Circulating HBV RNA and hepatitis B core-related antigen trajectories in persons with HIV/HBV coinfection and HBsAg loss on tenofovir therapy.

BACKGROUND: We evaluated long-term trajectories of circulating hepatitis B virus (HBV)-RNA and hepatitis B core-related antigen (HBcrAg) in persons with and without hepatitis B surface antigen (HBsAg) loss during tenofovir therapy in the Swiss HIV Cohort Study. METHODS: We included 29 persons with HIV (PWH) with HBsAg loss and 29 matched PWH without loss. We compared HBV-RNA and HBcrAg decline and assessed the cumulative proportions with undetectable HBV-RNA and HBcrAg levels during tenofovir therapy using Kaplan-Meier estimates. RESULTS: HBsAg loss occurred after a median of 4 years (IQR 1 - 8). All participants with HBsAg loss achieved suppressed HBV-DNA and undetectable HBV-RNA preceding undetectable qHBsAg levels, whereas 79% achieved negative HBcrAg. In comparison, 79% of the participants without HBsAg loss achieved undetectable HBV-RNA and 48% negative HBcrAg. After two years on tenofovir, an HBV RNA decline ≥1 log10 copies/ml had 100% sensitivity and 36.4% specificity for HBsAg loss, whereas an HBcrAg decline ≥1 log10 U/ml had 91.0% sensitivity and 64.5% specificity. CONCLUSIONS: HBV-RNA suppression preceded undetectable qHBsAg levels, and had high sensitivity but low specificity for HBsAg loss during tenofovir therapy in PWH. HBcrAg remained detectable in approximately 20% of persons with, and 50% of persons without HBsAg loss.

Quantification of circulating HBV RNA expressed from intrahepatic cccDNA in untreated and NUC treated patients with chronic hepatitis B.

OBJECTIVE: A convenient, reproducible biomarker of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) transcriptional activity is lacking. We measured circulating HBV RNA (cirB-RNA) in untreated and nucleos(t)ide analogues (NUC) treated chronic hepatitis B (CHB) patients to define its correlation with intrahepatic viral markers and HBV core-related antigen (HBcrAg). DESIGN: Paired liver biopsy and serum samples were collected from 122 untreated and 30 NUC-treated CHB patients. We measured cirB-RNA, HBV DNA, hepatitis B surface antigen (HBsAg), HBcrAg and alanine aminotransferase levels. cirB-RNA was quantified using an investigational HBV RNA assay for use on the cobas 6800 system. The test detects a region spanning the HBV canonical polyadenylation site. cccDNA and 3.5 kb RNA in liver tissue were assessed by quantitative PCR and droplet digital PCR. RESULTS: cirB-RNA was detectable in 100% of HBeAg(+) chronic hepatitis (CH), 57% and 14% of HBeAg(-) CH and chronic infection untreated patients and 47% of NUC-treated patients. cirB-RNA undetectability was associated with lower intrahepatic cccDNA transcriptional activity, as well as serum HBcrAg, but no significant differences in HBsAg, in both untreated and treated patients. In untreated HBeAg(-) patients, cirB-RNA correlated with intrahepatic 3.5 kb RNA and cccDNA transcriptional activity, serum HBV DNA and HBcrAg, but not with HBsAg or total cccDNA levels. Combined undetectability of both cirB-RNA and HBcrAg detection in untreated HBeAg(-) patients identified a subgroup with the lowest levels of intrahepatic transcriptionally active cccDNA. CONCLUSION: Our results support the usefulness of quantification of circulating HBV RNA expressed from cccDNA as an indicator of intrahepatic active viral reservoir in both untreated and NUC-treated CHB patients. TRIAL REGISTRATION NUMBER: NCT02602847.

TLR8 agonist selgantolimod regulates Kupffer cell differentiation status and impairs HBV entry into hepatocytes via an IL-6-dependent mechanism.

