Barbara Testoni

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.

Novel targets for hepatitis B virus therapy

Treatment with either pegylated interferon‐alpha (pegIFN‐α) or last generation nucleos(t)ide analogues (NAs) successfully leads to serum viral load suppression in most chronically infected hepatitis B (CHB) patients, but HBsAg loss is only achieved in 10% of the cases after a 5‐year follow‐up. Thus, therapy must be administered long‐term and it will not completely eliminate infection because of the persistent hepatitis B virus (HBV) minichromosome in infected cells, and cannot completely abolish the risk of developing severe sequelae such as cirrhosis and hepatocellular carcinoma. Recent progress in the development of in vitro and in vivo models of HBV infection have helped renew interest in the investigation of the viral life cycle, as well as specific virus–host cell interactions to identify new targets for the development of new antiviral drugs. This includes either direct inhibition of viral replication by targeting fundamental steps such as entry, cccDNA formation/stability, viral transcripts, capsid assembly and secretion or the manipulation of the host immune system for better defence against infection. Multiple strategies are currently under investigation, including boosting endogenous innate responses and/or restoring adaptive immunity via engineering of HBV‐specific T cells or via the use of inhibitors of negative regulators, as well as therapeutic vaccines. It is increasingly clear that multiple therapeutic strategies must be combined to reach a cure of HBV and that the definition of clinical, virological and immunological correlates for the management of treatment are urgently needed.

HBV cure: why, how, when?

Current HBV treatments control replication and liver disease progression in the vast majority of treated patients. However, HBV patients often require lifelong therapies due to the persistence of transcriptionally active viral cccDNA mini-chromosome in the nucleus, which is not directly targeted by current antiviral therapies. A true complete cure of HBV would require clearance of intranuclear cccDNA from all infected hepatocytes. An intermediate but still relevant step forward that would allow treatment cessation would be reaching a functional cure, equivalent to resolved acute infection, with a durable HBsAg loss±anti-HBs seroconversion, undetectable serum DNA and persistence of cccDNA in a transcriptionally inactive status. Recent advances in technologies and pharmaceutical sciences, including the cloning of the mayor HBV receptor (i.e. the NTCP transporter) and the development in vitro HBV infection models, have heralded a new horizon of innovative antiviral and immune-therapeutic approaches.

HDV RNA replication is associated with HBV repression and interferon-stimulated genes induction in super-infected hepatocytes

Hepatitis D virus (HDV) super-infection of Hepatitis B virus (HBV)-infected patients is the most aggressive form of viral hepatitis. HDV infection is not susceptible to direct anti-HBV drugs, and only suboptimal antiviral responses are obtained with interferon (IFN)-alpha-based therapy. To get insights on HDV replication and interplay with HBV in physiologically relevant hepatocytes, differentiated HepaRG (dHepaRG) cells, previously infected or not with HBV, were infected with HDV, and viral markers were extensively analyzed. Innate and IFN responses to HDV were monitored by measuring pro-inflammatory and interferon-stimulated gene (ISG) expression. Both mono- and super-infected dHepaRG cells supported a strong HDV intracellular replication, which was accompanied by a strong secretion of infectious HDV virions only in the super-infection setting and despite the low number of co-infected cells. Upon HDV super-infection, HBV replication markers including HBeAg, total HBV-DNA and pregenomic RNA were significantly decreased, confirming the interference of HDV on HBV. Yet, no decrease of circular covalently closed HBV DNA (cccDNA) and HBsAg levels was evidenced. At the peak of HDV-RNA accumulation and onset of interference on HBV replication, a strong type-I IFN response was observed, with interferon stimulated genes, RSAD2 (Viperin) and IFI78 (MxA) being highly induced. We established a cellular model to characterize in more detail the direct interference of HBV and HDV, and the indirect interplay between the two viruses via innate immune responses. This model will be instrumental to assess molecular and immunological mechanisms of this viral interference.

