Danni Colledge

Phenylpropenamide Derivatives AT-61 and AT-130 Inhibit Replication of Wild-Type and Lamivudine-Resistant Strains of Hepatitis B Virus In Vitro

ABSTRACT 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.

Cross-Resistance Testing of Antihepadnaviral Compounds Using Novel Recombinant Baculoviruses Which Encode Drug-Resistant Strains of Hepatitis B Virus

ABSTRACT Long-term nucleoside analog therapy for hepatitis B virus (HBV)-related disease frequently results in the selection of mutant HBV strains that are resistant to therapy. Molecular studies of such drug-resistant variants are clearly warranted but have been difficult to do because of the lack of convenient and reliable in vitro culture systems for HBV. We previously developed a novel in vitro system for studying HBV replication that relies on the use of recombinant baculoviruses to deliver greater than unit length copies of the HBV genome to HepG2 cells. High levels of HBV replication can be achieved in this system, which has recently been used to assess the effects of lamivudine on HBV replication and covalently closed circular DNA accumulation. The further development of this novel system and its application to determine the cross-resistance profiles of drug-resistant HBV strains are described here. For these studies, novel recombinant HBV baculoviruses which encoded the L526M, M550I, and L526M M550V drug resistance mutations were generated and used to examine the effects of these substitutions on viral sensitivity to lamivudine, penciclovir (the active form of famciclovir), and adefovir, three compounds of clinical importance. The following observations were made: (i) the L526M mutation confers resistance to penciclovir and partial resistance to lamivudine, (ii) the YMDD mutations M550I and L526M M550V confer high levels of resistance to lamivudine and penciclovir, and (iii) adefovir is active against each of these mutants. These findings are supported by the limited amount of clinical data currently available and confirm the utility of the HBV-baculovirus system as an in vitro tool for the molecular characterization of clinically significant HBV strains.

In vitro antihepadnaviral activities of combinations of penciclovir, lamivudine, and adefovir

ABSTRACT Penciclovir {9-[2-hydroxy-1-(hydroxymethyl)-ethoxymethyl]guanine [PCV]}, lamivudine ([−]-β-l-2′,3′-dideoxy-3′-thiacytidine [3TC]), and adefovir (9-[2-phosphonylmethoxyethyl]-adenine [PMEA]) are potent inhibitors of hepatitis B virus (HBV) replication. Lamivudine has recently received approval for clinical use against chronic human HBV infection, and both PCV and PMEA have undergone clinical trials against HBV in their respective prodrug forms {famciclovir and adefovir dipivoxil [bis-(POM)-PMEA]}. Since multidrug combinations are likely to be used to control HBV infection, investigation of potential interactions between PCV, 3TC, and PMEA is important. Primary duck hepatocyte cultures which were either acutely or congenitally infected with the duck hepatitis B virus (DHBV) were used to investigate in vitro interactions between PCV, 3TC, and PMEA. Here we show that the anti-DHBV effects of all the combinations containing PCV, 3TC, and PMEA are greater than that of each of the individual components and that their combined activities are approximately additive or synergistic. These results may underestimate the potential in vivo usefulness of PMEA-containing combinations, since there is evidence that PMEA has immunomodulatory activity and, at least in the duck model of chronic HBV infection, is capable of inhibiting DHBV replication in cells other than hepatocytes, the latter being unaffected by treatment with either PCV or 3TC. Further investigation of the antiviral activities of these drug combinations is therefore required, particularly since each of the component drugs is already in clinical use.

Stimulation of the interleukin-1 receptor and Toll-like receptor 2 inhibits hepatitis B virus replication in hepatoma cell lines in vitro.

