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Article InfoDespite the availability of a preventive vaccine and anti-viral treatments that arrest disease progression and reduce liver cancer risk, hepatitis B remains a major public health problem worldwide, on a similar scale of magnitude as human immunodeficiency virus (HIV), malaria and tuberculosis.1 According to the last Global Hepatitis Report 2017, 257 million persons live with chronic HBV infection, making it the most common chronic viral infection.2 HBV mortality (887,000 deaths/year) is mostly due to HBV-related cirrhosis and HBV-related liver cancer.Despite the availability of a preventive vaccine and anti-viral treatments that arrest disease progression and reduce liver cancer risk, hepatitis B remains a major public health problem worldwide, on a similar scale of magnitude as human immunodeficiency virus (HIV), malaria and tuberculosis.[url=]1[/url] According to the last Global Hepatitis Report 2017, 257 million persons live with chronic HBV infection, making it the most common chronic viral infection.[url=]2[/url] HBV mortality (887,000 deaths/year) is mostly due to HBV-related cirrhosis and HBV-related liver cancer. Currently, only 10% of chronically infected people have been diagnosed worldwide and only 1% are adequately treated. Available treatment options for chronic hepatitis B include pegylated interferon alpha2a (PegIFN) and nucleos(t)ide analogues (NUCs).[url=]3[/url] Loss of hepatitis B surface antigen (HBsAg) is considered a major treatment endpoint. However, only a limited number of patients achieve HBsAg loss and anti-HBs sero-conversion with PegIFN or NUC therapies.[url=]3[/url] Although HBsAg loss is a relevant clinical end-point and is considered to reflect a functional cure of HBV infection, it does not mean complete viral eradication.[url=][4][/url], [url=][5][/url] Indeed, HBV infection is a model of viral persistence as most infected patients never clear covalently closed circular DNA (cccDNA) and bear viral sequences integrated into the host DNA. Clinical resolution of infection requires the control of persisting viral genomes and residual infected cells by the immune system.
The key challenge for HBV drug development and HBV cure is to target the pool of (cccDNA), the persistent form of the virus in the nucleus of infected hepatocytes and to overcome the exhaustion of the effectors of adaptive immunity.[url=]6[/url] cccDNA is the template for the transcription of all viral mRNAs and the 3.5 Kb pregenomic RNA that serves as a template for viral genome replication within the intracytoplasmic nucleocapsids. Recent developments in our knowledge of HBV biology and in tools for molecular studies of HBV have created new possibilities to define novel therapeutic targets. Several new antiviral and immuno-modulatory compounds have reached preclinical and/or early clinical evaluation with the aim of silencing cccDNA and/or reducing the size of the cccDNA pool to achieve functional cure with finite treatment duration.[url=][4][/url], [url=][5][/url], [url=][6][/url]
Several definitions of cure have been agreed upon by the community which pertains to the degree of viral depletion that can be achieved.[url=]3[/url] These include: (i) Partial cure with detectable HBsAg but persistently undetectable HBV DNA in serum after completion of a finite course of treatment; (ii) Functional cure with sustained, undetectable HBsAg and HBV DNA in serum with or without seroconversion to anti-HBs antibody after completion of a finite course of treatment. In this situation, cccDNA may persist at low levels with a transcriptionally inactive status; (iii) Complete cure with undetectable HBsAg in serum and eradication of intrahepatic cccDNA associated with the disappearance of the risk of viral reactivation; (iv) Sterilizing cure with undetectable serum HBsAg and eradication of intrahepatic cccDNA and integrated viral sequences with a possible return to a status of a never infected liver. Functional cure is thought to be the most promising and attainable goal for future therapies in the near future.
Among the novel strategies that are being investigated to directly target the different steps of the viral life cycle and/or to enhance antiviral immunity, one promising approach is to decrease viral antigen expression and especially HBsAg to low or undetectable levels. To decrease HBsAg levels, several paths can be followed: i) elimination of cccDNA; ii) silencing the transcriptional activity of cccDNA; iii) targeting viral transcripts; iv) interfering with envelope protein translation, stability, or release (Fig. 1). Novel approaches to target the pool of established cccDNA include the stimulation of innate immunity pathways of infected hepatocytes that may lead to cccDNA degradation,[url=]7[/url] or by creating molecular damage of cccDNA through gene editing approaches.[url=]8[/url] Silencing cccDNA can be achieved with IFN alpha and other cytokines, but virus specific mechanisms are being explored to shut down its expression.[url=][4][/url], [url=][6][/url] In any case, these approaches still require a thorough pre-clinical evaluation to address hurdles regarding delivery, off-target effects, and specificity for infected cells.
