A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients.
Chronic hepatitis B virus (HBV) infection is one of the major causes of liver cirrhosis and liver cancer worldwide. Recommended treatment regimens of chronic hepatitis B based on interferon alpha and nucleot(s)ide analogues do not lead to the satisfactory results. Over the last 20 years, continuous efforts have been undertaken to develop new immunotherapeutic approaches for the treatment of chronic hepatitis B, however, without satisfactory results. We proposed here that the combination of potent antivirals with a prime-boost vaccination protocol that is inducing appropriate virus-specific T-cell responses may restore immune control over HBV. To test this hypothesis we performed a proof-of-principle experiment using woodchucks, a widely accepted animal model of chronic HBV infection. We pretreated animals with entecavir to suppress viral replication and immunized them by a prime-boost regimen with DNA vaccines expressing woodchuck hepatitis virus (WHV) surface and core antigens and adenoviral vectors expressing WHV core antigen. Consistent with our hypothesis, the combination therapy achieved a stronger antiviral effect than the monotherapy alone, leading to sustained immunological control of chronic WHV infection and viral clearance in some animals. These data are encouraging and implicate the feasibility and usefulness of the immunotherapeutic strategies for the treatment of chronically HBV-infected patients.
Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.
Chronic hepatitis B virus (HBV) infection is still one of the major public health problems. Two billion people worldwide have been infected with HBV, of whom more than 360 million developed chronic infection. Every year, approximately one million of these individuals will die from HBV-associated liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). Treatment of chronic hepatitis B remains a clinical challenge, and alternative strategies to treat chronic HBV infection are urgently needed. Here, we designed a new combination strategy to enhance the patient's own antiviral immune response and to achieve long-term viral suppression. The therapeutic effect of our combination therapy strategy for chronic hepadnaviral infection was tested in the woodchuck model. We demonstrated that our novel combination therapy could elicit potent antiviral immune response and achieved a strong antiviral effect, leading to sustained immunological control of chronic hepadnaviral infection and complete viral clearance in treated woodchucks. The results of this study may have an impact on clinical trials of the immunotherapy in chronically HBV-infected patients.
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.
Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.
MIV-210 is a prodrug of 3′-fluoro-2′,3′-dideoxyguanosine with high oral bioavailability in humans and potent activity against hepatitis B virus (HBV). Woodchucks infected with woodchuck hepatitis virus (WHV) represent an accurate model of HBV infection that is utilized for evaluation of the efficacy and safety of novel anti-HBV agents. Oral administration of MIV-210 at 20 or 60 mg/kg of body weight/day induced a rapid virological response in chronically infected woodchucks, reducing serum WHV DNA levels by 4.75 log10 and 5.72 log10, respectively, in 2 weeks. A progressive decline in WHV viremia occurred throughout the 10-week therapy, giving final reductions of 7.23 log10 and 7.68 log10 in the 20- and 60-mg/kg/day groups, respectively. Further, a daily dose of 10 mg/kg decreased the serum WHV load 400-fold after 4 weeks of treatment, and a dose of 5 mg/kg/day was sufficient to maintain this antiviral effect during the following 6-week period. MIV-210 at 20 or 60 mg/kg/day reduced the liver WHV DNA load 200- to 2,500-fold from pretreatment levels and, importantly, led to a 2.0 log10 drop in the hepatic content of WHV covalently closed circular DNA. The treatment with 60 mg/kg/day was well tolerated. Liver biopsy specimens obtained after the 10-week treatment with 20 or 60 mg/kg/day and after the 10-week follow-up showed hepatocyte and mitochondrial ultrastructures comparable to those in the placebo-treated group. It was concluded that MIV-210 is highly effective against chronic WHV infection. These findings, together with the previously demonstrated inhibitory activity of MIV-210 against lamivudine-, adefovir-, and entecavir-resistant HBV variants, make MIV-210 a highly valuable candidate for further testing as an agent against chronic hepatitis B.
