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The standard of care for the treatment of chronic hepatitis B (CHB) is typically lifelong treatment with nucleos(t)ide analogs (NAs), which suppress viral replication and provide long-term clinical benefits. However, infectious virus can still be detected in patients who are virally suppressed on NA therapy, which may contribute to the failure of these agents to cure most CHB patients. Accordingly, new antiviral treatment options are being developed to enhance the suppression of hepatitis B virus (HBV) replication in combination with NAs ("antiviral intensification"). Here, we describe GS-SBA-1, a capsid assembly modulator (CAM) belonging to class CAM-E, that demonstrates potent inhibition of extracellular HBV DNA

Background and aims GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus. Approach and results WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688, or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells and various other immune cell subsets may play a role in the antiviral response to GS-9688. Conclusions Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks; the identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect.

Development of curative therapies for chronic hepatitis B virus (HBV) infection will likely require new animal models. Here, we evaluate HBV infection in squirrel monkeys based on the high-sequence homology of the HBV receptor, Na+/taurocholate co-transporting peptide (NTCP), between humans and squirrel monkeys. HBV PreS1 peptide was examined for binding human and squirrel monkey NTCP. Immunodeficient Fah -/- , NOD, Rag1 -/- , Il2Rg null (FNRG) mice engrafted with human or squirrel monkey hepatocytes were challenged with HBV or Woolly Monkey HBV (WMHBV). In addition, adult squirrel monkeys were inoculated with HBV, WMHBV, adeno-associated virus containing an infectious genome of HBV (AAV-HBV), and AAV-WMHBV. Finally, neonate squirrel monkeys were assessed for the potential of chronic infection with WMHBV. PreS1 peptide efficiently bound to human and squirrel monkey NTCP but not to mouse or capuchin NTCP. FNRG mice engrafted with squirrel monkey hepatocytes were susceptible to infection by WMHBV but not human HBV. Similarly, adult squirrel monkeys could be infected with WMHBV but not human HBV, whereas chimeric mice engrafted with human hepatocytes were susceptible to HBV but not WMHBV. Infection of squirrel monkeys with AAV-WMHBV yielded maximum viremia of 108 genomes/mL with detectable virus for up to 8 months. Notably, covalently closed circular DNA was detected in the liver of these animals. Infection of neonates with WMHBV led to detectable viremia for up to 6 months. Conclusions: Adult and neonate squirrel monkeys exhibited prolonged WMHBV viremia lasting 6-8 months. This is greater than twice the duration of viremia achieved in other nonhuman primates and suggests that squirrel monkeys may be a suitable model for testing HBV therapeutics.

Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure.

Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, creating potentially oncogenic lesions that can lead to hepatocellular carcinoma (HCC). However, our current understanding of integrated HBV DNA architecture, burden, and transcriptional activity is incomplete due to technical limitations. A combination of genomics approaches was used to describe HBV integrations and corresponding transcriptional signatures in three HCC cell lines: huH-1, PLC/PRF/5, and Hep3B. To generate high-coverage, long-read sequencing data, a custom panel of HBV-targeting biotinylated oligonucleotide probes was designed. Targeted long-read DNA sequencing captured entire HBV integration events within individual reads, revealing that integrations may include deletions and inversions of viral sequences. Surprisingly, all three HCC cell lines contain integrations that are associated with host chromosomal translocations. In addition, targeted long-read RNA sequencing allowed for the assignment of transcriptional activity to specific integrations and resolved the contribution of overlapping HBV transcripts. HBV transcripts chimeric with host sequences were resolved in their entirety and often included >1,000 bp of host sequence. This study provides the first comprehensive description of HBV integrations and associated transcriptional activity in three commonly utilized HCC-derived cell lines. The application of novel methods sheds new light on the complexity of these integrations, including HBV bidirectional transcription, nested transcripts, silent integrations, and host genomic rearrangements. The observation of multiple HBV-associated chromosomal translocations gives rise to the hypothesis that HBV is a driver of genetic instability and provides a potential new mechanism for HCC development. IMPORTANCE HCC-derived cell lines have served as practical models to study HBV biology for decades. These cell lines harbor multiple HBV integrations and express only HBV surface antigen (HBsAg). To date, an accurate description of the integration burden, architecture, and transcriptional profile of these cell lines has been limited due to technical constraints. We have developed a targeted long-read sequencing assay that reveals the entire architecture of integrations in these cell lines. In addition, we identified five chromosomal translocations with integrated HBV DNA at the interchromosomal junctions. Incorporation of long-read transcriptome sequencing (RNA-Seq) data indicated that many integrations and translocations were transcriptionally silent. The observation of multiple HBV-associated translocations has strong implications regarding the potential mechanisms for the development of HBV-associated HCC.

Current therapies rarely cure chronic hepatitis B virus (HBV) infection due to the persistence of the viral episome, the covalently closed circular DNA (cccDNA), in hepatocytes. The hepatitis B virus core-related antigen (HBcrAg), a mixture of the viral precore/core gene products, has emerged as one potential marker to monitor the levels and activities of intrahepatic cccDNA. In this study, a comprehensive characterization of precore/core gene products revealed that HBcrAg components included the classical hepatitis B virus core antigen (HBc) and e antigen (HBeAg) and, additionally, the precore-related antigen, PreC, retaining the N-terminal signal peptide. Both HBeAg and PreC antigens displayed heterogeneous proteolytic processing at their C termini resulting in multiple species, which varied with viral genotypes. HBeAg was the predominant form of HBcrAg in HBeAg-positive patients. Positive correlations were found between HBcrAg and PreC, between HBcrAg and HBeAg, and between PreC and HBeAg but not between HBcrAg and HBc. Serum HBeAg and PreC shared similar buoyant density and size distributions, and both displayed density and size heterogeneity. HBc, but not HBeAg or PreC antigen, was found as the main component of capsids in DNA-containing or empty virions. Neither HBeAg nor PreC protein was able to form capsids in cells or in vitro under physiological conditions. In conclusion, our study provides important new quantitative information on levels of each component of precore/core gene products as well as their biochemical and biophysical characteristics, implying that each component may have distinct functions and applications in reflecting intrahepatic viral activities. IMPORTANCE Chronic hepatitis B virus (HBV) infection afflicts approximately 257 million people, who are at high risk of progressing to chronic liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. Current therapies rarely achieve cure of HBV infection due to the persistence of the HBV episome, the covalently closed circular DNA (cccDNA), in the nuclei of infected hepatocytes. Peripheral markers of cccDNA levels and transcriptional activities are urgently required to guide antiviral therapy and drug development. Serum hepatitis B core-related antigen (HBcrAg) is one such emerging peripheral marker. We have characterized the components of HBcrAg in HBV-infected patients as well as in cell cultures. Our results provide important new quantitative information on levels of each HBcrAg component, as well as their biochemical and biophysical characteristics. Our findings suggest that each HBcrAg component may have distinct functions and applications in reflecting intrahepatic viral activities.

