G-quadruplexes control hepatitis B virus replication by promoting cccDNA transcription and phase separation in hepatocytes.

Phase separation regulates fundamental processes in gene expression and is mediated by the local concentration of proteins and nucleic acids, as well as nucleic acid secondary structures such as G-quadruplexes (G4s). These structures play fundamental roles in both host gene expression and in viral replication due to their peculiar localisation in regulatory sequences. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is an episomal minichromosome whose persistence is at the basis of chronic infection. Identifying the mechanisms controlling its transcriptional activity is indispensable to develop new therapeutic strategies against chronic hepatitis B. The aim of this study was to determine whether G4s are formed in cccDNA and regulate viral replication. Combining biochemistry and functional studies, we demonstrate that cccDNA indeed contains ten G4s structures. Furthermore, mutations disrupting two G4s located in the enhancer I HBV regulatory region altered cccDNA transcription and viral replication. Finally, we showed for the first time that cccDNA undergoes phase separation in a G4-dependent manner to promote its transcription in infected hepatocytes. Altogether, our data give new insight in the transcriptional regulation of the HBV minichromosome that might pave the way for the identification of novel targets to destabilize or silence cccDNA.

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.

Helicases DDX5 and DDX17 promote heterogeneity in HBV transcription termination in infected human hepatocytes.

BACKGROUND & AIMS: Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. METHODS: To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro-infected hepatocytes and in chronically infected patients. RESULTS: The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. IMPACT AND IMPLICATIONS: HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.

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.

Hepatitis D virus interferes with hepatitis B virus RNA production via interferon-dependent and -independent mechanisms.

BACKGROUND & AIMS: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients. METHODS: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays. RESULTS: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2. CONCLUSIONS: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV. IMPACT AND IMPLICATIONS: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients.

Emerging anti-HDV drugs and HBV cure strategies with anti-HDV activity.

Hepatitis delta virus (HDV) is a satellite RNA virus that requires the presence of hepatitis B virus (HBV) for its replication. HDV/HBV co-infection is often associated with a faster disease progression of chronic hepatitis in comparison to HBV mono-infection. Therefore, the development of novel antiviral therapies targeting HDV represents a high priority and an urgent medical need. In this review, we summarize the ongoing efforts to evaluate promising HDV-specific drugs, such as lonafarnib (LNF), pegylated interferon lambda (PEG-IFN-λ) and their use as a combination therapy. Furthermore, we review the most recent developments in the area of anti-HBV drugs with potential effects against HDV, including therapeutic agents targeting hepatitis B surface antigen (HBsAg) expression, secretion and function. Finally, we consider the important insights that have emerged from the development of these potential antiviral strategies, as well as the intriguing questions that remain to be elucidated in this rapidly changing field.

The CCCTC-binding factor CTCF represses hepatitis B virus enhancer I and regulates viral transcription.

Hepatitis B virus (HBV) infection is of global importance with over 2 billion people exposed to the virus during their lifetime and at risk of progressive liver disease, cirrhosis and hepatocellular carcinoma. HBV is a member of the Hepadnaviridae family that replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate viral transcription. The chromatin-organising transcriptional insulator protein, CCCTC-binding factor (CTCF), has been reported to regulate transcription in a diverse range of viruses. We identified two conserved CTCF binding sites in the HBV genome within enhancer I and chromatin immunoprecipitation (ChIP) analysis demonstrated an enrichment of CTCF binding to integrated or episomal copies of the viral genome. siRNA knock-down of CTCF results in a significant increase in pre-genomic RNA levels in de novo infected HepG2 cells and those supporting episomal HBV DNA replication. Furthermore, mutation of these sites in HBV DNA minicircles abrogated CTCF binding and increased pre-genomic RNA levels, providing evidence of a direct role for CTCF in repressing HBV transcription.

Covalently closed circular DNA: The ultimate therapeutic target for curing HBV infections.