OBJECTIVE: Achieving HBV cure will require novel combination therapies of direct-acting antivirals and immunomodulatory agents. In this context, the toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has been investigated in preclinical models and clinical trials for chronic hepatitis B (CHB). However, little is known regarding its action on immune effectors within the liver. Our aim was to characterise the transcriptomic changes and intercellular communication events induced by SLGN in the hepatic microenvironment. DESIGN: We identified TLR8-expressing cell types in the human liver using publicly available single-cell RNA-seq data and established a method to isolate Kupffer cells (KCs). We characterised transcriptomic and cytokine KC profiles in response to SLGN. SLGN's indirect effect was evaluated by RNA-seq in hepatocytes treated with SLGN-conditioned media (CM) and quantification of HBV parameters following infection. Pathways mediating SLGN's effect were validated using transcriptomic data from HBV-infected patients. RESULTS: Hepatic TLR8 expression takes place in the myeloid compartment. SLGN treatment of KCs upregulated monocyte markers (eg, S100A12) and downregulated genes associated with the KC identity (eg, SPIC). Treatment of hepatocytes with SLGN-CM downregulated NTCP and impaired HBV entry. Cotreatment with an interleukin 6-neutralising antibody reverted the HBV entry inhibition. CONCLUSION: Our transcriptomic characterisation of SLGN sheds light into the programmes regulating KC activation. Furthermore, in addition to its previously described effect on established HBV infection and adaptive immunity, we show that SLGN impairs HBV entry. Altogether, SLGN may contribute through KCs to remodelling the intrahepatic immune microenvironment and may thus represent an important component of future combinations to cure HBV infection.

Single-cell landscape of functionally cured chronic hepatitis B patients reveals activation of innate and altered CD4-CTL-driven adaptive immunity.

BACKGROUND & AIMS: Hepatitis B surface antigen (HBsAg) loss or functional cure (FC) is considered the optimal therapeutic outcome for patients with chronic hepatitis B (CHB). However, the immune-pathological biomarkers and underlying mechanisms of FC remain unclear. In this study we comprehensively interrogate disease-associated cell states identified within intrahepatic tissue and matched PBMCs (peripheral blood mononuclear cells) from patients with CHB or after FC, at the resolution of single cells, to provide novel insights into putative mechanisms underlying FC. METHODS: We combined single-cell transcriptomics (single-cell RNA sequencing) with multiparametric flow cytometry-based immune phenotyping, and multiplexed immunofluorescence to elucidate the immunopathological cell states associated with CHB vs. FC. RESULTS: We found that the intrahepatic environment in CHB and FC displays specific cell identities and molecular signatures that are distinct from those found in matched PBMCs. FC is associated with the emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes, and an activated innate response represented by liver-resident natural killer cells, specific Kupffer cell subtypes and marginated neutrophils. Surprisingly, we found MHC class II-expressing hepatocytes in patients achieving FC, as well as low but persistent levels of covalently closed circular DNA and pregenomic RNA, which may play an important role in FC. CONCLUSIONS: Our study provides conceptually novel insights into the immuno-pathological control of HBV cure, and opens exciting new avenues for clinical management, biomarker discovery and therapeutic development. We believe that the discoveries from this study, as it relates to the activation of an innate and altered immune response that may facilitate sustained, low-grade inflammation, may have broader implications in the resolution of chronic viral hepatitis. IMPACT AND IMPLICATIONS: This study dissects the immuno-pathological cell states associated with functionally cured chronic hepatitis B (defined by the loss of HBV surface antigen or HBsAg). We identified the sustained presence of very low viral load, accessory antigen-presenting hepatocytes, adaptive-memory-like natural killer cells, and the emergence of helper CD4 T cells with cytotoxic or effector-like signatures associated with functional cure, suggesting previously unsuspected alterations in the adaptive immune response, as well as a key role for the innate immune response in achieving or maintaining functional cure. Overall, the insights generated from this study may provide new avenues for the development of alternative therapies as well as patient surveillance for better clinical management of chronic hepatitis B.

Long-term quantitative hepatitis B surface antigen (HBsAg) trajectories in persons with and without HBsAg loss on tenofovir-containing antiretroviral therapy.

OBJECTIVES: Improving the understanding of the patterns of quantitative hepatitis B surface antigen (qHBsAg) trajectories associated with HBsAg loss is important in light of novel anti-hepatitis B virus agents being developed. We evaluated long-term qHBsAg trajectories in persons with HIV and HBV during tenofovir-containing antiretroviral therapy in the Swiss HIV Cohort Study. METHODS: We included 29 participants with and 29 without HBsAg loss, defined as qHBsAg <0.05 IU/mL. We assessed qHBsAg decline during therapy in both groups and used agglomerative hierarchical clustering to identify different qHBsAg trajectory profiles in persons with HBsAg loss. RESULTS: The median follow-up time was 11.9 years (IQR 8.4-14.1), and the median time to HBsAg loss was 48 months (IQR 12-96). Among participants with HBsAg loss, 79% had a qHBsAg decline ≥1 log10 IU/mL 2 years after starting tenofovir. The trajectories in qHBsAg levels during tenofovir therapy were heterogeneous, characterized by five distinct profiles. Among participants without HBsAg loss, only 7% had a qHBsAg decline ≥1 log10 IU/ml after 2 years. CONCLUSIONS: Most persons with HIV who experienced HBsAg loss had an early decline in qHBsAg levels, with diverse trajectories during long-term tenofovir therapy. In persons without HBsAg loss, qHBsAg levels remained remarkably stable over time.

Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure.

OBJECTIVES: A major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA quantification is essential in preclinical and clinical studies. Southern blot (SB) permits cccDNA visualisation but lacks sensitivity and is very laborious. Quantitative PCR (qPCR) has no such limitations but inaccurate quantification due to codetection of viral replicative intermediates (RI) can occur. The use of different samples, preservation conditions, DNA extraction, nuclease digestion methods and qPCR strategies has hindered standardisation. Within the ICE-HBV consortium, available and novel protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures were compared in six laboratories to develop evidence-based guidance for best practices. DESIGN: Reference material (HBV-infected humanised mouse livers and HepG2-NTCP cells) was exchanged for cross-validation. Each group compared different DNA extraction methods (Hirt extraction, total DNA extraction with or without proteinase K treatment (+PK/-PK)) and nuclease digestion protocols (plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, exonucleases I/III). Samples were analysed by qPCR and SB. RESULTS: Hirt and -PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by qPCR. T5 and Exo I/III nucleases efficiently removed all RI forms. In contrast, PSD did not digest pf-rcDNA, but was less prone to induce cccDNA overdigestion. In stabilised tissues (eg, Allprotect), nucleases had detrimental effects on cccDNA. CONCLUSIONS: We present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.

External validation of the French alpha-fetoprotein model for hepatocellular carcinoma liver transplantation in a recent unicentric cohort - a retrospective study.

Prognostic models of liver transplantation (LT) for hepatocellular carcinoma (HCC) mainly derive from LT cohorts with numerous hepatitis C virus (HCV) patients. The AFP model, which is currently used in France to select LT candidates, was derived from a cohort of LT performed between 1988 and 2001, including a majority of HCV-positive recipients. The emergence of new direct-acting antiviral therapies and subsequent decrease of HCV incidence may change the generalizability of such models. We performed an external validation of the AFP model in a cohort of recipients transplanted between 2005 and 2018. Although multivariable analysis identified all three model's factors (AFP level, largest tumor size, number of nodules) as predictors of tumor recurrence, the AFP model showed poor discrimination and calibration in the present cohort. This poor performance could be related to significant differences between the derivation and the present cohort in terms of etiology, severity of underlying liver disease, tumor burden and differentiation, and use of neoadjuvant treatments. The present findings suggest that the decline of HCV-induced HCC among LT candidates may compromise the generalizability of the AFP model in more recent LT cohorts. Further studies are required for updating or building more robust prognostic models.

Hepatitis B protein HBx binds the DLEU2 lncRNA to sustain cccDNA and host cancer-related gene transcription.

OBJECTIVE: The HBV HBx regulatory protein is required for transcription from the covalently closed circular DNA (cccDNA) minichromosome and affects the epigenetic control of both viral and host cellular chromatin. DESIGN: We explored, in relevant cellular models of HBV replication, the functional consequences of HBx interaction with DLEU2, a long non-coding RNA (lncRNA) expressed in the liver and increased in human hepatocellular carcinoma (HCC), in the regulation of host target genes and the HBV cccDNA. RESULTS: We show that HBx binds the promoter region, enhances the transcription and induces the accumulation of DLEU2 in infected hepatocytes. We found that nuclear DLEU2 directly binds HBx and the histone methyltransferase enhancer of zeste homolog 2 (EZH2), the catalytic active subunit of the polycomb repressor complex 2 (PRC2) complex. Computational modelling and biochemical evidence suggest that HBx and EZH2 share two preferential binding sites in DLEU2 intron 1. HBx and DLEU2 co-recruitment on the cccDNA displaces EZH2 from the viral chromatin to boost transcription and viral replication. DLEU2-HBx association with target host promoters relieves EZH2 repression and leads to the transcriptional activation of a subset of EZH2/PRC2 target genes in HBV-infected cells and HBV-related HCCs. CONCLUSIONS: Our results highlight the ability of HBx to bind RNA to impact on the epigenetic control of both viral cccDNA and host genes and provide a new key to understand the role of DLEU2 and EZH2 overexpression in HBV-related HCCs and HBx contribution to hepatocytes transformation.

