Transgenic Expression of Entire Hepatitis B Virus in Mice Induces Hepatocarcinogenesis Independent of Chronic Liver Injury

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV (“preS2 mutant”) that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged—2-year-old—mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines—expressing either mutant or wildtype HBV—therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC

HBV quasispecies composition in Lamivudine-failed chronic hepatitis B patients and its influence on virological response to Tenofovir-based rescue therapy

The present study sought to evaluate the structure of HBV quasispecies in Lamivudine (LMV)-failed chronic hepatitis B (CHB) patients and its impact in defining the subsequent virological responses to Tenofovir (TDF)-based rescue-therapy. By analyzing HBV clones encompassing reverse transcriptase (RT) and surface (S) region from LMV-failed and treatment-naïve CHB patients, we identified 5 classical and 12 novel substitutions in HBV/RT and 9 substitutions in immune-epitopes of HBV/S that were significantly associated with LMV failure. In silico analysis showed spatial proximity of some of the newly-identified, mutated RT residues to the RT catalytic centre while most S-substitutions caused alteration in epitope hydrophobicity. TDF administration resulted in virological response in 60% of LMV-failed patients at 24-week but non-response in 40% of patients even after 48-weeks. Significantly high frequencies of 6 S-substitutions and one novel RT-substitution, rtH124N with 6.5-fold-reduced susceptibility to TDF in vitro, were noted at baseline in TDF non-responders than responders. Follow-up studies depicted greater evolutionary drift of HBV quasispecies and significant decline in frequencies of 3 RT and 6 S-substitutions in responder-subgroup after 24-week TDF-therapy while most variants persisted in non-responders. Thus, we identified the HBV-RT/S variants that could potentially predict unfavorable response to LMV/TDF-therapy and impede immune-mediated viral clearance

Cellular immunity in children with successful immunoprophylactic treatment for mother-to-child transmission of hepatitis B virus

Background: The administration of hepatitis B immunoglobulin followed by hepatitis B vaccine can result in a protective efficacy of almost 90% in mother-to-child transmission of hepatitis B virus (HBV). However, little is known about immunity against HBV infection in children after immunoprophylactic treatment. We tried to assess the association between T-cell responses and viremia in children after successful prophylactic treatment. Methods: Thirteen children and their 8 HBV carrier mothers (8 families), who were positive for human leukocyte antigen (HLA)-A24, were enrolled in this study. All of the 13 children received immunoprophylactic treatment and became negative for hepatitis B surface antigen (HBsAg) after birth. HBV-specific cytotoxic T lymphocyte (CTL) responses were evaluated using IFNγ - enzyme-linked immunosorbent spot (ELISPOT) and major histocompatibility complex class I peptide pentamer assays. Serum HBV DNA was measured by real-time PCR. Results: Significant HBV-specific T-cell responses were detected in 2 (15%) of the 13 children by ELISPOT. However, the frequency of HLA-A24-HBV-specific CTLs was very low in both HBV carrier mothers and children using pentamers. Of the 13 children, 4 (31%) were positive for serum HBV DNA. However, the levels of serum HBV DNA were 100 copies/ml or less. One of the 2 children in whom significant HBV-specific CTL responses were detectable was positive for serum HBV DNA. Conclusions: HBV core and polymerase-specific T-cell responses were detected and a low-dose viremia was observed in children after successful immunoprophylaxis treatment. Although the presence of viremia was not related to HBV-specific T-cell responses, CTLs might play a role in the control of HBV infection in children born to HBsAg-positive mothers after immunoprophylactic treatment. </p

The role of mutations in core protein of hepatitis B virus in liver fibrosis

The core protein of hepatitis B virus encompasses B- and T-cell immunodominant epitopes and subdivided into two domains: the N-terminal and the functional C-terminal consisted phosphorylation sites. Mutations of the core gene may change the conformation of the core protein or cause alteration of important epitopes in the host immune response. In this study twenty nine men (mean age 40 ± 9 years old) with chronic hepatitis B were recruited for direct sequencing of the core gene. Serum ALT and HBV DNA level were measured at the time of liver biopsy. The effects of core protein mutations on patients' characteristics and subsequently mutations in B cell, T helper and cytotoxic T lymphocyte (CTL) epitopes and also C-terminal domain of core protein on the activity of liver disease was evaluated. Liver fibrosis was significantly increased in patients with core protein mutation (1.0 ± 0.8 vs 1.9 ± 1.4 for mean stage of fibrosis P = 0.05). Mutations in CTL epitopes and in phosphorylation sites of C-terminal domain of core protein also were associated with higher liver fibrosis (P = 0.003 and P = 0.04; Fisher's exact test for both). Patients with mutation in C-terminal domain had higher serum ALT (62 ± 17 vs 36 ± 12 IU/l, p = 0.02). Patients with mutations in B cell and T helper epitopes did not show significant difference in the clinical features. Our data suggests that core protein mutations in CTL epitopes and C-terminal domain accompanied with higher stage of liver fibrosis may be due to alterations in the function of core protein

Complete hepatitis B virus genome analysis in HBsAg positive mothers and their infants with fulminant hepatitis B