Adefovir dipivoxil (ADV) and tenofovir disoproxil fumarate (TDF) are nucleotide analogs that inhibit the replication of wild-type hepatitis B virus (HBV) and lamivudine (3TC)-resistant virus in HBV-infected patients, including those who are coinfected with human immunodeficiency virus. The combination of ADV or TDF with other nucleoside analogs is a proposed strategy for managing antiviral drug resistance during the treatment of chronic HBV infection. The antiviral effect of oral ADV or TDF, alone or in combination with 3TC or emtricitabine (FTC), against chronic woodchuck hepatitis virus (WHV) infection was evaluated in a placebo-controlled study in the woodchuck, an established and predictive model for antiviral therapy. Once-daily treatment for 48 weeks with ADV plus 3TC or TDF plus FTC significantly reduced serum WHV viremia levels from the pretreatment level by 6.2 log10 and 6.1 log10 genome equivalents/ml serum, respectively, followed by TDF plus 3TC (5.6 log10 genome equivalents/ml), ADV alone (4.8 log10 genome equivalents/ml), ADV plus FTC (one survivor) (4.4 log10 genome equivalents/ml), TDF alone (2.9 log10 genome equivalents/ml), 3TC alone (2.7 log10 genome equivalents/ml), and FTC alone (2.0 log10 genome equivalents/ml). Individual woodchucks across all treatment groups also demonstrated pronounced declines in serum WHV surface antigen, characteristically accompanied by declines in hepatic WHV replication and the hepatic expression of WHV antigens. Most woodchucks had prompt recrudescence of WHV replication after drug withdrawal, but individual woodchucks across treatment groups had sustained effects. No signs of toxicity were observed for any of the drugs or drug combinations administered. In conclusion, the oral administration of 3TC, FTC, ADV, and TDF alone and in combination was safe and effective in the woodchuck model of HBV infection.
A novel l-nucleoside analog of deoxycytidine, 2′,3′-dideoxy-2′,3′-didehydro-β-l-5-fluorocytidine (β-l-Fd4C), was recently shown to strongly inhibit hepatitis B virus (HBV) replication in the 2.2.15 cell line. Therefore, its antiviral activity was evaluated in the duck HBV (DHBV) infection model. Using a cell-free system for the expression of the DHBV polymerase, β-l-Fd4C-TP exhibited a concentration-dependent inhibition of dCTP incorporation into viral minus-strand DNA with a 50% inhibitory concentration of 0.2 μM which was lower than that of other tested deoxycytidine analogs, i.e., lamivudine-TP, ddC-TP, and β-l-FddC-TP. Further analysis showed that β-l-Fd4C-TP is likely to be a competitive inhibitor of dCTP incorporation and to cause premature DNA chain termination. In primary duck hepatocyte cultures infected in vitro, β-l-Fd4C administration exhibited a long-lasting inhibitory effect on viral DNA synthesis but could not clear viral covalently closed circular DNA (CCC DNA). Results of short-term antiviral treatment in experimentally infected ducklings showed that β-l-Fd4C exhibited the most potent antiviral effect, followed by β-l-FddC, lamivudine, and ddC. Longer administration of β-l-Fd4C induced a sustained suppression of viremia (>95% of controls) and of viral DNA synthesis within the liver. However, the persistence of trace amounts of viral CCC DNA detected only by PCR was associated with a recurrence of viral replication after drug withdrawal. In parallel, β-l-Fd4C treatment suppressed viral antigen expression within the liver and decreased intrahepatic inflammation and was not associated with any sign of toxicity. Our data, therefore, demonstrate that in the duck model of HBV infection, β-l-Fd4C is a potent inhibitor of DHBV reverse transcriptase activity in vitro and suppresses viral replication in the liver in vivo.
The woodchuck (Marmota monax) has proven to be a suitable animal model for studying hepatitis B virus (HBV) infection owing to similarities in the course of infection between woodchuck hepatitis virus (WHV) in woodchucks and HBV in humans. (-)-beta-L-2',3'-Dideoxy-3'-thiacytidine (3TC; lamivudine) is a nucleoside analog which has demonstrated antiviral activity against HBV as well as human immunodeficiency virus (HIV). The purpose of the present investigation was to characterize the pharmacokinetics of 3TC following intravenous and oral administration of 20 mg of 3TC per kg of body weight to woodchucks. Following intravenous administration, the concentrations of 3TC in plasma declined, with a terminal half-life of 2.84 +/- 0.85 h (mean +/- standard deviation). The systemic clearance and steady-state volume of distribution of 3TC were 0.22 +/- 0.078 liters/h/kg and 0.75 +/- 0.13 liters/kg, respectively. The renal clearance of the nucleoside analog was 0.063 +/- 0.016 liters/h/kg. The oral bioavailability of 3TC ranged from 18 to 54%. Allometric relationships between pharmacokinetic parameters and body weight developed by Hussey et al. (E.K. Hussey, K.H. Donn, M.J. Daniel, S.T. Hall, A.J. Harker, and G.L. Evans, J. Clin. Pharmacol. 34:975-977, 1994) were augmented by including data from woodchucks, monkeys (S.M. Blaney, M.J. Daniel, A.J. Harker, K. Godwin, and F.M. Balis, Antimicrob. Agents Chemother. 39:2779-2782, 1995), and additional data from rats (P. Rajagopalan, L. Moore, C.K. Chu, R.F. Schinazi, and F.D. Boudinot, submitted for publication). Interspecies scaling of the pharmacokinetic parameters of 3TC demonstrated a good correlation between clearance (0.74 . W0.76 [where W is body weight]; r = 0.93; P < 0.025), apparent volume of distribution (1.62 . W0.81; r = 0.98; P < 0.005), and steady-state volume of distribution (1.09 . W0.94; r = 0.99; P < 0.05) and species body weight. The allometric relationships for clearance and volume of distribution at steady state predicted the observed pharmacokinetic parameters in humans quite well; however, the apparent volume of distribution was underestimated in humans. Thus, the pharmacokinetic data obtained with the woodchuck HBV animal model should be useful for designing clinical trials.
Treatment of hepatitis B virus carriers with the nucleoside analog lamivudine suppresses virus replication. However, rather than completely eliminating the virus, long-term treatment often ends in the outgrowth of drug-resistant variants. Using woodchucks chronically infected with woodchuck hepatitis virus (WHV), we investigated the consequences of combining lamivudine treatment with immunotherapy mediated by an adenovirus superinfection. Eight infected woodchucks were treated with lamivudine and four were infected with ∼1013 particles of an adenovirus type 5 vector expressing β-galactosidase. Serum samples and liver biopsies collected following the combination therapy revealed a 10- to 20-fold reduction in DNA replication intermediates in three of four woodchucks at 2 weeks after adenovirus infection. At the same time, covalently closed circular DNA (cccDNA) and viral mRNA levels both declined about two- to threefold in those woodchucks, while mRNA levels for gamma interferon and tumor necrosis factor alpha as well as for the T-cell markers CD4 and CD8 were elevated about twofold. Recovery from adenovirus infection was marked by elevation of sorbitol dehydrogenase, a marker for hepatocyte necrosis, as well as an 8- to 10-fold increase in expression of proliferating cell nuclear antigen, a marker for DNA synthesis, indicating significant hepatocyte turnover. The fact that replicative DNA levels declined more than cccDNA and mRNA levels following adenovirus infection suggests that the former decline either was cytokine induced or reflects instability of replicative DNA in regenerating hepatocytes. Virus titers in all four woodchucks were only transiently suppressed, suggesting that the effect of combination therapy is transient and, at least under the conditions used, does not cure chronic WHV infections.
Emtricitabine [(−)FTC] [(−)-β-2′,3′-dideoxy-5-fluoro-3′-thiacytidine] has been shown to be an effective inhibitor of hepatitis B virus (HBV) in cell culture, with a potency and selectivity that are essentially identical to those of lamivudine. The antiviral activity of oral administration of (−)FTC against WHV replication in chronically infected woodchucks, an established and predictive model for antiviral therapy against HBV, was examined in a placebo-controlled study. (−)FTC significantly reduced viremia and intrahepatic WHV replication in a dose-dependent manner that was comparable to the antiviral activity of lamivudine observed in previous studies conducted by our laboratories. No effect on the levels of hepatic WHV RNA or the levels of woodchuck hepatitis surface antigen or anti-woodchuck hepatitis surface and core antibodies in the serum of the treated animals was observed. No evidence of drug-related toxicity was observed in any of the animals treated.
(−)-β-d-2-Aminopurine dioxolane (APD) is a nucleoside prodrug that is efficiently converted to 9-(β-d-1,3-dioxolan-4-yl)guanine (DXG). DXG has antiviral activity in vitro against hepatitis B virus (HBV) but limited aqueous solubility, making it difficult to administer orally to HBV-infected individuals. APD is more water soluble than DXG and represents a promising prodrug for the delivery of DXG. A placebo-controlled, dose-ranging efficacy and pharmacokinetic study was conducted with woodchucks that were chronically infected with woodchuck hepatitis virus (WHV). APD was efficiently converted to DXG after oral and intravenous administrations of APD, with serum concentrations of DXG being higher following oral administration than following intravenous administration, suggestive of a considerable first-pass intestinal and/or hepatic metabolism. APD administered orally at 1, 3, 10, and 30 mg/kg of body weight per day for 4 weeks produced a dose-dependent antiviral response. Doses of 3 and 10 mg/kg/day reduced serum WHV viremia by 0.4 and 0.7 log10 copies/ml, respectively. The 30-mg/kg/day dose resulted in a more pronounced, statistically significant decline in serum WHV viremia of 1.9 log10 copies/ml and was associated with a 1.5-fold reduction in hepatic WHV DNA. Individual woodchucks within the highest APD dose group that had declines in serum WHV surface antigen levels, WHV viremia, and hepatic WHV DNA also had reductions in hepatic WHV RNA. There was a prompt recrudescence of WHV viremia following drug withdrawal. Therefore, oral administration of APD for 4 weeks was safe in the woodchuck model of chronic HBV infection, and the effect on serum WHV viremia was dose dependent.
Lamivudine [(−)-β-l-2′,3′-dideoxy-3′-thiacytidine] reduces woodchuck hepatitis virus (WHV) titers in the sera of chronically infected woodchucks by inhibiting viral DNA synthesis. However, after 6 to 12 months, WHV titers begin to increase toward pretreatment levels. Three WHV variants with mutations in the active site of the DNA polymerase gene are present at this time (W. S. Mason et al., Virology 245:18–32, 1998). We have asked if these mutant viruses were responsible for the lamivudine resistance and if their emergence caused an immediate rise in virus titers. Cell cultures studies implied that the mutants were resistant to lamivudine. Emergence of mutant WHV was not always associated, however, with an immediate rise in virus titers in the serum. One of the three types of mutant viruses became prominent in serum up to 7 months before titers in serum actually began to increase, at a time when wild-type virus was still predominant in the liver. The two other mutants did not show this behavior but were detected in serum and liver later, just at the time that virus titers began to rise. A factor linking all three mutants was that a similar duration of drug administration preceded the rise in titers, irrespective of which mutant ultimately prevailed. A simple explanation for these results is that the increase in virus titers following emergence of drug-resistant mutants can occur only as the preexisting wild-type virus is cleared from the hepatocyte population, allowing spread of the mutants. Thus, prolonged suppression of virus titers in the serum may sometimes be a measure of the stability of hepatocyte infection rather than of a successful therapeutic outcome.
Administration of either lamivudine (2′-deoxy-3′-thiacytidine) or l-FMAU (2′-fluoro-5-methyl-β-l-arabinofuranosyluracil) to woodchucks chronically infected with woodchuck hepatitis virus (WHV) induces a transient decline in virus titers. However, within 6 to 12 months, virus titers begin to increase towards pretreatment levels. This is associated with the emergence of virus strains with mutations of the B and C regions of the viral DNA polymerase (T. Zhou et al., Antimicrob. Agents Chemother. 43:1947–1954, 1999; Y. Zhu et al., J. Virol. 75:311–322, 2001). The present study was carried out to determine which of the mutants that we have identified conferred resistance to lamivudine and/or to l-FMAU. When inserted into a laboratory strain of WHV, each of the mutations, or combinations of mutations, of regions B and C produced a DNA replication-competent virus and typically conferred resistance to both nucleoside analogs in cell culture. Sequencing of the polymerase active site also occasionally revealed other mutations, but these did not appear to contribute to drug resistance. Moreover, in transfected cells, most of the mutants synthesized viral DNA nearly as efficiently as wild-type WHV. Computational models suggested that persistence of several of the WHV mutants as prevalent species in the serum and, by inference, liver for up to 6 months following drug withdrawal required a replication efficiency of at least 10 to 30% of that of the wild type. However, their delayed emergence during therapy suggested replication efficiency in the presence of the drug that was still well below that of wild-type WHV in the absence of the drug.
The (-) enantiomer of cis-5-fluoro-1l-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine [(-)-FTC)], a substituted oxathiolane compound with anti-hepatitis B virus activity in vitro, was assessed for its efficacy in woodchucks with naturally acquired woodchuck hepatitis virus (WHV) infection. Pharmacokinetics and in vitro anabolism were also determined. (-)-FTC was anabolized to the 5'-triphosphate in a dose-related fashion, reaching a maximum concentration at about 24 h in cultured woodchuck hepatocytes. Following administration of a dose of 10 mg/kg of body weight intraperitoneally (i.p.), the clearance of (-)-FTC from plasma was monoexponential, the terminal half-life was 3.76 +/- 1.4 h, and the systemic clearance was 0.12 +/- 0.06 liters/h/kg. The antiviral efficacy of (-)-FTC in the woodchuck model was assessed by quantitation of serum WHV DNA levels and by WHV particle-associated DNA polymerase activity at two dosages, 30 and 20 mg/kg given i.p. twice daily (b.i.d.), respectively. The level of WHV DNA in serum was reduced 20- to 150-fold (average, 56-fold) in the 30-mg/kg-b.i.d. treatment group and 6- to 49-fold (average, 27-fold) in the 20-mg/kg-b.i.d. treatment group. Viral DNA polymerase levels diminished accordingly. One week after treatment was discontinued, WHV levels returned to pretreatment levels in both studies. These animals were biopsied before and following treatment with 30 mg of (-)-FTC per kg. Their livers were characterized by a mild increase in cytoplasmic lipid levels, but this change was not associated with altered liver enzyme levels. Serum chemistry and hematology results were within the normal ranges for all treated animals. We conclude that (-)-FTC is a potent antihepadnaviral agent and that it has no detectable toxic effects in woodchucks when given for up to 25 days. Further development of (-)-FTC as an anti-hepatitis B virus therapy for patients is warranted.
Our aim was to evaluate the antiviral effect of a combination of two nucleoside reverse transcriptase inhibitors, emtricitabine (FTC) and clevudine (L-FMAU), with the addition of an adenovirus-driven delivery of recombinant gamma interferon (IFN-γ) in the woodchuck model of hepatitis B virus infection. Six woodchuck hepatitis virus (WHV)-infected woodchucks received L-FMAU (10 mg/kg) plus FTC (30 mg/kg) intraperitoneally for 8 weeks; six other animals received in addition an intravenous injection of a recombinant adenovirus vector expressing woodchuck IFN-γ (Ad-IFN) at weeks 4 and 8. In the control group, two animals received Ad-IFN alone, two received adenovirus vector expressing the green fluorescent protein reporter gene, and one remained untreated. In less than 2 weeks, all woodchucks that received L-FMAU plus FTC showed a rapid and marked inhibition of viral replication, with a 4-log10 drop in serum WHV DNA. In two animals, viremia remained suppressed for several months after the end of treatment. Similarly, a dramatic decrease in intrahepatic replicative intermediates of viral DNA was observed in the L-FMAU/FTC-treated groups. The additional administration of Ad-IFN led to increased inflammation in the liver but did not enhance the antiviral effect of the L-FMAU/FTC combination. In conclusion, therapies combining L-FMAU and FTC in WHV-infected woodchucks resulted in a potent and sustained antihepadnaviral effect both in the liver and in the blood circulation. However, no extra benefit of adding IFN-γ gene transduction to the L-FMAU/FTC combination could be detected.
Specific activation of T cells appears to be a prerequisite for viral clearance during hepatitis B virus (HBV) infection. The T-cell response to HBV core protein is essential in determining an acute or chronic outcome of HBV infection, but how this immune response contributes to the course of infection remains unclear. This is due to results obtained from humans, which are restricted to phenomenological observations occurring during the clinical onset after HBV infection. Thus, a useful animal model is needed. Characterization of the T-cell response to the core protein (WHcAg) of woodchuck hepatitis virus (WHV) in woodchucks contributes to the understanding of these mechanisms. Therefore, we investigated the response of woodchuck peripheral blood mononuclear cells (PBMCs) to WHcAg and WHcAg-derived peptides, using our 5-bromo-2'-deoxyuridine assay. We demonstrated WHcAg-specific proliferation of PBMCs and nylon wool-nonadherent cells from acutely WHV-infected woodchucks. Using a cross-reacting anti-human T-cell (CD3) antiserum, we identified nonadherent cells as woodchuck T cells. T-cell epitope mapping with overlapping peptides, covering the entire WHcAg, revealed T-cell responses of acutely WHV-infected woodchucks to peptide1-20, peptide100-119, and peptide112-131. Detailed epitope analysis in the WHcAg region from amino acids 97 to 140 showed that T cells especially recognized peptide97-110. Establishment of polyclonal T-cell lines with WHcAg or peptide97-110 revealed reciprocal stimulation by peptide97-110 or WHcAg, respectively. We vaccinated woodchucks with peptide97-110 or WHcAg to prove the importance of this immunodominant T-cell epitope. All woodchucks immunized with peptide97-110 or WHcAg were protected. Our results show that the cellular immune response to WHcAg or to one T-cell epitope protects woodchucks from WHV infection.
Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 1010 viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-γ) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-γ in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 1010 vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers.
Transient hepadnavirus infections can involve spread of virus to the entire hepatocyte population. In this situation hepatocytes present following recovery are derived from infected hepatocytes. During virus clearance antiviral cytokines are thought to block virus replication and formation of new covalently closed circular DNA (cccDNA), the viral transcriptional template. It remains unclear if existing cccDNA is eliminated noncytolytically or if hepatocyte death and proliferation, to compensate for killing of some of the infected hepatocytes, are needed to remove cccDNA from surviving infected hepatocytes. Interpreting the relationship between hepatocyte death and cccDNA elimination requires knowing both the amount of hepatocyte turnover and whether cccDNA synthesis is effectively blocked during the period of immune destruction of infected hepatocytes. We have addressed these questions by asking if treatment of woodchucks with the nucleoside analog inhibitor of viral DNA synthesis entecavir (ETV) reduced hepatocyte turnover during clearance of transient woodchuck hepatitis virus (WHV) infections. To estimate hepatocyte turnover, complexity analysis was carried out on virus-cell DNA junctions created by integration of WHV and present following recovery in the livers of WHV-infected control or ETV-treated woodchucks. We estimated that, on average, 2.2 to 4.8 times less hepatocyte turnover occurred during immune clearance in the ETV-treated woodchucks. Computer modeling of the complexity data suggests that mechanisms in addition to hepatocyte death were responsible for elimination of cccDNA during recovery from transient infections.
The woodchuck model of hepatitis B virus (HBV) infection displays many characteristics of human infection and has particular value for characterizing the host immune responses during the development of chronic infection. Using the newly developed custom woodchuck microarray platform, we compared the intrahepatic transcriptional profiles of neonatal woodchucks with self-limiting woodchuck hepatitis virus (WHV) infection to those woodchucks progressing to persistent WHV infection. This revealed that WHV does not induce significant intrahepatic gene expression changes during the early-acute stage of infection (8 weeks), suggesting it is a stealth virus. At the mid-acute phase of infection (14 weeks), resolution was associated with induction of a prominent cytotoxic T cell signature. Strikingly, this was accompanied by high level expression of PD-1 and various other inhibitory T cell receptors, which likely act to minimize liver damage by cytotoxic T cells during viral clearance. In contrast to the expression of perforin and other cytotoxic effector genes, the interferon-γ (IFN-γ) signaling response in the mid-acute phase was comparable to that in chronically infected adult animals. The absence of a strong IFN-α/β transcriptional response indicated that type I IFN is not a critical mediator of self-limiting infection. Nevertheless, a number of antiviral genes, including viperin, were differentially expressed during resolving infection, suggesting that a subset of IFN-stimulated genes (ISGs) may play a role in the control of WHV replication.
We identified new immune pathways associated with the clearance of hepadnavirus infection revealing novel molecular targets with potential for the therapeutic treatment of chronic hepatitis B.
HBV; WHV; stealth virus; PD-1; viperin
The antiviral efficacies and cytotoxicities of 2′,3′- and 4′-substituted 2′,3′-didehydro-2′,3′-dideoxycytidine analogs were evaluated. All compounds were tested (i) against a wild-type human immunodeficiency virus type 1 (HIV-1) isolate (strain xxBRU) and lamivudine-resistant HIV-1 isolates, (ii) for their abilities to inhibit hepatitis B virus (HBV) production in the inducible HepAD38 cell line, and (iii) for their abilities to inhibit bovine viral diarrhea virus (BVDV) production in acutely infected Madin-Darby bovine kidney cells. Some compounds demonstrated potent antiviral activities against the wild-type HIV-1 strain (range of 90% effective concentrations [EC90s], 0.14 to 5.2 μM), but marked increases in EC90s were noted when the compounds were tested against the lamivudine-resistant HIV-1 strain (range of EC90s, 53 to >100 μM). The β-l-enantiomers of both classes of compounds were more potent than the corresponding β-d-enantiomers. None of the compounds showed antiviral activity in the assay that determined their abilities to inhibit BVDV, while two compounds inhibited HBV production in HepAD38 cells (EC90, 0.25 μM). The compounds were essentially noncytotoxic in human peripheral blood mononuclear cells and HepG2 cells. No effect on mitochondrial DNA levels was observed after a 7-day incubation with the nucleoside analogs at 10 μM. These studies demonstrate that (i) modification of the sugar ring of cytosine nucleoside analogs with a 4′-thia instead of an oxygen results in compounds with the ability to potently inhibit wild-type HIV-1 but with reduced potency against lamivudine-resistant virus and (ii) the antiviral activity of β-d-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine against wild-type HIV-1 (EC90, 0.08 μM) and lamivudine-resistant HIV-1 (EC90 = 0.15 μM) is markedly reduced by introduction of a 3′-fluorine in the sugar (EC90s of compound 2a, 37.5 and 494 μM, respectively).
Induction of hepatitis B virus (HBV)-specific cytotoxic T cells by therapeutic immunization may be a strategy to treat chronic hepatitis B. In the HBV animal model, woodchucks, the application of DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen (WHcAg) in combination with antivirals led to the prolonged control of viral replication. However, it became clear that the use of more potent vaccines is required to overcome WHV persistence. Therefore, we asked whether stronger and more functional T-cell responses could be achieved using the modified vaccines and an optimized prime-boost vaccination regimen. We developed a new DNA plasmid (pCGWHc) and recombinant adenoviruses (AdVs) showing high expression levels of WHcAg. Mice vaccinated with the improved plasmid pCGWHc elicited a stronger WHcAg-specific CD8+ T-cell response than with the previously used vaccines. Using multicolor flow cytometry and an in vivo cytotoxicity assay, we showed that immunization in a DNA prime-AdV boost regimen resulted in an even more vigorous and functional T-cell response than immunization with the new plasmid alone. Immunization of naïve woodchucks with pCGWHc plasmid or AdVs induced a significant WHcAg-specific degranulation response prior to the challenge, this response had not been previously detected. Consistently, this response led to a rapid control of infection after the challenge. Our results demonstrate that high antigen expression levels and the DNA prime-AdV boost immunization improved the T-cell response in mice and induced significant T-cell responses in woodchucks. Therefore, this new vaccination strategy may be a candidate for a therapeutic vaccine against chronic HBV infection.
Gamma interferon (IFN-γ) is an important mediator with multiple functions in the host defense against viral infection. IFN-γ, in concert with tumor necrosis factor alpha (TNF-α), leads to a remarkable reduction of intrahepatic replication intermediates and specific mRNAs of hepatitis B virus (HBV) by a noncytolytic mechanism in the transgenic mouse model. Thus, it is rational to evaluate the potential value of IFN-γ for the treatment of chronic HBV infection. In the present study, we expressed recombinant woodchuck IFN-γ (wIFN-γ) in Escherichia coli and mammalian cells. wIFN-γ protected woodchuck cells against infection of murine encephalomyocarditis virus in a species-specific manner. It upregulated the mRNA level of the woodchuck major histocompatibility complex class I (MHC-I) heavy chain in permanent woodchuck WH12/6 cells and regulated differentially the gene expression. However, the level of the replication intermediates and specific RNAs of woodchuck hepatitis virus (WHV) in persistently WHV-infected primary woodchuck hepatocytes did not change despite a treatment with 1,000 U of wIFN-γ per ml or with a combination of wIFN-γ and woodchuck TNF-α. Rather, hepatocytes derived from chronic carriers had an elevated level of the MHC-I heavy-chain mRNAs, most probably due to the exposure to inflammatory cytokines in vivo. Treatment with high doses of wIFN-γ led to an abnormal cell morphology and loss of hepatocytes. Thus, wIFN-γ regulates the gene expression in woodchuck hepatocytes but could not deplete WHV replication intermediates and mRNAs in persistently infected hepatocytes. The cellular response to wIFN-γ may be changed in hepatocytes from chronically WHV-infected woodchucks. It should be clarified in the future whether the continuous exposure of hepatocytes to inflammatory cytokines or the presence of viral proteins leads to changes of the cellular response to wIFN-γ.
The Eastern woodchuck (Marmota monax) is naturally infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to the human hepatitis B virus (HBV). The woodchuck is used as an animal model for studying chronic hepatitis B (CHB) and HBV-associated hepatocellular carcinoma (HCC) in humans, but the lack of sequence information has hitherto precluded functional genomics analysis. To address this major limitation of the model, we report here the sequencing, assembly and annotation of the woodchuck transcriptome, together with the generation of custom woodchuck microarrays. Using this new platform, we characterized the transcriptional response to persistent WHV infection and WHV-induced HCC. This revealed that chronic WHV infection, like HBV, is associated with (i) a limited intrahepatic type I interferon response, (ii) intrahepatic induction of markers associated with T cell exhaustion, (iii) elevated levels of suppressor of cytokine signaling 3 (SOCS3) in the liver, and (iv) intrahepatic accumulation of neutrophils. Underscoring the translational value of the woodchuck model, this study also determined that WHV-induced HCC shares molecular characteristics with a subtype of human HCC with poor prognosis.
Our data establish the translational value of the woodchuck model and provide new insights into immune pathways which may play a role either in the persistence of HBV infection or the sequelae of CHB.
HBV; WHV; HCC; interferon; neutrophil
Binding sites for polymerized albumin on hepatitis B virus components were reported in human hepatitis B virus chronic carriers predominantly with active viral replication (HB e antigen positive). The presence of comparable albumin-binding sites in the woodchuck hepatitis virus (WHV) model was examined on WHV components obtained from woodchucks with active viral replication (DNA polymerase positive). Binding sites for polymerized woodchuck serum albumin were not detected on the intact WHV virion, on 22-nm woodchuck hepatitis surface antigen (WHsAg), or on WHsAg polypeptides. Woodchuck albumin was not detected in purified 22-nm WHsAg, and anti-albumin antibodies were not detected in WHV chronic-carrier woodchucks. Our results in the WHV model argue against a role for viral polyalbumin-binding sites in tissue- and host-specific virus infectivity.
The guanosine analogs BMS-200475 and lobucavir have previously been shown to effectively suppress propagation of the human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) in 2.2.15 liver cells and in the woodchuck animal model system, respectively. This repression was presumed to occur via inhibition of the viral polymerase (Pol) by the triphosphate (TP) forms of BMS-200475 and lobucavir which are both produced in mammalian cells. To determine the exact mode of action, BMS-200475–TP and lobucavir-TP, along with several other guanosine analog-TPs and lamivudine-TP were tested against the HBV, WHV, and duck hepatitis B virus (DHBV) polymerases in vitro. Estimates of the 50% inhibitory concentrations revealed that BMS-200475–TP and lobucavir-TP inhibited HBV, WHV, and DHBV Pol comparably and were superior to the other nucleoside-TPs tested. More importantly, both analogs blocked the three distinct phases of hepadnaviral replication: priming, reverse transcription, and DNA-dependent DNA synthesis. These data suggest that the modest potency of lobucavir in 2.2.15 cells may be the result of poor phosphorylation in vivo. Kinetic studies revealed that BMS-200475–TP and lobucavir-TP competitively inhibit HBV Pol and WHV Pol with respect to the natural dGTP substrate and that both drugs appear to bind to Pol with very high affinities. Endogenous sequencing reactions conducted in replicative HBV nucleocapsids suggested that BMS-200475–TP and lobucavir-TP are nonobligate chain terminators that stall Pol at sites that are distinct yet characteristically two to three residues downstream from dG incorporation sites.