS2 peptides, which were shown to stimulate the clones. Thus, a preS2 epitope can represent a target Ag for liver-infiltrating T cells, which could kill the hepatocytes expressing the Ag plus the appropriate MHC molecule.     

自然人群乙型肝炎病毒慢性感染者HBV DNA的流行病学分析

目的了解寿光市自然人群乙型肝炎病毒(hepatitis B virus,HBV)慢性感染者HBV DNA的流行病学特征,为乙型肝炎的防治提供依据。方法采集2012年寿光市3个镇街道居民体检发现的HBs Ag阳性者静脉血5 m L,以实时荧光定量聚合酶链反应(FQ-PCR)法检测血清HBV DNA载量,分析HBV DNA与性别、年龄、有无乙肝/肝癌家族史、密切接触史、输血史等因素的相关性。结果在2 026例HBs Ag阳性感染者中,HBV DNA阳性率为49.41%(1 001/2 026),HBV DNA载量的平均值为6.27×107拷贝/m L。其中男性HBV DNA阳性率为53.28%(585/1 098),女性为44.83%(416/928),差异有统计学意义(χ2=14.370,P<0.01);HBV DNA均值男性为(6.72×107±2.07×108)拷贝/m L,女性为(5.56×107±1.44×108)拷贝/m L,差异无统计学意义(t=0.447,P>0.05)。HBVDNA阳性率与年龄呈负相关(r=-0.983),与HBV感染的主要影响因素乙肝/肝癌家族史、密切接触史、输血史等均无关。结论 HBV DNA阳性率与慢性HBV感染者的性别、年龄有关,与HBV感染的主要影响因素无关。     

Occult Hepatitis B virus (HBV) infection and challenges for hepatitis elimination: A literature review

Occult hepatitis B infection (OBI) is characterized by the detection of hepatitis B virus (HBV) DNA in serum or liver but negativity for hepatitis B surface antigen. OBI, which is thought to be maintained by host, immunological, viral and/or epigenetic factors, is one of the most challenging clinical features in the study of viral hepatitis. Currently, there is no validated detection test for OBI. It is believed that OBI is widely distributed throughout the world, with a higher prevalence in populations at high-risk HBV, but the detailed worldwide prevalence patterns are unknown. We conducted a survey of recently published studies on OBI rates across all continents. High prevalence rates of OBI are observed in some specific groups, including patients with hepatitis C virus, human immunodeficiency virus co-infection or hepatocellular carcinoma. In 2016, the World Health Organization adopted strategies to eliminate viral hepatitis by 2030, but the difficulties in detecting and treating OBI currently challenge this goal. Subjects with OBI can transmit HBV, and episodes of reactivation can occur. Further studies to understanding the mechanisms that drive the development of OBI are needed and can contribute to efforts at eliminating viral hepatitis.     

Increased oxidative damage of RNA in liver injury caused by hepatitis B virus (HBV) infection

To evaluate the urinary levels of 8-oxo-7,8-dihydro-2′deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) in liver injury patients with hepatitis B virus (HBV) infection and to explore the relationship between urinary 8-oxo-dGsn or 8-oxo-Gsn and degree of liver damage. We enrolled 138 liver injury patients with HBV infection and 169 age- and sex-matched healthy controls in this study. A sensitive and accurate isotope-diluted liquid chromatograph mass spectrometer/mass spectrometer (LC-MS/MS) method was used to measure the urinary levels of 8-oxo-Gsn and 8-oxo-dGsn. Simultaneously, pathological analysis of liver biopsy tissues was carried out, and immunohistochemistry was carried out for 8-oxo-Guo, 8-oxo-dGuo and MTH1 protein in some liver injury tissues. We analysed the correlation between the degrees of inflammation and fibrosis and levels of 8-oxo-Gsn and 8-oxo-dGsn. We also analysed the levels of urinary 8-oxo-Gsn and 8-oxo-dGsn with clinical data of HBeAg, HBsAg, and HBV genotype and detected the levels of plasma aspartate aminotransferase, alanine aminotransferase (AST), platelet, alkaline phosphatase, prothrombin time (PT) and HBV DNA, and calculated the aspartate amino transferase-to–platelet ratio index (APRI) score. Nonparametric correlations were used to evaluate the correlation between 8-oxo-Gsn, 8-oxo-dGsn or APRI and various laboratory biochemical indicators. Results showed that the levels of urinary 8-oxo-Gsn and 8-oxo-dGsn in patients with liver injury were significantly higher than those of healthy controls (both p?p?=?.013, p?=?.026 and p?=?.049). The receiver operating characteristic curves of 8-oxo-Gsn were 0.696 (0.632–0.759) and 0.731 (0.672–0.790) for inflammatory activity and fibrosis, respectively. Patients with higher levels of urinary 8-oxo-Gsn are more likely to have a high degree of fibrosis and urinary 8-oxo-Gsn may have a great potential in assessing liver fibrosis.     

Emergence of and takeover by hepatitis B virus (HBV) with rearrangements in the pre-S/S and pre-C/C genes during chronic HBV infection.