BACKGROUND Toll-like receptors (TLRs) are a key component of the innate immune system and TLR2 has been shown to be involved in the immunopathogenesis of hepatitis B virus (HBV) infection in vivo. We investigated the role of TLR2 stimulation of virus-infected hepatocyte cell lines as a potential antiviral mechanism in vitro. METHODS The hepatoblastoma cell line HepG2 was transduced with recombinant HBV baculoviruses and the hepatoma cell line Huh-7 was transiently transfected with complimentary DNA clones of HBV. HBV viral replication was quantified after stimulation with interleukin (IL)-1beta and Pam-2-Cys, a synthetic TLR2 ligand, by measuring intracellular core-associated single-stranded HBV DNA using Southern blot hybridization, as well as viral nucleocapsid formation using a non-denaturing immunoblot method. RESULTS Stimulation of both cell lines in vitro with IL-1beta and Pam-2-Cys, both known to induce expression of the pro inflammatory cytokines tumour necrosis factor-alpha and IL-8 via a nuclear factor-kappaB dependent pathway, resulted in the inhibition of HBV DNA replication in the transduced HepG2 cells by up to 90% and nucleocapsid formation in the transiently transfected Huh-7 cells by up to 30%, when compared with mock-treated cells. CONCLUSIONS Hepatoma cell lines expressed functional IL-1 receptor and TLR2 receptors, which when stimulated led to a signalling cascade that inhibited HBV replication. These data support an active role for hepatocytes in inhibiting HBV replication and provide a rationale for the development of TLR agonists as potentially novel antiviral agents.

Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody.

The Hepatitis B virus precore protein is processed in the endoplasmic reticulum (ER) into secreted hepatitis B e antigen (HBeAg), which acts as an immune tolerogen to establish chronic infection. Downregulation of secreted HBeAg should improve clinical outcome, as patients who effectively respond to current treatments (IFN-α) have significantly lower serum HBeAg levels. Here, we describe a novel reagent, a single variable domain (V(NAR)) of the shark immunoglobulin new antigen receptor (IgNAR) antibodies. V(NAR)s possess advantages in stability, size (~14 kDa) and cryptic epitope recognition compared to conventional antibodies. The V(NAR) domain displayed biologically useful affinity for recombinant and native HBeAg, and recognised a unique conformational epitope. To assess therapeutic potential in targeting intracellular precore protein to reduce secreted HBeAg, the V(NAR) was engineered for ER-targeted in vitro delivery to function as an intracellular antibody (intrabody). In vitro data from HBV/precore hepatocyte cell lines demonstrated effective intrabody regulation of precore/HBeAg.

Identification of a novel hepatitis B virus precore/core deletion mutant in HIV/hepatitis B virus co-infected individuals.

Objectives:Although HAART has resulted in improved health outcomes for most HIV-infected individuals, liver failure has emerged as a major cause of morbidity and mortality in people co-infected with hepatitis B virus (HBV). In HBV mono-infected individuals, core deletion mutants are associated with more aggressive liver disease. As HIV accelerates HBV liver disease progression, we hypothesized that HIV-HBV co-infected individuals have increased frequency of core mutations including deletions. To test this hypothesis, we have analysed genome-length sequences of HBV DNA from patients both prior to and during antiviral therapy. Setting:Prospective HIV/HBV co-infected cohort study. Methods:Genomic length HBV DNA was amplified by PCR from the serum samples of ten HIV/HBV co-infected individuals and five HBV mono-infected individuals prior to the commencement of lamivudine therapy and again after nine to 74 months of treatment. The complete genomes were sequenced and in order to further analyse some mutations, their frequency was determined in additional HIV/HBV co-infected and HBV mono-infected individuals. Results:A novel –1G mutation was identified in the HBV precore and overlapping core genes that truncated the deduced precore/core proteins. The mutant genome was the dominant species in some HIV/HBV co-infected individuals and was more prevalent in HIV/HBV co-infected individuals than HBV mono-infected individuals. The mutation was also associated with high HBV DNA concentrations in HIV/HBV co-infected individuals. Additional mutations were identified in the core/precore and polymerase genes and regulatory regions. Conclusion:Mutations in the HBV core and precore genes may be contributing to disease pathogenesis in HIV/HBV co-infected individuals.

Downregulation of Interleukin-18-Mediated Cell Signaling and Interferon Gamma Expression by the Hepatitis B Virus e Antigen

ABSTRACT The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection. IMPORTANCE It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.