Nucleos(t)ide analogs are standard-of-care for the treatment of chronic hepatitis B and can effectively reduce hepatitis B virus (HBV) replication but rarely leads to cure. Nucleos(t)ide analogs do not directly eliminate the viral episome, therefore treatment cessation typically leads to rapid viral rebound. While treatment is effective, HBV DNA is still detectable (although not quantifiable) in the periphery of the majority of nucleos(t)ide analog treated HBV patients, even after prolonged treatment. Addressing whether the detectable HBV DNA represents infectious virus is a key unknown and has important implications for the development of a curative treatment for HBV. The minimum HBV genome equivalents required to establish infection in human liver chimeric mice was determined by titration of HBV patient sera and the infectivity in chimeric mice of serum from patients (n = 7) suppressed to the limit of detection on nucleos(t)ide analog therapy was evaluated. A minimum of 5 HBV genome equivalents were required to establish infection in the chimeric mice, confirming this model has sufficient sensitivity to determine whether serum from virally suppressed patients contains infectious virus. Strikingly, serum from 75% (n = 3 out of 4) of nucleos(t)ide-treated HBV patients with DNA that was detectable, but below the lower limit of quantitation, also established infection in the chimeric mice. These results demonstrate that infectious virus is still present in some HBV patients on suppressive nucleos(t)ide therapy. This residual virus may support viral persistence via continuous infection and explain the ongoing risk for HBV-related complications despite long-term suppression on therapy. Thus, additional treatment intensification may facilitate HBV cure.

Current approved therapies for Hepatitis B Virus (HBV) effectively control viral replication but are rarely curative. Patients on suppressive therapy still harbor cccDNA and cessation of therapy leads to rapid viral rebound 1-3. Even after long-term therapy of nucleos(t)ide analog (NA) treatment, the majority of chronic hepatitis B (CHB) patients have detectable but not quantifiable HBV DNA 4. While these data suggest viral suppression is not absolute with current NA therapies, it has not been determined whether the detectable HBV DNA represents infectious virus. Here, we show that serum from patients on suppressive therapy with viral loads below the limit of quantitation still contain infectious circulating virus. We used a chimeric mouse model with a humanized liver to demonstrate HBV-infected human serum can establish infection similar to that in humans. We determined the limit of detection of the model using diluted patient serum, then measured the infectivity of serum from patients suppressed on therapy. Our results support the hypothesis that residual virus can serve as a reservoir to maintain a chronic infection in patients on therapy. These results highlight the urgent need for orthogonal therapies to eliminate low levels of circulating infectious virus which may minimize ongoing chromosomal damage to the liver and potentially lead to a cure.

Background & Aims GS-9620, an oral agonist of toll-like receptor 7, is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the chimpanzee and woodchuck models of CHB. Herein, we investigated the immunomodulatory mechanisms underlying these antiviral effects. Methods Archived liver biopsies and paired peripheral blood mononuclear cell samples from a previous chimpanzee study were analyzed by RNA sequencing, quantitative reverse transcription PCR, immunohistochemistry (IHC) and in situ hybridization (ISH). Results GS-9620 treatment of CHB chimpanzees induced an intrahepatic transcriptional profile significantly enriched with genes associated with hepatitis B virus (HBV) clearance in acutely infected chimpanzees. Type I and II interferon, CD8+ T cell and B cell transcriptional signatures were associated with treatment response, together with evidence of hepatocyte death and liver regeneration. IHC and ISH confirmed an increase in intrahepatic CD8+ T cell and B cell numbers during treatment, and revealed that GS-9620 transiently induced aggregates predominantly comprised of CD8+ T cells and B cells in portal regions. There were no follicular dendritic cells or IgG-positive cells in these lymphoid aggregates and very few CD11b+ myeloid cells. There was no change in intrahepatic natural killer cell number during GS-9620 treatment. Conclusion The antiviral response to GS-9620 treatment in CHB chimpanzees was associated with an intrahepatic interferon response and formation of lymphoid aggregates in the liver. Our data indicate these intrahepatic structures are not fully differentiated follicles containing germinal center reactions. However, the temporal correlation between development of these T and B cell aggregates and the antiviral response to treatment suggests they play a role in promoting an effective immune response against HBV. Lay summary New therapies to treat chronic hepatitis B (CHB) are urgently needed. In this study we performed a retrospective analysis of liver and blood samples from a chimpanzee model of CHB to help understand how GS-9620, a drug in clinical trials, suppressed hepatitis B virus (HBV). We found that the antiviral response to GS-9620 was associated with accumulation of immune cells in the liver that can either kill cells infected with HBV or can produce antibodies that may prevent HBV from infecting new liver cells. These findings have important implications for how GS-9620 may be used in patients and may also help guide the development of new therapies to treat chronic HBV infection.

The COBAS TaqMan assay has a lower limit of quantification (LLOQ) of 169 HBV copies/mL and a lower limit of detection (LLOD) of 58 copies/mL. HBV DNA below the TaqMan LLOQ is classified as target not detected (TND) (

The NS3 protease inhibitor (PI) GS-9256 has demonstrated antiviral activity in a monotherapy study and in combination with other DAAs for treatment of chronic hepatitis C virus (HCV) infection. The resistance profile of GS-9256 was investigated in a phase 1 monotherapy study of patients with HCV genotype (GT) 1 infection. No PI resistance associated substitutions (RASs) at positions 36, 155, 156, 168 and 170 were observed at baseline by population sequencing (15% cutoff) in the 54 patients enrolled in the study, however the PI RAS Q80K were detected in 41% of patients at baseline. In patients who received 75 mg of the investigational protease inhibitor (PI) GS-9256 BID, 300 mg of GS-9256 QD and 200 mg of GS-9256 BID for three days, NS3 RASs (A156V, R155K, D168G/E/N/V) were observed in 9/21, 3/7 and 8/8 post-treatment, respectively. Q80K was not selected in any patients post-treatment. The mean maximal viral load response was −3.0 ± 0.42 log10 IU/mL HCV RNA in the 200 mg BID cohort. In more than 50% of the patients with RASs detected at Day 4, mutations were no longer detectable by population sequencing at Day 14. One patient had the R155K mutation persist to Week 24. Phenotypic analyses showed that substitutions at R155, A156 and D168 significantly reduced susceptibility to GS-9256. In conclusion, NS3 PI RASs were rapidly selected in the majority of patients receiving GS-9256 as monotherapy, despite undetectable levels at baseline. The R155, A156 and D168 substitutions identified in patients confer reduced susceptibility to GS-9256 and other PIs in vitro.