Update of the statements on biology and clinical impact of occult hepatitis B virus infection.

In October 2018 a large number of international experts with complementary expertise came together in Taormina to participate in a workshop on occult hepatitis B virus infection (OBI). The objectives of the workshop were to review the existing knowledge on OBI, to identify issues that require further investigation, to highlight both existing controversies and newly emerging perspectives, and ultimately to update the statements previously agreed in 2008. This paper represents the output from the workshop.

Serum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients.

BACKGROUND & AIMS: It has been proposed that serum hepatitis B core-related antigen (HBcrAg) reflects intrahepatic covalently closed circular (ccc)DNA levels. However, the correlation of HBcrAg with serum and intrahepatic viral markers and liver histology has not been comprehensively investigated in a large sample. We aimed to determine if HBcrAg could be a useful therapeutic marker in patients with chronic hepatitis B. METHODS: HBcrAg was measured by chemiluminescent enzyme immunoassay in 130 (36 hepatitis B e antigen [HBeAg]+ and 94 HBeAg-) biopsy proven, untreated, patients with chronic hepatitis B. HBcrAg levels were correlated with: a) serum hepatitis B virus (HBV)-DNA, quantitative hepatitis B surface antigen and alanine aminotransferase levels; b) intrahepatic total (t)HBV-DNA, cccDNA, pregenomic (pg)RNA and cccDNA transcriptional activity (defined as pgRNA/cccDNA ratio); c) fibrosis and necroinflammatory activity scores. RESULTS: HBcrAg levels were significantly higher in HBeAg+ vs. HBeAg- patients and correlated with serum HBV-DNA, intrahepatic tHBV-DNA, pgRNA and cccDNA levels, and transcriptional activity. Patients who were negative for HBcrAg (<3 LogU/ml) had less liver cccDNA and lower cccDNA activity than the HBcrAg+ group. Principal component analysis coupled with unsupervised clustering identified that in a subgroup of HBeAg- patients, higher HBcrAg levels were associated with higher serum HBV-DNA, intrahepatic tHBV-DNA, pgRNA, cccDNA transcriptional activity and with higher fibrosis and necroinflammatory activity scores. CONCLUSIONS: Our results indicate that HBcrAg is a surrogate marker of both intrahepatic cccDNA and its transcriptional activity. HBcrAg could be useful in the evaluation of new antiviral therapies aiming at a functional cure of HBV infection either by directly or indirectly targeting the intrahepatic cccDNA pool. LAY SUMMARY: Hepatitis B virus causes a chronic infection which develops into severe liver disease and liver cancer. The viral covalently closed circular DNA (cccDNA) is responsible for the persistence of the infection in hepatocytes. To better manage patient treatment and follow-up, and to develop new antiviral treatments directly targeting the intrahepatic pool of cccDNA, serum surrogate markers reflecting the viral activity in the liver are urgently needed. In this work, we demonstrate that quantification of hepatitis B core-related antigen in serum correlates with cccDNA amount and activity and could be used to monitor disease progression.

PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The covalently closed circular DNA (cccDNA) minichromosome, which serves as the template for the transcription of viral RNAs, plays a key role in viral persistence. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation of cccDNA-bound histone 3 (H3) and H4, the potential contributions of histone methylation and related host factors remain obscure. Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as an effective restrictor of HBV transcription and replication. In cell culture-based models for HBV infection and in liver tissues of patients with chronic HBV infection, we found that symmetric dimethylation of arginine 3 on H4 on cccDNA was a repressive marker of cccDNA transcription and was regulated by PRMT5 depending on its methyltransferase domain. Moreover, PRMT5-triggered symmetric dimethylation of arginine 3 on H4 on the cccDNA minichromosome involved an interaction with the HBV core protein and the Brg1-based human SWI/SNF chromatin remodeler, which resulted in down-regulation of the binding of RNA polymerase II to cccDNA. In addition to the inhibitory effect on cccDNA transcription, PRMT5 inhibited HBV core particle DNA production independently of its methyltransferase activity. Further study revealed that PRMT5 interfered with pregenomic RNA encapsidation by preventing its interaction with viral polymerase protein through binding to the reverse transcriptase-ribonuclease H region of polymerase, which is crucial for the polymerase-pregenomic RNA interaction. CONCLUSION: PRMT5 restricts HBV replication through a two-part mechanism including epigenetic suppression of cccDNA transcription and interference with pregenomic RNA encapsidation; these findings improve the understanding of epigenetic regulation of HBV transcription and host-HBV interaction, thus providing new insights into targeted therapeutic intervention. (Hepatology 2017;66:398-415).