The hepatitis B e antigen suppresses IL-1β-mediated NF-κB activation in hepatocytes

Summary.  Previous clinical studies have demonstrated an association between the hepatitis B e antigen and Toll‐like receptor (TLR) expression and signalling. Therefore, the aim of this study was to develop an in vitro assay to measure the effect of hepatitis B virus proteins, including the precore protein, on signalling mediated by members of the Toll‐like/interleukin 1 (TIR) superfamily, by measuring NF‐κB promoter activity. The basal level of NF‐κB reporter activity was measured in three hepatocyte cell lines (Huh7, HepG2 and PH5CH8) and one kidney cell line (HEK293) using a luciferase assay. All cell lines were virtually refractory to stimulation with lipopolysaccharide; however, PH5CH8 cells had a robust activation of NF‐κB in response to IL‐1β stimulation, with ∼40‐fold higher activation than the unstimulated control, a higher degree of activation than that observed in either Huh7 and HepG2, or HEK293 and HEK293‐TLR2 cells. In PH5CH8 cells transfected with pCI expression constructs and stimulated with IL‐1β, we showed that the precursor form of the precore protein, p25, inhibits NF‐κB activation by up to 30% and the cytosolic form, p22, inhibits NF‐κB activation by 70%. The core protein, p21, which shares significant homology with the precore protein except for a 10‐amino acid extension at the N‐terminus, had no effect on NF‐κB activation. We hypothesize that the inhibition of IL‐1β‐mediated NF‐κB activation by the precore protein may be a mechanism that allows the virus to persist, suggesting a role for the pool of precore protein that remains intracellular.

Deep sequencing shows that HBV basal core promoter and precore variants reduce the likelihood of HBsAg loss following tenofovir disoproxil fumarate therapy in HBeAg-positive chronic hepatitis B.

Objective Hepatitis B e antigen (HBeAg) seroconversion and hepatitis B surface antigen (HBsAg) loss are important clinical outcomes for patients with chronic hepatitis B (CHB) treated with antiviral therapy. To date, there have been few studies that have evaluated viral sequence markers predicting serological response to nucleos(t)ide analogue (NA) treatment. Design We used next-generation sequencing (NGS) and quantitative HBV serology (HBeAg and HBsAg) to identify viral sequence markers associated with serological response to long-term tenofovir disoproxil fumarate therapy among HBeAg-positive patients. In the GS-US-174-0103 study, approximately half the patients seroconverted to anti-HBe by week 192 and 11% of patients exhibited HBsAg loss, the closest outcome to functional cure. The frequency of HBV variants that have previously been associated with HBV clinical outcomes was evaluated. HBV viral diversity in baseline sequences generated by NGS was calculated using Shannon entropy. Results NGS analysis of HBV sequences from 157 patients infected with genotypes A to D showed the frequency of variants in the basal core promoter (BCP) and precore (PC) regions varied by genotype and that these mutations were associated with the absence of HBsAg loss. This was the case even when mutations were present at frequencies below the threshold of detection by population sequencing. Increased viral diversity across the HBV genome as determined by NGS was also associated with reduced likelihood of HBsAg loss. Conclusion Patients with detectable BCP and/or PC variants and higher viral diversity have a lower probability of HBsAg loss during long-term NA therapy. Strategies to achieve functional cure of HBV infection through combination therapy should consider using NGS to stratify patients according to BCP/PC sequence. Consideration should also be given to earlier initiation of therapy prior to the emergence of BCP/PC variants. Trial registration number NCT00116805; Post result.

Single-molecule sequencing reveals complex genomic variation of hepatitis B virus during 15 years of chronic infection following liver transplantation.

ABSTRACT Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV), replicates via an RNA intermediate and is error prone, leading to the rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome generated via splicing (spHBV variants). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. spHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from samples, including the liver explant, longitudinally collected from a subject with CHB over a 15-year period after liver transplantation. By employing novel bioinformatics methods, this analysis showed that the dynamics of the viral population across a period of changing treatment regimens was complex. The spHBV variants detected in the liver explant remained present posttransplantation, and a highly diverse novel spHBV population as well as variants with multiple deletions in the pre-S genes emerged. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occurred with known drug resistance-associated mutations highlights the relevance of using full-genome deep sequencing and supports the hypothesis that drug resistance involves interactions across the full length of the HBV genome. IMPORTANCE Single-molecule sequencing allowed the characterization, in unprecedented detail, of the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimens. This analysis also showed the rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open-source bioinformatics tools with the capacity to easily identify potential spliced variants from deep sequencing data are freely available.