Current antiviral therapies, such as pegylated interferon-α and nucleos(t)ide analogues, effectively improve the quality of life of patients with chronic hepatitis B. However, they can only control the infection rather than curing infected hepatocytes. Complete HBV cure is hampered by the lack of therapies that can directly affect the viral minichromosome (in the form of covalently closed circular DNA [cccDNA]). Approaches currently under investigation in early clinical trials are aimed at achieving a functional cure, defined as the loss of HBsAg and undetectable HBV DNA levels in serum. However, achieving a complete HBV cure requires therapies that can directly target the cccDNA pool, either via degradation, lethal mutations or functional silencing. In this review, we discuss cutting-edge technologies that could lead to non-cytolytic direct cccDNA targeting and cure of infected hepatocytes.

Viral and immune factors associated with successful treatment withdrawal in HBeAg-negative chronic hepatitis B patients.

BACKGROUND & AIMS: Factors associated with a successful outcome upon nucleos(t)ide analogue (NA) treatment withdrawal in HBeAg-negative chronic hepatitis B (CHB) patients have yet to be clarified. The objective of this study was to analyse the HBV-specific T cell response, in parallel with peripheral and intrahepatic viral parameters, in patients undergoing NA discontinuation. METHODS: Twenty-seven patients without cirrhosis with HBeAg-negative CHB with complete viral suppression (>3 years) were studied prospectively. Intrahepatic HBV-DNA (iHBV-DNA), intrahepatic HBV-RNA (iHBV-RNA), and covalently closed circular DNA (cccDNA) were quantified at baseline. Additionally, serum markers (HBV-DNA, HBsAg, HBV core-related antigen [HBcrAg] and HBV-RNA) and HBV-specific T cell responses were analysed at baseline and longitudinally throughout follow-up. RESULTS: After a median follow-up of 34 months, 22/27 patients (82%) remained off-therapy, of whom 8 patients (30% of the total cohort) lost HBsAg. Baseline HBsAg significantly correlated with iHBV-DNA and iHBV-RNA, and these parameters were lower in patients who lost HBsAg. All patients had similar levels of detectable cccDNA regardless of their clinical outcome. Patients achieving functional cure had baseline HBsAg levels ≤1,000 IU/ml. Similarly, an increased frequency of functional HBV-specific CD8+ T cells at baseline was associated with sustained viral control off treatment. These HBV-specific T cell responses persisted, but did not increase, after treatment withdrawal. A similar, but not statistically significant trend, was observed for HBV-specific CD4+ T cell responses. CONCLUSIONS: Decreased cccDNA transcription and low HBsAg levels are associated with HBsAg loss upon NA discontinuation in patients with HBeAg-negative CHB. The presence of functional HBV-specific T cells at baseline are associated with a successful outcome after treatment withdrawal. LAY SUMMARY: Nucleos(t)ide analogue therapy can be discontinued in a high proportion of chronic hepatitis B patients without cirrhosis. The strength of HBV-specific immune T cell responses may contribute to successful viral control after antiviral treatment interruption. Our comprehensive study provides in-depth data on virological and immunological factors than can help guide individualised therapy in patients with chronic hepatitis B.

HBV 2021: New therapeutic strategies against an old foe.

Hepatitis B virus (HBV) affects more than 250 million people worldwide, and is one of the major aetiologies for the development of cirrhosis and hepatocellular carcinoma (HCC). In spite of universal vaccination programs, HBV infection is still a public health problem, and the limited number of available therapeutic approaches complicates the clinical management of these patients. Thus, HBV infection remains an unmet medical need that requires a continuous effort to develop new individual molecules, treatment combinations and even completely novel therapeutic strategies to achieve the goal of HBV elimination. The following review provides an overview of the current situation in chronic HBV infection, with an analysis of the scientific rationale of certain clinical interventions and, more importantly, explores the most recent developments in the field of HBV drug discovery.

Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum.