Genome-wide identification of direct HBx genomic targets.

BACKGROUND: The Hepatitis B Virus (HBV) HBx regulatory protein is required for HBV replication and involved in HBV-related carcinogenesis. HBx interacts with chromatin modifying enzymes and transcription factors to modulate histone post-translational modifications and to regulate viral cccDNA transcription and cellular gene expression. Aiming to identify genes and non-coding RNAs (ncRNAs) directly targeted by HBx, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) to analyse HBV recruitment on host cell chromatin in cells replicating HBV. RESULTS: ChIP-Seq high throughput sequencing of HBx-bound fragments was used to obtain a high-resolution, unbiased, mapping of HBx binding sites across the genome in HBV replicating cells. Protein-coding genes and ncRNAs involved in cell metabolism, chromatin dynamics and cancer were enriched among HBx targets together with genes/ncRNAs known to modulate HBV replication. The direct transcriptional activation of genes/miRNAs that potentiate endocytosis (Ras-related in brain (RAB) GTPase family) and autophagy (autophagy related (ATG) genes, beclin-1, miR-33a) and the transcriptional repression of microRNAs (miR-138, miR-224, miR-576, miR-596) that directly target the HBV pgRNA and would inhibit HBV replication, contribute to HBx-mediated increase of HBV replication. CONCLUSIONS: Our ChIP-Seq analysis of HBx genome wide chromatin recruitment defined the repertoire of genes and ncRNAs directly targeted by HBx and led to the identification of new mechanisms by which HBx positively regulates cccDNA transcription and HBV replication.

Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B.

BACKGROUND & AIMS: Hepatitis B virus (HBV) persistence and the pathobiology of chronic HBV (CHB) infections result from the interplay between viral replication and host immune responses. We aimed to comprehensively analyse the expression of intrahepatic host genes as well as serum and liver HBV markers in a large cohort of untreated CHB patients. METHODS: One-hundred and five CHB patients untreated at the time of liver biopsy (34 HBeAg[+] and 71 HBeAg[-]) were analysed for the intrahepatic expression profile of 67 genes belonging to multiple innate immunity pathways. Results were correlated to serological (quantification of HBsAg [qHBsAg] and HBV DNA) and intrahepatic viral markers (total HBV DNA, pre-genomic RNA and covalently closed circular HBV DNA). RESULTS: Intrahepatic gene expression profiling revealed a strong downregulation of antiviral effectors, interferon stimulated genes, Toll-like and pathogen recognition receptor pathways in CHB patients as compared to non-infected controls, which was not directly correlated to HBV replication. A subset of genes [CXCL10, GBP1, IFITM1, IFNB1, IL10, IL6, ISG15, TLR3, SOCS1, SOCS3] was more repressed in HBeAg(-) respect to HBeAg(+) patients (median of serum HBV DNA 7.9×103vs. 7.9×107IU/ml, respectively). Notably, HBeAg(-) patients with lower qHBsAg (<5×103IU/ml) showed a relief of repression of genes belonging to multiple pathways. CONCLUSIONS: Our results show a strong impairment of innate immune responses in the liver of CHB patients. The association of low levels of qHBsAg with gene repression, if confirmed, might prove useful for the identification of patients who would most benefit from immune-modulators and/or HBsAg targeting agents as strategies to restore immune responsiveness. LAY SUMMARY: Chronic hepatitis B virus (HBV) infections represent a major public health problem worldwide. Over 200 million people are chronically infected and at risk of developing chronic hepatitis, liver cirrhosis and cancer. Our work aimed to understand the molecular consequences of chronic hepatitis B in the infected liver. It was conducted in a large cohort of untreated chronically infected HBV patients and analysed the expression of immunity and liver disease-related genes in the liver, with respect to markers of viral replication and persistence. Our results indicate that chronic HBV infection has a suppressive effect on immune responses, which was more pronounced with high levels of hepatitis B virus surface antigen (HBsAg). These data provide novel insight into the mechanisms of HBV persistence in the liver and suggest that approaches aimed at reducing HBsAg levels, may restore immune responsiveness against the virus.

Direct-acting antiviral therapy decreases hepatocellular carcinoma recurrence rate in cirrhotic patients with chronic hepatitis C.

BACKGROUND AND AIMS: Arrival of direct-acting antiviral agents against hepatitis C virus with high-sustained virological response rates and very few side effects has drastically changed the management of hepatitis C virus infection. The impact of direct-acting antiviral exposure on hepatocellular carcinoma recurrence after a first remission in patients with advanced fibrosis remains to be clarified. METHODS: 68 consecutive hepatitis C virus patients with a first hepatocellular carcinoma diagnosis and under remission, subsequently treated or not with a direct-acting antiviral combination, were included. Clinical, biological and virological data were collected at first hepatocellular carcinoma diagnosis, at remission and during the surveillance period. RESULTS: All patients were cirrhotic. Median age was 62 years and 76% of patients were male. Twenty-three patients (34%) were treated with direct-acting antivirals and 96% of them achieved sustained virological response. Median time between hepatocellular carcinoma remission and direct-acting antivirals initiation was 7.2 months (IQR: 3.6-13.5; range: 0.3-71.4) and median time between direct-acting antivirals start and hepatocellular carcinoma recurrence was 13.0 months (IQR: 9.2-19.6; range: 3.0-24.7). Recurrence rate was 1.7/100 person-months among treated patients vs 4.2/100 person-months among untreated patients (P=.008). In multivariate survival analysis, the hazard ratio for hepatocellular carcinoma recurrence after direct-acting antivirals exposure was 0.24 (95% confidence interval: 0.10-0.55; P<.001). CONCLUSIONS: Hepatocellular carcinoma recurrence rate was significantly lower among patients treated with direct-acting antivirals compared with untreated patients. Given the potential impact of our observation, large-scale prospective cohort studies are needed to confirm these results.

Ribavirin restores IFNα responsiveness in HCV-infected livers by epigenetic remodelling at interferon stimulated genes.

OBJECTIVES: Caveats in the understanding of ribavirin (RBV) mechanisms of action has somehow prevented the development of better analogues able to further improve its therapeutic contribution in interferon (IFN)-based and direct antiviral agent-based regimens for chronic HCV or other indications. Here, we describe a new mechanism by which RBV modulates IFN-stimulated genes (ISGs) and contributes to restore hepatic immune responsiveness. DESIGN: RBV effect on ISG expression was monitored in vitro and in vivo, that is, in non-transformed hepatocytes and in the liver of RBV mono-treated patients, respectively. Modulation of histone modifications and recruitment of histone-modifying enzymes at target promoters was analysed by chromatin immunoprecipitation in RBV-treated primary human hepatocytes and in patients' liver biopsies. RESULTS: RBV decreases the mRNA levels of several abnormally preactivated ISGs in patients with HCV, who are non-responders to IFN therapy. RBV increases G9a histone methyltransferase recruitment and histone-H3 lysine-9 dimethylation/trimethylation at selected ISG promoters in vitro and in vivo. G9a pharmacological blockade abolishes RBV-induced ISG downregulation and severely impairs RBV ability to potentiate IFN antiviral action and induction of ISGs following HCV infection of primary human hepatocytes. CONCLUSIONS: RBV-induced epigenetic changes, leading to decreased ISG expression, restore an IFN-responsive hepatic environment in patients with HCV, which may also prove useful in IFN-free regimens.

Mechanisms of HBV-induced hepatocellular carcinoma.

Hepatitis B virus (HBV) contributes to hepatocellular carcinoma (HCC) development through direct and indirect mechanisms. HBV DNA integration into the host genome occurs at early steps of clonal tumor expansion and induces both genomic instability and direct insertional mutagenesis of diverse cancer-related genes. Prolonged expression of the viral regulatory protein HBx and/or altered versions of the preS/S envelope proteins dysregulates cell transcription and proliferation control and sensitizes liver cells to carcinogenic factors. Accumulation of preS1 large envelope proteins and/or preS2/S mutant proteins activates the unfold proteins response, that can contribute to hepatocyte transformation. Epigenetic changes targeting the expression of tumor suppressor genes occur early in the development of HCC. A major role is played by the HBV protein, HBx, which is recruited on cellular chromatin and modulates chromatin dynamics at specific gene loci. Compared with tumors associated with other risk factors, HBV-related tumors have a higher rate of chromosomal alterations, p53 inactivation by mutations and overexpression of fetal liver/hepatic progenitor cells genes. The WNT/ß-catenin pathway is also often activated but HBV-related tumors display a low rate of activating ß-catenin mutations. HBV-related HCCs may arise on non-cirrhotic livers, further supporting the notion that HBV plays a direct role in liver transformation by triggering both common and etiology specific oncogenic pathways in addition to stimulating the host immune response and driving liver chronic necro-inflammation.