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.
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.
Novel targets for hepatitis B virus therapy Testoni, Barbara; Durantel, David; Zoulim, Fabien
Liver international,
January 2017, 2017-01-00, 20170101, 2017-01, Volume:
37, Issue:
S1
Journal Article
Peer reviewed
Open access
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.
Summary
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.
Background & Aims The outcome of hepatitis B virus (HBV) infection may be influenced by early interactions between the virus and hepatocyte innate immune responses. To date, the study of such ...interactions during the very early step of infection has not been adequately investigated. Methods We used the HepaRG cell line, as well as primary human hepatocytes to analyze, within 24 h of exposure to HBV, either delivered by a physiologic route or baculovirus vector (Bac-HBV), the early modulation of the expression of selected antiviral/pro-inflammatory cytokines and interferon stimulated genes. Experiments were also performed in the presence or absence of innate receptor agonists to investigate early HBV-induced blockade of innate responses. Results We show that hepatocytes themselves could detect HBV, and express innate genes when exposed to either HBV virions or Bac-HBV. Whereas Bac-HBV triggered a strong antiviral cytokine secretion followed by the clearance of replicative intermediates, a physiologic HBV exposure led to an abortive response. The early inhibition of innate response by HBV was mainly evidenced on Toll-like receptor 3 and RIG-I/MDA5 signaling pathways upon engagement with exogenous agonist, leading to a decreased expression of several pro-inflammatory and antiviral cytokine genes. Finally, we demonstrate that this early inhibition of dsRNA-mediated response is due to factor(s) present in the HBV inoculum, but not being HBsAg or HBeAg themselves, and does not require de novo viral protein synthesis and replication. Conclusions Our data provide strong evidence that HBV viral particles themselves can readily inhibit host innate immune responses upon virion/cell interactions, and may explain, at least partially, the “stealthy” character of HBV.
Chronic hepatitis B (CHB) virus infection is a major public health burden and the leading cause of hepatocellular carcinoma. Despite the efficacy of current treatments, hepatitis B virus (HBV) cannot ...be fully eradicated due to the persistence of its minichromosome, or covalently closed circular DNA (cccDNA). The HBV community is investing large human and financial resources to develop new therapeutic strategies that either silence or ideally degrade cccDNA, to cure HBV completely or functionally. cccDNA transcription is considered to be the key step for HBV replication. Transcription not only influences the levels of viral RNA produced, but also directly impacts their quality, generating multiple variants. Growing evidence advocates for the role of the co-transcriptional regulation of HBV RNAs during CHB and viral replication, paving the way for the development of novel therapies targeting these processes. This review focuses on the mechanisms controlling the different co-transcriptional processes that HBV RNAs undergo, and their contribution to both viral replication and HBV-induced liver pathogenesis.
Hepatitis B virus (HBV) remains a significant cause of mortality and morbidity worldwide, since chronic HBV infection is associated with elevated risk of cirrhosis and hepatocellular carcinoma. ...Current licensed therapies against HBV efficiently suppress viral replication; however, they do not have significant effects on the intrahepatic covalently closed circular DNA (cccDNA) of the viral minichromosome responsible for viral persistence. Thus, life-long treatment is required to avoid viral rebound. There is a significant need for novel therapies that can reduce, silence or eradicate cccDNA, thus preventing HBV reemergence after treatment withdrawal. In this review, we discuss the latest developments and applications of gene editing and related approaches for directly targeting HBV DNA and, more specifically, cccDNA in infected hepatocytes.