Background GS-9256 is an inhibitor of HCV NS3 protease with a macrocyclic structure and novel phosphinic acid pharmacophore. Methods Key preclinical properties of GS-9256 including in vitro antiviral activity, cross-resistance and pharmacokinetic properties were investigated in non-human species. Results In genotype (GT) 1b Huh-luc cells with a replicon encoding luciferase, GS-9256 had a mean 50% effective concentration (EC50) value of 20.0 nM, with minimal cytotoxicity. Antiviral activity was similar in a number of additional GT1b and GT1a replicon cell lines. Similar potency was observed in chimeric replicons encoding the NS3 protease of GT1 clinical isolates. GS-9256 was less active in GT2a replicon cells (14.2-fold increase in EC50). Additive to synergistic in vitro antiviral activity was observed when GS-9256 was combined with other agents including interferon-α, ribavirin, NS5B polymerase inhibitors GS-6620 and tegobuvir, as well as the NS5A inhibitor ledipasvir. GS-9256 retained wild-type activity against all tested NS5B and NS5A inhibitor resistance mutations. GS-9256 was metabolically stable in microsomes and hepatocytes of tested species, including rodents, dogs and humans. GS-9256 had high bioavailability in mice (near 100%) and moderate bioavailability in rats (14%), dogs (21%) and monkeys (14%). Elimination half-lives were approximately 2 h in mice, 0.6 h in rats, 5 h in dogs and 4 h in monkey. A study in bile duct-cannulated rats indicated that the major route of elimination is through biliary excretion of unmetabolized GS-9256. Conclusions GS-9256 showed a favourable preclinical profile supportive of clinical development for the treatment of chronic HCV infection in GT1 patients.

Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro . Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors. IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancer worldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have developed a mammalian cell-free system in which HBc is expressed at physiological (low) concentrations and assembles into capsids under near-physiological conditions. In this cell-free system, as in mammalian cells, capsid assembly depends on the C-terminal domain (CTD) of HBc, in contrast to other assembly systems in which HBc assembles into capsids independently of the CTD under conditions of nonphysiological protein and salt concentrations. Furthermore, the phosphorylation state of the CTD regulates capsid assembly and RNA encapsidation in the cell-free system in a manner similar to that seen in mammalian cells. This system will facilitate detailed studies on capsid assembly and RNA encapsidation under physiological conditions and identification of antiviral agents that target HBc.

Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC 50 ) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC 50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC 50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC 50 values equivalent to those for the wild type.

Background & Aims GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. Methods Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. Results GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors – although not APOBEC3A or the Smc5/6 complex – and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. Conclusions Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. Lay summary GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.

The C-terminal domain (CTD) of hepadnavirus core protein is involved in multiple steps of viral replication. In particular, the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging into immature nucleocapsids (NCs) and the early stage of viral DNA synthesis. For the avian hepadnavirus duck hepatitis B virus (DHBV), CTD is dephosphorylated subsequently to facilitate the late stage of viral DNA synthesis and to stabilize NCs containing mature viral DNA. The role of CTD phosphorylation in virion secretion, if any, has remained unclear. Here, the CTD from the human hepatitis B virus (HBV) was found to be dephosphorylated in association with NC maturation and secretion of DNA-containing virions, as in DHBV. In contrast, the CTD in empty HBV virions (i.e., enveloped capsids with no RNA or DNA) was found to be phosphorylated. The potential role of CTD dephosphorylation in virion secretion was analyzed through mutagenesis. For secretion of empty HBV virions, which is independent of either viral RNA packaging or DNA synthesis, multiple substitutions in the CTD to mimic either phosphorylation or dephosphorylation showed little detrimental effect. Similarly, phospho-mimetic substitutions in the DHBV CTD did not block the secretion of DNA-containing virions. These results indicate that CTD dephosphorylation, though associated with NC maturation in both HBV and DHBV, is not essential for the subsequent NC-envelope interaction to secrete DNA-containing virions, and the CTD state of phosphorylation also does not play an essential role in the interaction between empty capsids and the envelope for secretion of empty virions. IMPORTANCE The phosphorylation state of the C-terminal domain (CTD) of hepatitis B virus (HBV) core or capsid protein is highly dynamic and plays multiple roles in the viral life cycle. To study the potential role of the state of phosphorylation of CTD in virion secretion, we have analyzed the CTD phosphorylation state in complete (containing the genomic DNA) versus empty (genome-free) HBV virions. Whereas CTD is unphosphorylated in complete virions, it is phosphorylated in empty virions. Mutational analyses indicate that neither phosphorylation nor dephosphorylation of CTD is required for virion secretion. These results demonstrate that while CTD dephosphorylation is associated with HBV DNA synthesis, the CTD state of phosphorylation may not regulate virion secretion.

Background GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays. Methods Potency of the compounds against NS3 protease and selectivity against a panel of mammalian proteases were determined through steady-state enzyme kinetics. Binding kinetics were determined using stopped-flow techniques. Dissociation rates were measured using dilution methods. Results GS-9256 and vedroprevir had measured Ki values of 89 pM and 410 pM, respectively, against genotype 1b NS3 protease; Ki values were higher against genotype 2a (2.8 nM and 39 nM) and genotype 3 proteases (104 nM and 319 nM) for GS-9256 and vedroprevir, respectively. Selectivity of GS-9256 and vedroprevir was > 10,000-fold against all tested off-target proteases. Association rate constants of 4 × 105 M− 1 s− 1 and 1 × 106 M− 1 s− 1, respectively, were measured, and dissociation rate constants of 4.8 × 10− 5 s− 1 and 2.6 × 10− 4 s− 1 were determined. Conclusions GS-9256 and vedroprevir are potent inhibitors of NS3 protease with high selectivity against off-target proteases. They have rapid association kinetics and slow dissociation kinetics. General Significance The NS3 protease is a key drug target for the treatment of hepatitis C. The potency, selectivity, and binding kinetics of GS-9256 and vedroprevir constitute a biochemical profile that supports the evaluation of these compounds in combination with other direct-acting antivirals in clinical trials for hepatitis C.