BACKGROUND: After perinatal transmission of hepatitis B virus, infants of anti-HBe positive HBsAg carrier mothers may develop fulminant hepatitis B. Previously it has been suggested, that fulminant hepatitis B in adults was associated with specific mutations in the HBV-genome. The aim of this study was to investigate, whether specific viral variants are associated with fulminant hepatitis B in young infants. METHODS: The complete HBV-genomes of five mothers and their infants with fulminant hepatitis were isolated from the sera, amplified and directly sequenced. RESULTS: Between 6 and 43 base pair exchanges between the HBV genomes of the infants and their mothers were identified. The mutations spread over the entire virus genome. Nucleotide exchanges in the basic core promotor and precore region were identified in all cases. A heterogeneous virus population was detected in four mothers. CONCLUSIONS: Many new mutations were proved to emerge during fulminant hepatitis B in infants, who had been perinatally infected. HBeAg negative variants were the predominant population in all children, whereas these mutants could only be detected as subpopulations in four mothers. The data suggest that the selection of a specific HBeAg negative viral strain may be associated with the development of fulminant hepatitis B in children

HBsAg Inhibits the Translocation of JTB into Mitochondria in HepG2 Cells and Potentially Plays a Role in HCC Progression

Background and Aims: The expression of the jumping translocation breakpoint (JTB) gene is upregulated in malignant liver tissues; however, JTB is associated with unbalanced translocations in many other types of cancer that suppress JTB expression. No comprehensive analysis on its function in human hepatocellular carcinoma (HCC) has been performed to date. We aimed to define the biological consequences for interaction between JTB and HBsAg in HCC cell lines. Methods: We employed the stable transfection to establish small HBsAg expressing HepG2 cell line, and stably silenced the JTB expression using short hairpin RNA in HepG2 cell line. The effects of JTB and small HBsAg in vitro were determined by assessing cell apoptosis and motility. Results: Silencing of JTB expression promoted cancer cell motility and reduced cell apoptosis, which was significantly enhanced by HBs expression. Expression of HBsAg inhibited the translocation of JTB to the mitochondria. Furthermore, silencing of the JTB resulted in an increase in the phosphorylation of p65 in HepG2 cells and HepG2-HBs cells, whereas HBsAg expression decreased the phosphorylation of p65. The silencing of JTB in HepG2-HBs cells conferred increased advantages in cell motility and anti-apoptosis. Conclusion: HBsAg inhibited the translocation of JTB to the mitochondria and decreased the phosphorylation of p65 through the interaction with JTB, After JTB knockdown, HBsAg exhibited a stronger potential to promote tumor progression. Our data suggested that JTB act as a tumor suppressor gene in regards to HBV infection and its activation might be applied as a therapeutic strategy for in control of HBV related HCC development.National Natural Science Foundation of China [30971362, 81072013]; Fundamental Research Funds for the Central Universities in China [2010111082]; Key Projects for Technology Plan of Fujian Province in China [2009D020]; Foundation of Health Bureau of Fujian in China [2007CXB8, 3502z20077046]; Foundation of Health Bureau of Xiamen in China [2007CXB8, 3502z20077046

Protection of Hepatocytes from Cytotoxic T Cell Mediated Killing by Interferon-Alpha

&lt;p&gt;Background: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV) infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL) to recognise and kill hepatocytes under cytokine stimulation.&lt;/p&gt; &lt;p&gt;Methods/Principle Findings: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-α treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-α stimulated hepatocyte lines were still able to present antigen and induce IFN-γ expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-α, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor–proteinase inhibitor 9 (PI-9). PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection.&lt;/p&gt; &lt;p&gt;Conclusion/Significance: IFN-α induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.&lt;/p&gt

Analysis of FOXP3+ Regulatory T Cells That Display Apparent Viral Antigen Specificity during Chronic Hepatitis C Virus Infection

We reported previously that a proportion of natural CD25+ cells isolated from the PBMC of HCV patients can further upregulate CD25 expression in response to HCV peptide stimulation in vitro, and proposed that virus-specific regulatory T cells (Treg) were primed and expanded during the disease. Here we describe epigenetic analysis of the FOXP3 locus in HCV-responsive natural CD25+ cells and show that these cells are not activated conventional T cells expressing FOXP3, but hard-wired Treg with a stable FOXP3 phenotype and function. Of ∼46,000 genes analyzed in genome wide transcription profiling, about 1% were differentially expressed between HCV-responsive Treg, HCV-non-responsive natural CD25+ cells and conventional T cells. Expression profiles, including cell death, activation, proliferation and transcriptional regulation, suggest a survival advantage of HCV-responsive Treg over the other cell populations. Since no Treg-specific activation marker is known, we tested 97 NS3-derived peptides for their ability to elicit CD25 response (assuming it is a surrogate marker), accompanied by high resolution HLA typing of the patients. Some reactive peptides overlapped with previously described effector T cell epitopes. Our data offers new insights into HCV immune evasion and tolerance, and highlights the non-self specific nature of Treg during infection

Disruption of TLR3 Signaling Due to Cleavage of TRIF by the Hepatitis A Virus Protease-Polymerase Processing Intermediate, 3CD

Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3Cpro, that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3Cpro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3Cpro and downstream 3Dpol sequence, but not 3Dpol polymerase activity. Cleavage occurs at two non-canonical 3Cpro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3Dpol sequence modulates the substrate specificity of the upstream 3Cpro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3Cpro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate