A novel orally available small molecule that inhibits hepatitis B virus expression
H. MuellerSteffen WildumSouphalone LuangsayJohanna WaltherAnaïs LopezPhilipp TropbergerGiorgio OttavianiWenzhe LuNeil ParrottJitao David ZhangRoland SchmuckiTomáš RačekJean-Christophe HoflackErich KuengFloriane PointXue ZhouGuido SteinerMarc LütgehetmannGianna RappTassilo VolzMaura DandriYang SongJohn A. T. YoungHassan Javanbakht
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Highlights•RG7834 is a novel orally bioavailable HBV viral gene expression inhibitor.•The effect of RG7834 on HBV expression levels is highly virus-specific.•The unique antiviral profile is differentiated from that of nucleos(t)ide analogues.•Combination with entecavir and/or PegIFNα improves the antiviral profile and potency.Graphical abstractAbstractBackground & AimsThe hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834).MethodsRG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir.ResultsUnlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09 log10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice.ConclusionWe have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues.Lay summaryWe discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses.Keywords:
Viremia
cccDNA
Entecavir
Hepatitis B
Introduction: We previously demonstrated that 48 weeks of ADV therapy resulted in a significant median 73% reduction of serum HBsAg (Werle, Gastroenterology 2004). Changes in HBsAg were significantly and positively correlated with changes in intrahepatic cccDNA, total intrahepatocellular HBV DNA and serum HBV DNA. The number of HBcAg positive hepatocytes remained stable. Aim: To determine the reduction of serum HBsAg titer from baseline to end-of- treatment in CH-B patients undergoing antiviral combination therapy. Methods: 21/26 patients with HBeAg positive and negative CH-B completed a 48-week course of treatment with ADV and PegIFN in a single center pilot study as of September 2004. HBsAg was quantified in patient sera by ELISA using the Monolisa® AgHBs Ultra Kit (Biorad, France) with purified HBsAg as the standard (Hytest, Finland). Intrahepatic ccc- and HBV DNA was quantified as described earlier. Results: At baseline, median serum HBsAg titer was 257µg/ml. After 48 weeks of combination therapy median serum HBsAg titer decreased to 136µg/ml, a 47% reduction. cccDNA decreased from median 6 copies/hepatocyte to 0.2 copies/hepatocyte (–1.46log). Total intrahepatocellular HBV DNA was reduced by –2.1log10, serum HBV DNA decreased by–4.7log10. Changes in HBsAg were positively correlated with changes in total intracellular HBV DNA and serum HBV DNA. Conclusions: 48 weeks of combination therapy with PegIFN and ADV results in an approx. 50% decrease in serum HBsAg in chronic hepatitis B patients. Earlier studies suggested the decline in HBsAg titer during antiviral therapy with a nucleotide analogue due to the reduced reservoir of transcriptionally active cccDNA rather than to the loss of infected hepatocytes. Further analysis of this non-cytolytical mechanism for the clearance of HBV-infection is currently under way.
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Adefovir
HBcAg
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Pegylated interferon
Hepatitis B
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In the era of antiviral therapy, the main goal of treatment has shifted from the persistent inhibition of hepatitis B virus (HBV) replication to the pursuit of serological clearance of HBs surface antigen (HBsAg). Based on the life cycle of HBV, HBsAg originates from covalently closed circular DNA (cccDNA) and integrated HBV DNA, thus reflecting their transcriptional activity. Complete HBsAg loss may mean elimination or persistent inactivity of the HBV genome including cccDNA and integrated HBV DNA. HBsAg loss improves the recovery of abnormal immune function, which in turn, may further promote the clearance of residual viruses. Combined with functional cure and the great improvement of clinical outcomes, the continuous seroclearance of high-sensitivity quantitative HBsAg may represent the complete cure of chronic hepatitis B (CHB). For many other risk factors besides HBV itself, patients with HBsAg loss still need regular monitoring. In this review, we summarized the evolution of CHB treatment, the origin of serum HBsAg, the pattern of HBsAg seroclearance, and the effect of HBsAg loss on immune function and disease outcomes. In addition, we discuss the significance of high-sensitivity HBsAg detection and its possibility as a surrogate of complete cure.