Treatment with GS-9669, a novel nonnucleoside inhibitor (site II) of hepatitis C virus (HCV) nonstructural 5B (NS5B) polymerase, resulted in significant antiviral activity in HCV genotype (GT) 1 patients dosed at 50 and 500 mg once daily (QD) and at 50, 100, and 500 mg twice daily (BID) for 3 days. This report characterizes the virologic resistance to GS-9669 in vitro and in GT1 HCV-infected patients from a phase I clinical study. An in vitro resistance selection study with GS-9669 revealed substitutions at several NS5B residues that conferred resistance. The M423 variants were selected at low drug concentrations (5× the 50% effective concentration [EC 50 ]), and the L419, R422, and I482 variants were selected at higher drug concentrations (20× the EC 50 ). During the phase I clinical study, substitutions at NS5B residues 419, 422, and 486 were the predominant changes associated with GS-9669 monotherapy. Substitutions at position 423 were observed only in GT1a patients in the low-dose groups (50 and 100 mg BID). Interestingly, four HCV patients had substitutions at position 423 at baseline. Consistent with the low resistance level at this position, three patients with M423I or M423V at baseline achieved >2-log 10 reductions of HCV RNA when treated with 100 mg BID or with 500 mg QD or BID of GS-9669. The fourth patient, who had the M423V substitution at baseline, had a 4.4-log 10 reduction of HCV RNA with 500 mg BID of GS-9669. Phenotypic analyses demonstrated that the viral isolates with multiple GS-9669 resistance-associated variants have reduced susceptibility to GS-9669 and lomibuvir (VX-222) but are not cross-resistant to other classes of HCV inhibitors. (This study has been registered at ClinicalTrials.gov under registration no. NCT01431898.)

We describe here the anti-HBV activity of natural and synthetic retinoids in primary human hepatocytes (PHHs). The most potent compounds inhibited HBsAg, HBeAg, viral RNA and DNA production by HBV infected cells with EC 50 values ranging from 0.4 to 2.6 μM. The activity was independent of PHH donor and HBV genotype used in testing. 13-cis retinoic acid (Accutane) was selected for further evaluation in the PXB chimeric mouse model of HBV infection at doses allowing to achieve Accutane peak serum concentrations near its antiviral EC 90 and exposures ∼5-fold higher than a typical clinical dose. While these supraclinical exposures of 100 mg/kg/day were well-tolerated by regular Balb/c mice, PXB mice were more sensitive and even a lower those of 60 mg/kg/day led to significant weight loss. Despite dosing at this maximal tolerated dose for 28 days, Accutane failed to show any anti-HBV activity. RAR target engagement was verified using transcriptome analysis of liver samples from treated versus vehicle groups. However, gene expression changes in PXB liver samples were vastly muted when compared to the in vitro PHH system. When comparing transcriptional changes associated with the conditioning of fresh hepatocytes toward enabling HBV infection, we also observed a large number of changes. Noticeably, a significant number of genes that were up- or down-regulated by the conditioning process were down- or up-regulated by HBV infected PHH treatment with Accutane, respectively. While the lack of efficacy in the PXB model may have many explanations, the observed, opposing transcriptional changes upon conditioning PHH and treating these cultured, HBV-infected PHH with Accutane allow for the possibility that the PHH system may yield artificial anti-HBV hits.

The development of JFH1 based intergenotypic recombinants that exploit the unique replication characteristics of JFH1 has made it possible to study infectious hepatitis C virus (HCV) encoding the structural genes of additional HCV genotypes. To facilitate the study of 1b structural proteins, we aimed to develop a robust 1b/2a chimera encoding a humanized Renilla luciferase reporter gene (1b/2a hRluc). The unadapted genome replicated efficiently but produced very low titers of infectious virus. Adaptation by continuous passage over a novel Huh-7 Lunet clone improved viral titers approximately 100-fold but caused an unexpected decline in luciferase activity, limiting the utility of the reporter-containing virus. Genotypic analysis revealed 17 adenosine to guanosine (A to G) nucleotide mutations in the luciferase gene and two potential adaptive mutations. To overcome the problems of low viral titers and editing of the luciferase gene during viral adaptation, six adaptive mutations previously identified in a non-reporter 1b/2a HCV genome were introduced into the 1b/2a hRluc genome. This resulted in the immediate production of high-titer viral stocks (approximately 1000-fold greater than the parental virus) that could efficiently infect naïve cells and generate robust luciferase signals. The improved sensitivity of the luciferase reporter also facilitated time of addition studies validating the utility of this system for characterizing the early steps of HCV infection. Thus, the development of the 1b/2a hRluc reporter virus described here provides a versatile tool for discovery of inhibitors targeting the early steps of the viral life cycle and genotype 1b structural genes.

Background GS-9451 is a novel inhibitor of the HCV NS3/4A protease and demonstrates potent in vitro suppression of HCV genotype 1 replicons. Methods The safety, pharmacokinetics and antiviral efficacy of GS-9451 were evaluated in a Phase I study in treatment-naive, HCV genotype-1-infected patients. Patients were randomized to 3 days of once-daily dosing with placebo ( n=8) or GS-9451 60 mg ( n=8 genotype 1a), 200 mg ( n=8 genotype 1a; n=8 genotype 1b) or 400 mg ( n=9 genotype 1a). Plasma samples were collected up to and on day 14 for pharmacokinetic evaluation, serum HCV RNA quantitation and NS3 sequencing. Results No patients interrupted or discontinued dosing because of an adverse event. The median (range) maximal HCV RNA reductions from baseline were -0.88 (-1.24– -0.64), -3.19 (-3.31– -2.94) and -3.64 (-4.08– -3.54) log10 IU/ml in genotype 1a patients receiving 60, 200 and 400 mg/day GS-9451, respectively, and -3.48 (-3.54– -3.03) log10 IU/ml in genotype 1b patients receiving GS-9451 200 mg/day. Median half-life ranged from 14 to 17 h. Day 3 mean concentration at the end of dosing interval was 5.5- and 17-fold above protein-binding adjusted mean 50% effective inhibitory concentration in 200 mg and 400 mg cohorts, respectively. No resistance mutations were detected with GS-9451 60 mg/day. In the 200 mg/day or 400 mg/day groups, predominant mutations were NS3 R155 (R155K) in genotype 1a patients and D168 (D168E, D168V and D168G) in genotype 1b patients. Conclusions GS-9451 was well-tolerated. During 3 days of monotherapy, GS-9451 200 mg/day or 400 mg/day demonstrated potent antiviral activity in both HCV genotype 1a- and 1b-infected patients. GS-9451 is currently being evaluated in combination regimens with and without pegylated interferon-α.

The discovery of GS-9451 is reported. Modification of the P3 cap and P2 quinoline with a series of solubilizing groups led to the identification of potent HCV NS3 protease inhibitors with greatly improved pharmacokinetic properties in rats, dogs and monkeys.

Clinical trials of direct-acting antiviral agents in patients chronically infected with hepatitis C virus (HCV) have demonstrated that viral resistance is detected rapidly during monotherapy. In patients, HCV does not exist as a single, genetically homogenous virus but rather as a population of variants termed “quasispecies.” Preexisting variants resistant to specific antiviral drugs, overlooked in traditional hit-to-lead discovery efforts, may be responsible for these poor clinical outcomes. To enable real-time studies of resistance emergence in live cells, we established fluorescent protein-labeled HCV replicon cell lines. We validated these cell lines by demonstrating that antiviral susceptibility and the selection of signature resistance mutations for various drug classes are similar to traditional replicon cell lines. By quantifying the kinetics and uniformity of replication within colonies of drug-resistant fluorescent replicon cells, we showed that resistance emerged from a single cell and preexisted in a treatment-naive replicon population. Within this population, we determined the relative frequency of preexisting replicons capable of establishing foci during treatment with distinct antivirals. By measuring relative frequency as a function of dose, we quantitatively ranked distinct antiviral molecules on the basis of their distinct barriers to resistance. These insights into RNA virus quasispecies structure provide guidance for selecting clinical drug concentrations and selecting antiviral drug combinations most likely to suppress resistance.

A potent and novel class of product-like inhibitors of the HCV NS3 protease was discovered by employing a phosphinic acid as a carboxylate isostere. The replicon activity and pharmacokinetic profile of this series of compounds was optimized by exploring the substitution of the phosphinic acid, as well as conformationally constraining these compounds through macrocyclization. The syntheses and preliminary biological evaluation of these phosphinic acids is described.

Treatment of patients infected with hepatitis C virus (HCV) with direct acting antivirals can lead to the emergence of drug-resistant variants that may pose a long-term threat to viral eradication. HCV replicons have been used to select resistance mutations; however, genotype 2a JFH-1-based viruses provide the opportunity to perform resistance selection in a bona fide infection system. In this study, we used a tissue culture-adapted J6/JFH-1 virus to select resistance to the NS3 protease inhibitors BILN-2061 and VX-950. Lunet-CD81 cells were infected with J6/JFH-1 virus and maintained in the presence of inhibitors until high-titer viral supernatant was produced. Viral supernatants were passaged over naive cells at escalating drug concentrations, and the resulting viruses were then characterized. Three NS3 resistance mutations were identified in BILN-2061-resistant viruses: A156G, D168A, and D168V. Interestingly, D168A, D168V, and A156T/V, but not A156G, were selected in parallel using a genotype 2a replicon. For VX-950, the T54A and A156S NS3 resistance mutations were identified in the virus selections, whereas only A156T/V emerged in genotype 2a replicon selections. Of note, VX-950 resistance mutations selected using the 2a virus (T54A and A156S) were also observed during VX-950 clinical studies in genotype 2 patients. We also performed viral fitness evaluations and determined that the mutations selected in the viral system did not confer marked reductions in virus production kinetics or peak titers. Overall, the HCV infection system is an efficient tool for drug resistance selections and has advantages for the rapid identification and characterization of clinically relevant resistance mutations.

The hepatitis C virus infection system represents an important new tool for drug discovery. In this study, we compared the in vitro antiviral efficacy of several NS3 and NS5B inhibitors in genotype 1a, 1b, and 2a replicons and in the 2a infectious virus system. The nucleoside inhibitor 2'-C-methyl adenosine showed similar efficacy in each system tested. Three non-nucleoside inhibitors had small differences in potency between genotype 1a and 1b. In contrast, there was a dramatic loss of potency for these non-nucleoside inhibitors in the genotype 2a replicon, 2a infectious virus, and 2a NS5B biochemical assays. The protease inhibitor BILN-2061 had similar efficacy against 1a and 1b replicons but was 61-109-fold less potent against the 2a replicon and virus, respectively. VX-950, a covalent protease inhibitor, had similar efficacy (3-fold changes in EC(50)) regardless of genotype or subtype. Importantly, we observed a significant correlation (p0.0001) in antiviral potency between the 2a replicon and 2a infectious virus for all classes of compounds tested.

A novel class of phosphonate derivatives was designed to mimic the interaction of product-like carboxylate based inhibitors of HCV NS3 protease. A phosphonic acid (compound 2) was demonstrated to be a potent HCV NS3 protease inhibitor, and a potential candidate for treating HCV infection. The syntheses and preliminary biological evaluation of this phosphonate class of inhibitor are described.

Hepatitis C virus (HCV) protease inhibitors targeting HCV NS3 can efficiently suppress HCV replication. However, the selection of resistance has been observed both in vitro and in vivo. Here, we describe a new method for efficient analysis of the drug susceptibility of the NS3 protease genes from patient isolates. Luciferase-reporter 1b replicon shuttle vectors that allow cloning of either the HCV full-length NS3/4A gene or the NS3 protease domain gene only were created. Initially, chimeric replicons carrying patient-derived full-length NS3/4A failed to replicate in cell culture. However, the poor replication efficiency of the NS3/4A shuttle vector was enhanced by approximately 100-fold when the NS3 helicase domains of clinical isolates were substituted for that of the 1b Con1 lab strain. Chimeric replicons carrying only the patient-derived NS3 protease domains replicated at levels sufficient for phenotypic analysis in 20/20 clinical isolates. EC 50 values for the NS3 inhibitor BILN-2061 ranged from 0.2 to 1.1 nM for 20 genotype 1 patient isolates. Significantly reduced susceptibility to BILN-2061 was observed with mutant/wild type mixtures of 5%/95% for the D168V or 50%/50% for A156T resistance mutations in the NS3. These shuttle vectors can be used to evaluate candidate drugs and assist in the development of new drugs targeting the NS3 protease.

There are now five nucleoside/nucleotide analogs approved for the treatment of chronic hepatitis B (CHB) including three agents approved in the United States and/or European Union in the past three years. Each of these drugs has demonstrated short-term benefits in patients including histologic improvement, HBeAg seroconversion, suppression of hepatitis B virus (HBV) DNA, and alanine aminotransferase (ALT) normalization. However, long-term therapy is required in most patients and the five approved agents differ with respect to resistance profile and ability to achieve complete antiviral suppression. Lamivudine was the first approved agent, but its use leads to frequent antiviral resistance. Adefovir dipivoxil has a superior first line resistance profile and is fully active against lamivudine-resistant HBV. Newer agents including tenofovir disoproxil fumarate, entecavir, and telbivudine offer greater potency than lamivudine and adefovir dipivoxil. However, telbivudine resistance rates are comparatively high and both telbivudine and entecavir have decreased efficacy against lamivudine-resistant HBV. Tenofovir disoproxil fumarate, the most recently approved nucleotide (2008 in the European Union, and United States), is highly potent in both treatment-naïve and treatment-experienced patients. Overall, this class of compounds presents the opportunity to achieve complete antiviral suppression in the majority of patients, at least in the short-term. The challenge is how to best use these drugs long-term to minimize antiviral resistance and maintain maximal antiviral suppression, which is anticipated to make the greatest impact on limiting advanced complications of CHB.

Analysis of the replication and drug resistance of patient serum hepatitis B virus (HBV) populations can contribute to the therapeutic management of chronic hepatitis B. We developed a procedure for cloning serum HBV quasispecies populations and for phenotypic analysis of the cloned populations for in vitro drug susceptibility. Equivalent sequences were compared to the respective serum HBV DNAs of the cloned quasispecies by population sequencing. Analysis of individual clones revealed that each population contained a diversity of HBV quasispecies. Furthermore, secreted HBV in the supernatant following transfection of the quasispecies populations remained mostly unchanged from the respective input populations. HBV obtained from patients who had developed resistance to adefovir or lamivudine, as demonstrated by development of the rtA181V or rtL180M/M204V mutations in HBV polymerase, respectively, were tested. Phenotypic analysis demonstrated that a population containing the HBV rtA181V mutation showed a 2.9-fold increase in the 50% effective concentration (EC50) for adefovir compared to the wild-type baseline isolate, while the lamivudine-resistant HBV quasispecies population showed a >1,000-fold increase in the lamivudine EC50. In summary, a strategy of cloning full genome HBV quasispecies populations from patient sera was developed, which could provide a useful tool in clinical HBV drug resistance phenotyping and studies of the evolution of clinical viral species.

Background Adefovir dipivoxil is a nucleotide prodrug approved for the treatment of chronic hepatitis B. During clinical trials, ADV-associated mutations were observed in 0, 3, 11, 18 and 29% of patients after 48, 96, 144, 192 and 240 weeks of therapy, respectively. Hepatitis B virus (HBV) polymerase mutations associated with virological breakthrough to ADV include rtA181V and rtN236T, which occur alone or in combination. The rtA181T mutation has also been observed at low frequency, alone or in combination with rtN236T. Methods To investigate the in vitro activity of adefovir and other anti-HBV agents against these mutants, we generated five stable cell lines that each expressed one of the following HBV mutants: rtN236T, rtA181V, rtA181V+rtN236T, rtA181T+rtN236T and rtA181T. Using these cell lines, we quantified in vitro changes in drug susceptibility for eight nucleotide/nucleoside analogues. Results The rtN236T mutant had 7-fold resistance to adefovir but remained sensitive to entecavir, telbivudine and torcitabine (≤3.2-fold reduced susceptibility). The A181V mutant had 4.3-fold resistance to adefovir and had reduced susceptibility to multiple other agents ranging from 3.2-fold (tenofovir) to >191-fold (clevudine). The A181V+rtN236T double mutant was the most highly resistant showing 18-fold resistance to adefovir and higher levels of resistance to other tested drugs with the exception of tenofovir (10-fold reduced susceptibility). Our results and preliminary clinical data suggest that patients with rtN236T or rtA181V remain susceptible to tenofovir, entecavir and lamivudine. Further clinical data are necessary to precisely define in vitro cutoffs indicative of clinically-relevant resistance, particularly for drugs in development such as emtricitabine, telbivudine, torcitabine and clevudine.

Tenofovir disoproxil fumarate (TDF) is a nucleotide analogue approved for treatment of human immunodeficiency virus (HIV) infection. TDF also has been shown in vitro to inhibit replication of wild-type hepatitis B virus (HBV) and lamivudine-resistant HBV mutants and to inhibit lamivudine-resistant HBV in patients and HBV in patients coinfected with the HIV. Data on the in vivo efficacy of TDF against wild-type virus in non-HIV-coinfected or lamivudine-naïve chronic HBV-infected patients are lacking in the published literature. The antiviral effect of oral administration of TDF against chronic woodchuck hepatitis virus (WHV) infection, an established and predictive animal model for antiviral therapy, was evaluated in a placebo-controlled, dose-ranging study (doses, 0.5 to 15.0 mg/kg of body weight/day). Four weeks of once-daily treatment with TDF doses of 0.5, 1.5, or 5.0 mg/kg/day reduced serum WHV viremia significantly (0.2 to 1.5 log reduction from pretreatment level). No effects on the levels of anti-WHV core and anti-WHV surface antibodies in serum or on the concentrations of WHV RNA or WHV antigens in the liver of treated woodchucks were observed. Individual TDF-treated woodchucks demonstrated transient declines in WHV surface antigen serum antigenemia and, characteristically, these woodchucks also had transient declines in serum WHV viremia, intrahepatic WHV replication, and hepatic expression of WHV antigens. No evidence of toxicity was observed in any of the TDF-treated woodchucks. Following drug withdrawal there was prompt recrudescence of WHV viremia to pretreatment levels. It was concluded that oral administration of TDF for 4 weeks was safe and effective in the woodchuck model of chronic HBV infection.