Challenges to a Cure for HBV Infection

Current first-choice treatments for chronic hepatitis B are able to efficiently induce viral suppression in the majority of patients, but life-long therapy is needed to maintain infection under control due to their inability to eliminate the virus from infected hepatocytes. The residual viral replication and antigen production in most patients under treatment substantially contributes to the residual risk of hepatocarcinogenesis. New therapeutic approaches are needed to overcome hepatitis B virus persistence in the infected cells, or at least to control its transcriptional and replicative activity. In this review, the authors discuss the key points of the viral life cycle and host immune responses that need to be addressed to achieve a “functional cure,” or even a “complete cure,” allowing a finite duration of treatment and preventing virus reactivation and liver disease progression in a significantly higher number of patients than what is currently attained.

Genome-wide identification of direct HBx genomic targets

BackgroundThe 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.ResultsChIP-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.ConclusionsOur 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.

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).

Development of hepatocellular carcinoma in chronic hepatitis B patients with advanced fibrosis is independent of viral genotype

Hepatitis B is leading cause of liver related morbidity in Asia with predominant genotypes B and C in East‐Asia. Data on Serum, intrahepatic viral‐markers, and long‐term follow‐up of prevalent genotypes (GT) B and C in patients with biopsy proven advanced fibrosis are sparse. To compare serum, intrahepatic viral‐markers and development of hepatocellular carcinoma (HCC) in GT‐B and C in patients with advanced fibrosis (Ishak ≥ 4). Sixty‐three treatment‐naïve patients identified with advanced fibrosis on liver‐biopsy performed between 1998 and 2000 at Singapore General Hospital. FFPE tissue was available for 59 patients and serum for 42 patients. HBV‐DNA was quantified in serum and liver while qHBsAg quantified in serum. Patients were followed‐up till December 2015. The median age was 47 ± 16 years, with 77.7% males. About 19 were GT‐B, 43 patients were GT‐C, and 1 had both GT‐B and C. Mean follow‐up was 13.5 years. The median serum HBV‐DNA was 6.25 ± 2.17 and 6.58 ± 1.85 log IU/ml, serum HBsAg was 3.29 ± 0.80 and 3.45 ± 1.85 log IU/ml, and intrahepatic HBV‐DNA was 0.52 ± 3.73 copies/cell and 0.4 ± 1.37 copies/cell in the GT‐B and C, respectively (P > 0.1 in all). Complete cirrhosis (Ishak‐6) was present in 47.6%, Ishak‐5 fibrosis in 33.3%, and Ishak‐4 fibrosis in 19% at recruitment. On follow‐up HCC developed in 8/43 in GT‐C and in 3/19 GT‐B (P = 0.86). Advanced age and cirrhosis were significant factors for development of HCC. No difference in serum HBV‐DNA, qHBsAg or intrahepatic HBV‐DNA was seen in the two genotypes. HCC development seen over long‐term follow‐up was independent of genotypes in patients with advanced fibrosis. J. Med. Virol. 89:845–848, 2017.

Targeting a phospho-STAT3-miRNAs pathway improves vesicular hepatic steatosis in an in vitro and in vivo model

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease. Although genetic predisposition and epigenetic factors contribute to the development of NAFLD, our understanding of the molecular mechanism involved in the pathogenesis of the disease is still emerging. Here we investigated a possible role of a microRNAs-STAT3 pathway in the induction of hepatic steatosis. Differentiated HepaRG cells treated with the fatty acid sodium oleate (fatty dHepaRG) recapitulated features of liver vesicular steatosis and activated a cell-autonomous inflammatory response, inducing STAT3-Tyrosine-phosphorylation. With a genome-wide approach (Chromatin Immunoprecipitation Sequencing), many phospho-STAT3 binding sites were identified in fatty dHepaRG cells and several STAT3 and/or NAFLD-regulated microRNAs showed increased expression levels, including miR-21. Innovative CARS (Coherent Anti-Stokes Raman Scattering) microscopy revealed that chemical inhibition of STAT3 activity decreased lipid accumulation and deregulated STAT3-responsive microRNAs, including miR-21, in lipid overloaded dHepaRG cells. We were able to show in vivo that reducing phospho-STAT3-miR-21 levels in C57/BL6 mice liver, by long-term treatment with metformin, protected mice from aging-dependent hepatic vesicular steatosis. Our results identified a microRNAs-phosphoSTAT3 pathway involved in the development of hepatic steatosis, which may represent a molecular marker for both diagnosis and therapeutic targeting.