BACKGROUND & AIMS: Covalently closed circular DNA (cccDNA) is the episomal form of the HBV genome that stably resides in the nucleus of infected hepatocytes. cccDNA is the template for the transcription of 6 major viral RNAs, i.e. preC, pg, preS1/2, S and HBx RNA. All viral transcripts share the same 3' end and are all to various degrees subsets of each other. Especially under infection conditions, it has been difficult to study in depth the transcription of the different viral transcripts. We thus wanted to develop a method with which we could easily detect the full spectrum of viral RNAs in any lab. METHODS: We set up an HBV full-length 5'RACE (rapid amplification of cDNA ends) method with which we measured and characterized the full spectrum of viral RNAs in cell culture and in chronically infected patients. RESULTS: In addition to canonical HBx transcripts coding for full-length X, we identified shorter HBx transcripts potentially coding for short X proteins. We showed that interferon-ß treatment leads to a strong reduction of preC and pgRNAs but has only a moderate effect on the other viral transcripts. We found pgRNA, 1 spliced pgRNA variant and a variety of HBx transcripts associated with viral particles generated by HepAD38 cells. The different HBx RNAs are both capped and uncapped. Lastly, we identified 3 major categories of circulating RNA species in patients with chronic HBV infection: pgRNA, spliced pgRNA variants and HBx. CONCLUSIONS: This HBV full-length 5'RACE method should significantly contribute to the understanding of HBV transcription during the course of infection and therapy and may guide the development of novel therapies aimed at targeting cccDNA. LAY SUMMARY: Especially under infection conditions, it has been difficult to study the different hepatitis B virus transcripts in depth. This study introduces a new method that can be used in any standard lab to discriminate all hepatitis B viral transcripts in cell culture and in the serum of patients.

Predictors of hepatitis B surface antigen loss, relapse and retreatment after discontinuation of effective oral antiviral therapy in noncirrhotic HBeAg-negative chronic hepatitis B.

Reliable predictors of outcomes after treatment discontinuation in HBeAg-negative chronic hepatitis B (CHB) patients have not been established. We investigated the role of hepatitis B surface antigen (HBsAg), interferon-inducible protein-10 (IP10) and hepatitis B core-related antigen (HBcrAg) serum levels as predictors of HBsAg loss, relapse and retreatment in noncirrhotic HBeAg-negative CHB patients who discontinued long-term antiviral therapy. All HBsAg-positive (n = 57) patients of the prospective DARING-B study were included and followed monthly for 3 months, every 2/3 months until month-12 and every 3/6 months thereafter. HBsAg, IP10 and HBcrAg levels were measured by enzyme immunoassays, and SCALE-B score was calculated. Twelve patients achieved HBsAg loss before retreatment with 18-month cumulative incidence of 25%. Independent predictors of HBsAg loss were baseline HBsAg and month-1 IP10 levels. Of 10 patients with baseline HBsAg ≤100 IU/mL, 70% cleared HBsAg and 10% required retreatment. Of 23 patients with baseline HBsAg >1000 IU/mL, 4% cleared HBsAg and 43% required retreatment. Of 24 patients with intermediate baseline HBsAg (100-1000 IU/mL), 17% cleared HBsAg and 21% required retreatment; in this subgroup, month-1 IP10 was significantly associated with HBsAg loss, which occurred in 30% and 7% of cases with IP10 >150 and ≤150 pg/mL, respectively. Baseline HBcrAg was undetectable in all patients who cleared HBsAg and was associated with retreatment. SCALE-B was associated with HBsAg loss but not with relapse or retreatment. In conclusion, HBsAg, IP10 and HBcrAg serum levels can be useful for the decisions and management of treatment discontinuation in noncirrhotic Caucasian patients with HBeAg-negative CHB.

Can we cure hepatitis B virus with novel direct-acting antivirals?

Current treatments against chronic hepatitis B (CHB) include pegylated interferon alpha (Peg-IFNα) and nucleos(t)ide analogs (NAs), the latter targeting the viral retrotranscriptase, thus inhibiting de novo viral production. Although these therapies control infection and improve the patient's quality of life, they do not cure HBV-infected hepatocytes. A complete HBV cure is currently not possible because of the presence of the stable DNA intermediate covalently closed circular DNA (cccDNA). Current efforts are focused on achieving a functional cure, defined by the loss of Hepatitis B surface antigen (HBsAg) and undetectable HBV DNA levels in serum, and on exploring novel targets and molecules that are in the pipeline for early clinical trials. The likelihood of achieving a long-lasting functional cure, with no rebound after therapy cessation, is higher using combination therapies targeting different steps in the hepatitis B virus (HBV) replication cycle. Novel treatments and their combinations are discussed for their potential to cure HBV infection, as well as exciting new technologies that could directly target cccDNA and cure without killing the infected cells.

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.