Hepatitis B virus (HBV) is currently viewed as a stealth virus that does not elicit innate immunity in vivo. This assumption has not yet been challenged in vitro because of the lack of a relevant ...cell culture system. The HepaRG cell line, which is physiologically closer to differentiated hepatocytes and permissive to HBV infection, has opened new perspectives in this respect.HBV baculoviruses were used to initiate an HBV replication in both HepG2 and HepaRG cells. To monitor HBV replication, the synthesis of encapsidated DNA, and secretion of hepatitis B surface antigen (HBsAg), was respectively analyzed by southern blot and enzyme‐linked immunosorbent assay. The induction of a type I interferon (IFN) response was monitored by targeted quantitative reverse transcription polymerase chain reaction (qRT‐PCR), low‐density arrays, and functional assays. The invalidation of type I IFN response was obtained by either antibody neutralization or RNA interference. We demonstrate that HBV elicits a strong and specific innate antiviral response that results in a noncytopathic clearance of HBV DNA in HepaRG cells. Challenge experiment showed that transduction with Bac‐HBV‐WT, but not with control baculoviruses, leads to this antiviral response in HepaRG cells, whereas no antiviral response is observed in HepG2 cells. Cellular gene expression analyses showed that IFN‐β and other IFN‐stimulated genes were up‐regulated in HepG2 and HepaRG cells, but not in cells transduced by control baculoviruses. Interestingly, a rescue of viral replication was observed when IFN‐β action was neutralized by antibodies or RNA interference of type I IFN receptor. Conclusion: Our data suggest that a strong HBV replication is able to elicit a type I IFN response in HepaRG‐transduced cells. (HEPATOLOGY 2009.)
Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and ...CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) delivery system in HBV-infected HepG2-NTCP cells. HBV parameters after Cas9 editing were analyzed. Southern blot (SB) analysis and DNA/RNA sequencing (DNA/RNA-seq) were performed to determine the consequences of cccDNA editing and transcriptional activity of mutated cccDNA. Treatment of infected cells with HBV-specific gRNAs showed that CRISPR-Cas9 can efficiently affect HBV replication. The appearance of episomal HBV DNA variants after dual gRNA treatment was observed by PCR, SB analysis, and DNA/RNA-seq. These transcriptionally active variants are the products of simultaneous Cas9-induced double-strand breaks in two target sites, followed by repair and religation of both short and long fragments. Following suppression of HBV DNA replicative intermediates by nucleoside analogs, mutations and formation of smaller transcriptionally active HBV variants were still observed, suggesting that established cccDNA is accessible to CRISPR-Cas9 editing. Targeting HBV DNA with CRISPR-Cas9 leads to cleavage followed by appearance of episomal HBV DNA variants. Effects induced by Cas9 were sustainable after RNP degradation/loss of detection, suggesting permanent changes in the HBV genome instead of transient effects due to transcriptional interference.
Hepatitis B virus infection can develop into chronic infection, cirrhosis, and hepatocellular carcinoma. Treatment of chronic hepatitis B requires novel approaches to directly target the viral minichromosome, which is responsible for the persistence of the disease. Designer nuclease approaches represent a promising strategy to treat chronic infectious diseases; however, comprehensive knowledge about the fate of the HBV minichromosome is needed before this potent tool can be used as a potential therapeutic approach. This study provides an in-depth analysis of CRISPR-Cas9 targeting of HBV minichromosome.
NF-Y is a transcription factor that recognizes with high specificity and affinity the widespread CCAAT box promoter element. It is formed by three subunits: NF-YA and the NF-YB/NF-YC- heterodimer ...containing histone fold domains (HFDs). We previously identified a large NF-Y gene family in Arabidopsis thaliana, composed of 29 members, and characterized their expression patterns in various plant tissues.
We used yeast Two-hybrids assays (Y2H), pull-down and Electrophoretic Mobility Shift Assay (EMSA) in vitro experiments with recombinant proteins to dissect AtNF-YB/AtNF-YC interactions and DNA-binding with different AtNF-YAs.
Consistent with robust conservation within HFDs, we show that heterodimerization is possible among all histone-like subunits, including the divergent and related LEC1/AtNF-YB9 and L1L/AtNF-YB6 required for embryo development. DNA-binding to a consensus CCAAT box was investigated with specific AtNF-YB/AtNF-YC combinations and observed with some, but not all AtNF-YA subunits.
Our results highlight (i) the conserved heterodimerization capacity of AtNF-Y histone-like subunits, and (ii) the different affinities of AtNF-YAs for the CCAAT sequence. Because of the general expansion of NF-Y genes in plants, these results most likely apply to other species.
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