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배경/목적: 과거에는 B형 간염바이러스의 표면항원(HBsAg)의 소실이 HBV 감염으로부터의 회복을 의미해 왔으나 최근 분자생물학적 진단 방법의 발달로 인하여 적은 양이지만 HBV DNA가 간을 비롯한 체내에 존재한다는 것이 확인 되었다. 하지만 그 기전에 대해서는 아직까지 불분명한 상태이므로 본 연자 등은 추적 관찰 중 HBsAg이 소실된 환자들의 조직을 분자 생물학적 실험을 통해 분석하였다. 대상과 방법: 총 430명의 만성 HBsAg 보유자로부터 항원이 소실된 22명의 간조직을 얻어 분석하였고 이중 3명은 항원 소실 전후의 조직을 얻을 수 있었다. 22명중 2명은 인터페론을, 1명은 라미부딘을 치료 받았었다. 모든 간조직에서 HBV DNA를 추출하여 major hydrophilic region(MHR)과 HBsAg부위를 control vector에 cloning하였다. 자동염기서열분석법으로 S gene의 돌연변이와 아형을 확인하고 HepG2 cell에 transfection시킨 후 in vitro transcription/translation을 통한 HBsAg의 정량적 분석과 ELISA를 이용한 HBsAg/anti-HBs binding assay를 시행하였다. 또한 PCR방법을 통해 core promoter/precore 변이를 알아보았다. Real-time PCR은 HBV의 replicative form인 cccDNA의 정량을 위하여 시행되었다. 결과: Real-time PCR 결과 모든 조직에서 HBV의 cccDNA가 검출되어 혈청 내에서 HBsAg이 검출 되지 않는다 해도 간 조직에는 바이러스의 복제가 미약하나마 이루어지고 있다는 것을 알 수 있었다. MHR의 염기서열분석에서 T123S, M123I/N, I126L,C139R 그리고 D144E 변이가 6명에서 발견되었는데 특히 C139R 또는 D144E의 변이를 가진 군은 간세포내에 cccDNA가 많이 존재함에도 불구하고 낮은 항원성을 나타내었는데 이는 a-determinant 돌연변이로 인한 anti-HBs와 결합력 감소에 의한 것으로 보였다. 인터페론을 투여한 환자 중 한명은 225번의 아미노산인 티로신의 결손으로 HBsAg의 세포질내로 배출이 감소되어 HBsAg 검출이 안 되었음을 확인하였다. 라미부딘 투여 환자에서는 간세포에서 낮은 양의 cccDNA가 검출되어 실제로 HBsAg분비가 거의 소실 되었음을 알 수 있었다. Core promoter의 전사 인자 결합 부위에 A1762T와 G1764A변이는 혈청 HBeAg감소와 세포내 cccDNA감소와 동반되었는데 HBsAg소실과는 연관성이 없었다. 흥미롭게도 HBV 아형의 분석결과 HBsAg소실 환자들의 대부분이 ayw형 이었고 우리나라에서 흔한 adr형은 관찰되지 않았다. 결론: 만성 B형 간염 환자에서 HBsAg이 소실된 경우, HBV의 복제능 상실 외에도 돌연변이에 의한 HBsAg 항원성의 변화, HBsAg의 세포질내로 분비 장애가 항원 검출에 영향을 줄 수 있음을 확인하였다.
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As alternative indexes of hepatitis B virus (HBV), co-valently closed circular DNA (cccDNA) transcriptional activity, hepatitis B surface antigen (HBsAg), hepatitis B core-related antigen (HBcrAg), and peripheral blood RNA known as pgRNA, have been advocated as novel serum markers for prediction of prognosis and treatment response in chronic hepatitis B (CHB).
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Entecavir
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Antiviral Therapy
Hepatitis B
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Therapy with nucleic acid polymers (NAPs), tenofovir disoproxil fumarate (TDF), and pegylated interferon (pegIFN) achieve high rates of HBsAg loss/seroconversion and functional cure in chronic hepatitis B virus (HBV) infection. The role of hepatitis B surface antigen (HBsAg) seroconversion and inactivation of covalently closed circular DNA (cccDNA) in establishing functional cure were examined. Archived serum from the REP 401 study was analyzed using the Abbott ARCHITECT HBsAg NEXT assay (Chicago, IL), Abbott research use–only assays for HBsAg immune complexes (HBsAg ICs), circulating HBV RNA, and the Fujirebio assay for hepatitis B core‐related antigen (HBcrAg; Malvern, PA). HBsAg became < 0.005 IU/mL in 23 participants during NAP exposure, which persisted in all participants with functional cure. HBsAg IC declined during lead‐in TDF monotherapy and correlated with minor declines in HBsAg. Following the addition of NAPs and pegIFN, minor HBsAg IC increases (n = 13) or flares (n = 2) during therapy were not correlated with HBsAg decline, hepatitis B surface antibody (anti‐HBs) titers, or alanine aminotransferase. HBsAg IC universally declined during follow‐up in participants with virologic control or functional cure. Universal declines in HBV RNA and HBcrAg during TDF monotherapy continued with NAP + pegIFN regardless of therapeutic outcome. At the end of therapy, HBV RNA was undetectable in only 5 of 14 participants with functional cure but became undetectable after removal of therapy in all participants with functional cure. Undetectable HBV RNA at the end of therapy in 5 participants was followed by relapse to virologic control or viral rebound. Conclusion: Anti‐HBs‐independent mechanisms contribute to HBsAg clearance during NAP therapy. Inactivation of cccDNA does not predict functional cure following NAP‐based therapy; however, functional cure is accompanied by persistent inactivation of cccDNA. Persistent HBsAg loss with functional cure may also reflect reduction/clearance of integrated HBV DNA. Clinicaltrials.org number NCT02565719.
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When antiretroviral therapy (ART) is administered for long periods to HIV-1-infected patients, most achieve viral loads that are "undetectable" by standard assay methods (ie, HIV-1 RNA <50 copies/mL). Despite sustaining viral loads lower than the level of detection, a number of patients experience unexplained episodes of transient viremia or viral "blips." We propose that transient activation of the immune system by infectious agents may explain these episodes of viremia. Using 2 different mathematical models, one in which blips arise because of target cell activation and subsequent infection and another in which latent cell activation generates blips, we establish a nonlinear (power law) relationship between blip amplitude and viral load (under ART) that suggest blips should be of lower amplitude, and thus harder to detect, as increasingly potent therapy is used. This effect can be more profound than is predicted by simply lowering the baseline viral load from which blips originate. Finally, we suggest that sporadic immune activation may elevate the level of chronically infected cells and replenish viral reservoirs, including the latent cell reservoir, providing a mechanism for recurrent viral blips and low levels of viremia under ART.
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The focus of hepatitis B functional cure, defined as sustained loss of hepatitis B virus (HBV) surface antigen (HBsAg) and HBV DNA from blood, is on eliminating or silencing the intranuclear template for HBV replication, covalently closed circular DNA (cccDNA). However, HBsAg also derives from HBV DNA integrated into the host genome (iDNA). Little is known about the contribution of iDNA to circulating HBsAg with current therapeutics. We applied a multiplex droplet digital PCR assay to demonstrate that iDNA is responsible for maintaining HBsAg quantities in some individuals. Using paired bulk liver tissue from 16 HIV/HBV-coinfected persons on nucleos(t)ide analog (NUC) therapy, we demonstrate that people with larger HBsAg declines between biopsies derive HBsAg from cccDNA, whereas people with stable HBsAg levels derive predominantly from iDNA. We applied our assay to individual hepatocytes in paired tissues from 3 people and demonstrated that the individual with significant HBsAg decline had a commensurate loss of infected cells with transcriptionally active cccDNA, while individuals without HBsAg decline had stable or increasing numbers of cells producing HBsAg from iDNA. We demonstrate that while NUC therapy may be effective at controlling cccDNA replication and transcription, innovative treatments are required to address iDNA transcription that sustains HBsAg production.
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