Toll-like receptor 3 downregulation is an escape mechanism from apoptosis during hepatocarcinogenesis.

BACKGROUND & AIMS: Low levels of toll-like receptor 3 (TLR3) in patients with hepatocellular carcinoma (HCC) are associated with poor prognosis, primarily owing to the loss of inflammatory signaling and subsequent lack of immune cell recruitment to the liver. Herein, we explore the role of TLR3-triggered apoptosis in HCC cells. METHODS: Quantitative reverse transcription PCR, western blotting, immunohistochemistry and comparative genomic hybridization were used to analyze human and mouse HCC cell lines, as well as surgically resected primary human HCCs, and to study the impact of TLR3 expression on patient outcomes. Functional analyses were performed in HCC cells, following the restoration of TLR3 by lentiviral transduction. The role of TLR3-triggered apoptosis in HCC was analyzed in vivo in a transgenic mouse model of HCC. RESULTS: Lower expression of TLR3 in tumor compared to non-tumor matched tissue was observed at both mRNA and protein levels in primary HCC, and was predictive of shorter recurrence-free survival after surgical resection in both univariate (hazard ratio [HR] 1.79; 95% CI 1.04-3.06; p = 0.03) and multivariate analyses (HR 1.73; CI 1.01-2.97; p = 0.04). Immunohistochemistry confirmed frequent downregulation of TLR3 in human and mouse primary HCC cells. None of the 6 human HCC cell lines analyzed expressed TLR3, and ectopic expression of TLR3 following lentiviral transduction not only restored the inflammatory response but also sensitized cells to TLR3-triggered apoptosis. Lastly, in the transgenic mouse model of HCC, absence of TLR3 expression was accompanied by a lower rate of preneoplastic hepatocyte apoptosis and accelerated hepatocarcinogenesis without altering the tumor immune infiltrate. CONCLUSION: Downregulation of TLR3 protects transforming hepatocytes from direct TLR3-triggered apoptosis, thereby contributing to hepatocarcinogenesis and poor patient outcome. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a heterogeneous disease associated with a poor prognosis. In patients with HCC, TLR3 downregulation is associated with reduced survival. Herein, we show that the absence of TLR3 is associated with a lower rate of apoptosis, and subsequently more rapid hepatocarcinogenesis, without any change to the immune infiltrate in the liver. Therefore, the poor prognosis associated with low TLR3 expression in HCC is likely linked to tumors ability to escape apoptosis. TLR3 may become a promising therapeutic target in TLR3-positive HCC.

Biological basis for functional cure of chronic hepatitis B.

Chronic hepatitis B (CHB) infection affects over 250 millon people worldwide and 800000 are expected to die yearly due to the development of hepatocellular carcinoma (HCC). Current antiviral therapies include nucleoside analogs (NAs) that target the viral retrotranscriptase inhibiting de novo viral production. Pegylated interferon (Peg-IFN) is also effective in reducing the viral DNA load in serum. However, both treatments remain limited to control the infection, aiming for viral suppression and improving the quality of life of the infected patients. Complete cure is not possible due to the presence of the stable DNA intermediate covalently closed circular DNA (cccDNA). Attempts to achieve a functional cure are thus ongoing and novel targets and molecules, together with different combination therapies are currently in the pipeline for early clinical trials. In this review we discuss novel treatments both targeting directly and indirectly cccDNA. As we gain knowledge in the Hepatitis B virus (HBV) transcriptional control, and newer technologies emerge that could potentially allow the destruction of cccDNA, exciting new possibilities for curative therapies are discussed.

Late hepatitis B reactivation following direct-acting antiviral-based treatment of recurrent hepatitis C in an anti-HBc-positive liver transplant recipient.

Direct-acting antivirals (DAAs) have changed the landscape of hepatitis C virus (HCV) treatment, but chronic hepatitis C (CHC) remains a leading indication for liver transplantation (LT). Hepatitis B virus (HBV) reactivation has been reported in HBV-HCV-coinfected patients treated with DAAs. We report on a case of late HBV reactivation after DAA-based treatment of recurrent hepatitis C in an antibody against hepatitis B core antigen (anti-HBc)-positive LT recipient. (Hepatology 2018;67:791-793).