Several next-generation nucleoside and nucleotide analogues are currently in clinical development for the treatment of chronic hepatitis B. However, the efficacy of newer agents against lamivudine-resistant hepatitis B virus (HBV) has not been fully explored. To investigate this in vitro, we generated novel stable cell lines expressing HBV encoding the four major patterns of lamivudine resistance mutations (rtL180M+rtM204V, rtV173L+rtL180M+rtM204V, rtM204I and rtL180M+ rtM204I). Using these cell lines, we assessed the susceptibility of all four strains of lamivudine-resistant HBV to eleven nucleoside analogues in various stages of clinical development. Our studies indicate that lamivudine-resistant HBV remain sensitive to acyclic phosphonate nucleotides (adefovir, tenofovir, and alamifovir), have reduced susceptibility to entecavir, and have high-level cross-resistance to all L-nucleosides tested including emtricitabine, telbivudine, clevudine, and torcitabine.

BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a unique episomal replicative intermediate responsible for persistent infection of hepatocytes. Technical constraints have hampered the direct study of cccDNA maintenance and clearance mechanisms in patients. The aim of this study was to develop a sensitive and specific assay for quantifying cccDNA in biopsy samples from chronic hepatitis B patients during different natural history phases and in patients undergoing antiviral therapy. METHODS: Intrahepatic cccDNA levels were quantified by a specific real-time PCR assay. Ninety-eight liver biopsy samples from patients in the major phases of the natural history of chronic hepatitis B and 32 pairs of samples from patients receiving adefovir dipivoxil (ADV) therapy were assessed. RESULTS: cccDNA was detected, at levels ranging over 3 orders of magnitude, in patients in different phases of the natural history of chronic hepatitis B. cccDNA levels were strongly correlated with levels of total intracellular HBV DNA and serum HBV DNA. Forty-eight weeks of ADV therapy resulted in a significant 0.8 log decrease in cccDNA copies/cell. Changes in cccDNA were correlated with a similar reduction in serum HBsAg titer but not with a decrease in the number of HBV antigen-positive cells during ADV treatment.CONCLUSIONS: cccDNA persists throughout the natural history of chronic hepatitis B, even in patients with serologic evidence of viral clearance. Long-term ADV therapy significantly decreased cccDNA levels by a primarily noncytolytic mechanism.

Analyses of drug susceptibility and replication capacity for clinical HBV isolates have been hampered by the limitations of available in vitro culture systems. Site-directed mutagenesis has been used to study the effects of point mutations in recombinant laboratory HBV strains, however, the validity of such analyses are compromised since mutations are removed from their natural genetic context. Here we report the development of a new plasmid vector that facilitates the cloning and expression of full-length HBV genomes amplified from the sera of chronic hepatitis B patients. Using this vector, we cloned a total of 28 full-length HBV isolates from nine different patients. The majority of cloned HBV genomes ( approximately 70%) replicated in vitro and were suitable for further phenotypic characterization. Adefovir susceptibility was measured for clones from all nine patients. IC(50) values were similar to those previously obtained with standard laboratory HBV strains and did not vary significantly between individual patient isolates (mean IC(50)=0.24+/-0.08 microM). The vector described here enables the efficient phenotypic analysis of full-length HBV isolates from patients and will be useful in future studies including resistance surveillance, cross-resistance analyses, and novel drug-discovery.

Therapy of chronic hepatitis B virus (HBV) infection with the polymerase inhibitor lamivudine frequently is associated with the emergence of viral resistance. Genotypic changes in the YMDD motif (reverse transcriptase [rt] mutations rtM204V/I) conferred resistance to lamivudine as well as reducing the in vitro replication efficiency of HBV. A second mutation, rtL180M, was previously reported to partially restore replication fitness as well as to augment drug resistance in vitro. Here we report the functional characterization of a third polymerase mutation (rtV173L) associated with resistance to lamivudine and famciclovir. rtV173L was observed at baseline in 9 to 22% of patients who entered clinical trials of adefovir dipivoxil for the treatment of lamivudine-resistant HBV. In these patients, rtV173L was invariably found as a third mutation in conjunction with rtL180M and rtM204V. In vitro analyses indicated that rtV173L did not alter the sensitivity of wild-type or lamivudine-resistant HBV to lamivudine, penciclovir, or adefovir but instead enhanced viral replication efficiency. A molecular model of HBV polymerase indicated that residue rtV173 is located beneath the template strand of HBV nucleic acid near the active site of the reverse transcriptase. Substitution of leucine for valine at this residue may enhance polymerization either by repositioning the template strand of nucleic acid or by affecting other residues involved in the polymerization reaction. Together, these results suggest that rtV173L is a compensatory mutation that is selected in lamivudine-resistant patients due to an enhanced replication phenotype.

Background & aims: Adefovir dipivoxil effectively inhibits both hepatitis B virus (HBV) replication and disease activity in patients with chronic hepatitis B. Resistance to treatment was not observed in 2 recent large placebo-controlled 48-week studies with this drug. The aim of this study was to characterize adefovir resistance in a patient who developed clinical and virologic evidence of breakthrough during a 96-week course of treatment. Methods: HBV DNA was PCR amplified and sequenced. Phenotypic studies used patient-derived HBV as well as specific mutations created by site-directed mutagenesis of a HBV/baculovirus recombinant. Results: Following the commencement of treatment with adefovir dipivoxil, the patient initially responded with a 2.4 log 10 decrease in serum HBV DNA and normalization of alanine aminotransaminase levels by week 16. During the second year of treatment, however, serum HBV DNA rose progressively, eventually returning to near-pretreatment levels. This increase in viral replication was associated with a marked increase in alanine aminotransferase and mild changes in bilirubin, albumin, and prothrombin time. Comparison of pretreatment and posttreatment HBV DNA by polymerase chain reaction sequencing identified a novel asparagine to threonine mutation at residue rt236 in domain D of the HBV polymerase. In vitro testing of a laboratory strain encoding the rtN236T mutation and testing of patient-derived virus confirmed that the rtN236T substitution caused a marked reduction in susceptibility to adefovir. Conclusions: The development of this novel mutation in the HBV polymerase confers resistance to adefovir dipivoxil. The patient responded to subsequent lamivudine therapy, achieving normalization of alanine aminotransferase and a significant decrease in serum HBV DNA.