We have shown, by analyzing serial serum samples from a chronic hepatitis B virus (HBV) carrier, the emergence of HBV DNA molecules with nucleotide rearrangements in the pre-S/S and pre-C/C genes. Serum samples were obtained at four different times (1983, 1985, 1988, and 1989) from an HBsAg- and HBeAg-positive carrier with chronic hepatitis. The polymerase chain reaction was used to amplify the pre-S/S and pre-C/C genes. The amplified products were cloned, and 8 to 10 independent clones were sequenced. In 1983 and 1985 only one type of HBV DNA molecule was observed. Nucleotide divergence relative to the adw2 subtype was 4.7, 7.2, and 1.6%, for the pre-S1, pre-S2, and S regions, respectively, and 2.2 and 3.9% for the pre-C and C regions, respectively. In 1988 and 1989, HBV DNA forms with marked rearrangements of both the pre-S/S and pre-C/C regions were evidenced. In the pre-S/S region, they comprised two distinct HBV DNA molecules. The first showed nucleotide divergence of 20.4, 14.8, and 3.3% for the pre-S1, pre-S2, and S regions when compared with the adw2 sequence. In addition, nucleotide deletions in the pre-S1 region led to the appearance of a stop codon. The second was created by recombination between the original and mutated HBV DNA. In the pre-C/C region, the mutated viral DNA showed 11.7% divergence when compared with the adw2 sequence. A point mutation led to the creation of a stop codon in the pre-C region, together with an insertion of 36 nucleic acids in the core gene. Most of this DNA insertion was identical to that reported in an independent HBV isolate but showed no significant homology with known sequences. Semiquantitative estimation of the proportion of wild-type and mutated HBV DNA molecules showed a marked increase in the mutated forms during the period of follow-up. Sucrose gradient analysis indicated that the defective HBV DNA molecules were present in circulating virions. Western immunoblot analysis showed the appearance of modified translation products. Our findings thus indicate the emergence of and gradual takeover by mutated HBV DNA forms during the HBV chronic carrier state. The rearrangements we observed in the pre-S/S and pre-C/C genes might lead to changes in the immunogenicity of the viral particles and thus affect the clearance of the virus by the immune system.     

A revised evolutionary history of hepatitis B virus (HBV)

Previous studies of the evolutionary history of hepatitis B virus (HBV) have been compromised by intergenotype recombination and complex patterns of nucleotide substitution, perhaps caused by differential selection pressures. We examined the phylogenetic distribution of recombination events among human HBV genotypes and found that genotypes A plus D, and genotypes B plus C, had distinct patterns of recombination suggesting differing epidemiological relationships among them. By analyzing the nonoverlapping regions of the viral genome we found strong bootstrap support for some intergenotypic groupings, with evidence of a division between human genotypes A–E from the viruses sampled from apes and human genotype F. However, the earliest events in the divergence of HBV remain uncertain. These uncertainties could not be explained by differential selection pressures, as the ratio of nonsynonymous-to-synonymous substitutions (d _N/d _S) did not vary extensively among lineages and there is no strong evidence for positive selection across the whole tree. Finally, we provide a new estimate of the mean substitution rate in HBV, 4.2 × 10⁻⁵, which suggests that divergence of HBV in humans and apes has occurred only in the last 6000 years.     

The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice.

In this study, we examined the ability of the hepatitis B virus (HBV) precore, envelope, and X gene products to modulate HBV replication in the livers of transgenic mice that replicate the virus. Hepatic HBV replication was not affected by overexpression of the envelope or X gene products when these animals were crossed with transgenic mice that express the corresponding viral genes in the hepatocyte. Overexpression of the precore protein, however, eliminated nucleocapsid particles from the cytoplasm of the hepatocytes and abolished HBV replication without affecting the hepatic steady-state content of pregenomic HBV RNA. These observations suggest that the precore protein can exert a dominant negative effect on HBV replication, presumably at the level of nucleocapsid particle maturation or stability, suggesting an important role for this enigmatic viral protein in the HBV life cycle.     

Anti-idiotype modulation of the in vitro immune response to hepatitis B surface antigen (HBsAg) following remote infection by hepatitis B virus in man

An antigen-inhibitable Ab-2 that exhibits internal image activity will selectively stimulate the in vitro production of anti-HBs in individuals with remotely established immunity to hepatitis B virus. This response is seen (1) in the absence of a polyconal increase in total IgG, (2) with the F(ab')2 component of the Ab-2, (3) in cultures depleted of T-cells, and (4) in the absence of stimulation by antigen. This observation demonstrates that the Ab-2-mediated stimulation of specific IgG production may be an important regulatory function in man.     

The relationship between core promoter mutation of hepatitis B virus, viral load and hepatitis B e antigen status in chronic hepatitis B patients

The aim of this study is to detect the possible association of hepatitis B virus (HBV) core mutation, hepatitis B e antigen (HBeAg) status and the viral load in chronic hepatitis B (CHB) patients. Sixty-six patients with CHB were enrolled. Hepatitis markers and hepatitis C virus antibody (HCV-Ab) were tested using micro particle enzyme immunoassay kits. Viral load was measured by real-time polymerase chain reaction (PCR) and the mutation was analyzed by nested PCR followed by restriction fragment length polymorphism. Most of CHB patients were HBeAg (-ve). The HBeAg status did not have an influence on the presence or absence of T1762/A1764 mutation. HBV-DNA serum level was not significantly different in patients with core mutation and patients without core mutation in HBeAg (-ve) group, while in HBeAg (+ve) group HBV-DNA serum level was significantly higher in patients with core mutation. This study reports the predominance of HBeAg (-ve) and HBV core promoter mutation.