Key obstacles to be overcome to attain HBV cure (adapted from Testoni et al.[url=]6[/url]). cccDNA viral minichromosome persistence and transcriptional activity are at the basis of HBV replication and antigen production. HBV DNA integration in the host genome is not supposed to contribute to virion production, but there are several lines of evidence that HBsAg may also originate from integrated sequences. During chronic infection, there is a progressive impairment in HBV specific T cell function, due to multiple mechanisms, including prolonged exposure to secreted HBV antigens, a deficient activation of innate immune cells and defective antigen presentation by infected cells, and the restoration of a tolerogenic environment in the infected liver, due to immune-modulatory cells and secretion of tolerogenic molecules. To decrease HBsAg levels and potentially restore innate and adaptive immunity, several paths can be followed: i) targeting cccDNA through elimination or silencing its transcriptional activity; ii) targeting viral transcripts via antisense oligonucleotides or small interfering RNAs; iii) interfering with envelope protein processing and half-life in the blood. cccDNA, covalently closed circular DNA; DC, dendritic cell; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; IL-10, interleukin-10; NK, natural killer; PD-1, programmed cell death 1; PD-L1, programmed cell death ligand-1; pgRNA, pregenomic RNA; rcDNA, relaxed circular DNA; TGFβ, transforming growth factor-β.
Targeting HBsAg, the protein itself, has been evaluated with therapeutic monoclonal antibodies to neutralize or titrate HBsAg from the bloodstream or to inhibit HBsAg secretion from infected hepatocytes, for instance with nucleic acid polymers.[url=]9[/url] Both approaches are under pre-clinical and clinical evaluation.
A lot of interest has also emerged regarding post-transcriptional silencing of viral gene expression, using antisense oligonucleotides, small interfering RNA (siRNA). Their ability to inhibit viral gene expression and replication has been extensively evaluated in vitro and in animal models, and clinical trials are underway to assess their clinical relevance.[url=][10][/url], [url=][11][/url] Different methodologies to deliver in vivo HBV-specific siRNA to infected hepatocytes are now used in clinical studies.[url=]10[/url] A recent study demonstrated that HBsAg was expressed not only from the episomal cccDNA minichromosome, but also from transcripts arising from HBV DNA integrated into the host genome, which had an impact on siRNA effect since target sequences were affected by integration. These results will be important to consider for the design of novel siRNA and for endpoint expectations of new therapies.[url=]12[/url]
In this issue of Journal of Hepatology, the investigators from Roche report the discovery of a novel orally bioavailable small molecule inhibitor of HBV gene expression (RG7834).[url=]13[/url] RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV infection assays in HepaRG cells and primary human hepatocytes, and in HBV-infected urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model, either alone or in combination with entecavir. The results showed that unlike NUC therapies, which reduce viremia but do not lead to a reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Time course RNA-seq analysis revealed a selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV infected humanized mice with RG7834 led to a significant HBsAg reduction whereas entecavir had no significant effect on HBsAg levels. Combination of RG7834, entecavir and PegIFNα led to significant reductions of both HBV DNA and HBsAg levels in mice with humanized liver. This is a very interesting observation of an HBV inhibitor with a novel mode of action. Based on the results, it was suggested that this compound does not interfere with transcription factors gene expression. Results of RNAseq and northern blot analysis studies suggested that RG7834 may directly or indirectly modify viral RNAs and promote their degradation. The preference for degradation of subgenomic instead of pregenomic is intriguing and deserves further study to elucidate the exact mechanism of action of RG7834. It will be interesting to see how this compound interferes with viral RNA processing within infected cells.[url=]14[/url] A recent study showed that a dihydroquinolizinone compound induced an accelerated viral RNA degradation which was dependent on the HBV post-transcriptional regulatory element[url=]15[/url]and may have a similar activity as RG7834. Another point of interest will be to see if this compound can target subgenomic transcripts expressed from both cccDNA and integrated viral genomes, to overcome one of the issues related to siRNA, as discussed earlier. Considering its oral delivery and its high selectivity, i.e. its enantiomer was shown to be inactive, its potential for clinical application appears promising.
However, consistent with the presumed mode of action, this compound did not affect the pool of intrahepatic cccDNA when administered in mono- or combination therapy in immunodeficient mice, and treatment cessation was associated with viral rebound. The main issue with this compound and the majority of direct acting antivirals that are currently developed for HBV is that they would be mainly suppressive, unless they are associated with the reconstitution of anti-HBV immunity.[url=][4][/url], [url=][6][/url]
Several clinical studies have shown that the intrahepatic expression of innate immunity pathways and the ability to induce expression of interferon stimulated genes in peripheral blood mononuclear cells are altered more in patients with higher levels of HBsAg expression.[url=][16][/url], [url=][17][/url] Although, the situation seems less clear with respect to the effect of antigen reduction on T cell recovery,[url=]18[/url] it is tempting to hypothesize that the combination of viral gene expression inhibitor, with strategies to restore innate and/or adaptive immunity might turn out to be a cornerstone for successful therapies aiming at curing HBV infection. As several direct acting antivirals aiming at decreasing HBsAg levels and immunomodulatory agents are entering clinical evaluation, trials of combination therapy accompanied by virological and immunological studies will be instrumental to address this question.[url=]19[/url]
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