Background & aims: Hepatitis B virus (HBV) genotype may influence disease progression and antiviral response. We therefore analyzed the frequency and distribution of genotypes in patients from 2 multinational phase III studies of adefovir dipivoxil. Antiviral efficacy of adefovir dipivoxil 10-mg therapy was examined with respect to HBV genotype, hepatitis B e antigen (HBeAg) serostatus, and race. Methods: HBV genotypes were assigned by phylogenetic analyses of DNA sequences amplified from baseline serum samples (n = 694). Results: Patients from Asia/Oceania were infected predominantly with genotypes B and C, whereas patients from Western European countries were infected predominantly with genotypes A and D. In Mediterranean countries, genotype D was dominant. The most common genotype in North America was C, followed by A, B, and D. Regardless of location, Asian patients were infected predominantly with genotypes B or C, whereas Caucasian patients were infected predominantly with A or D. There were significant differences in the baseline serum HBV-DNA levels of patients infected with different HBV genotypes regardless of HBeAg serostatus. Forty-eight weeks of adefovir dipivoxil 10-mg therapy resulted in potent reductions in serum HBV DNA with no significant differences based on genotype, HBeAg status, or race; similarly, there was no statistical difference in HBeAg seroconversion rates between genotypes in these patients. Conclusions: HBV genotypes were distributed asymmetrically with respect to race, geography, and HBeAg status. Forty-eight weeks of adefovir dipivoxil therapy resulted in significant decreases in serum HBV-DNA levels in patients regardless of HBV genotype, HBeAg status, or race.

Seven hundred nucleoside treatment-naive patients were enrolled in two phase 3 trials of adefovir dipivoxil (ADV) for the treatment of chronic hepatitis B. To monitor for the emergence of potential adefovir resistance mutations over the first 48 weeks, all intent-to-treat patients (467 ADV-treated and 228 placebo patients) were included in a prospectively defined, treatment-blinded, virology substudy. The study protocol mandated genotypic analysis for all patients with detectable hepatitis B virus (HBV) DNA by Roche Amplicor polymerase chain reaction (PCR) at baseline and week 48, and in vitro phenotypic analyses for patients with conserved site substitutions in HBV polymerase or 1.0 log(10) or greater increase in HBV DNA from nadir. Paired sequences of the entire HBV reverse transcriptase were obtained for 271 ADV-treated and 227 placebo patients by using a sequencing method that detects down to 30% of minor species present within mixtures. Four substitutions (rtS119A, rtH133L, rtV214A, and rtH234Q) developed once each at conserved sites in HBV polymerase in 4 ADV-treated patients. Seven conserved site substitutions developed in 6 placebo patients. HBV mutants encoding the 4 substitutions that emerged in ADV-treated patients remained fully susceptible to adefovir in vitro. Furthermore, these 4 ADV-treated patients had HBV-DNA reductions of 3.3 to 5.9 log(10) copies/mL by week 48 with no rebound. All other substitutions occurred at very low frequencies (

The phenylpropenamide derivatives AT-61 and AT-130 are nonnucleoside analogue inhibitors of hepatitis B virus (HBV) replication. They inhibited the replication of wild-type HBV with 50% inhibitory concentrations of 21.2 ± 9.5 and 2.40 ± 0.92 μM, respectively, compared to 0.064 ± 0.020 μM lamivudine. There were no significant differences in sensitivity between wild-type and nucleoside analogue-resistant (rtL180M, rtM204I, and rtL180M + rtM204V) HBV.

Current therapies for chronic hepatitis B virus (HBV) infection do not provide adequate long-term control of viral replication in the majority of patients. Monotherapy with nucleoside analogs, such as lamivudine and famciclovir, is effective for short periods but results in the emergence of drug-resistant HBV in a substantial number of patients within 1 year of therapy. Adefovir dipivoxil (ADV) has demonstrated clinical activity against wild-type and lamivudine-resistant HBV, but it is unclear whether resistance mutations will emerge after long-term therapy with this drug. To determine whether extended treatment with ADV led to the emergence of drug-resistant populations of HBV, we analyzed virus isolated from patients currently enrolled in a long-term open-label study. The reverse transcriptase domain of HBV polymerase was amplified and sequenced from patients that had received a cumulative exposure of up to 60 weeks of ADV. During our analyses, several previously unreported amino acid substitutions were observed in the reverse transcriptase domain of HBV. Importantly, none of the observed mutations occurred in more than 1 patient, nor were they associated with an adefovir-resistant phenotype in vitro. Furthermore, none of the patients from whom these mutant viruses were isolated had evidence of virologic rebound. In conclusion, these results, although based on a limited number of patients, suggest that treatment with ADV does not lead to the emergence of resistant virus after up to 60 weeks of therapy.

Despite the availability of prophylactic vaccines lamivudine and IFN-alpha, chronic hepatitis B remains an enormous global health problem. Several promising nucleosides/nucleotides are undergoing clinical trials, including adefovir dipivoxil, the latter of which is active against lamivudine-resistant hepatitis B virus (HBV). In addition to nucleosides/nucleotides, it will be important to develop new agents with different modes of action. Novel small molecule inhibitors, as well as gene therapy approaches, have produced encouraging results in vitro and in animal models. Additional immunomodulatory therapies, including thymosin-alpha 1, IL-12 and several therapeutic vaccines, are also being explored. Combination therapy with multiple nucleosides/nucleotides and other agents will play an important role in the treatment of hepatitis and may help achieve complete viral suppression, host-mediated elimination of infected cells and lasting immunity.

The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known. In this study we characterized viral kinetics and the host response during HBV infection of primary human hepatocytes (PHH) to address these unresolved questions. We determined that Smc5/6 localizes with Nuclear Domain 10 (ND10) in PHH. Co-localization has functional implications since depletion of ND10 structural components alters the nuclear distribution of Smc6 and induces HBV gene expression in the absence of HBx. We also found that HBV infection and replication does not induce a prominent global host transcriptional response in PHH, either shortly after infection when Smc5/6 is present, or at later times post-infection when Smc5/6 has been degraded. Notably, HBV and an HBx-negative virus establish high level infection in PHH without inducing expression of interferon-stimulated genes or production of interferons or other cytokines. Our study also revealed that Smc5/6 is degraded in the majority of infected PHH by the time cccDNA transcription could be detected and that HBx RNA is present in cell culture-derived virus preparations as well as HBV patient plasma. Collectively, these data indicate that Smc5/6 is an intrinsic antiviral restriction factor that suppresses HBV transcription when localized to ND10 without inducing a detectable innate immune response. Our data also suggest that HBx protein may be initially expressed by delivery of extracellular HBx RNA into HBV-infected cells.