Chronic Woodchuck Hepatitis Virus Infection Research Articles

Application of the woodchuck animal model for the treatment of hepatitis B virus-induced liver cancer.

This review describes woodchucks chronically infected with the woodchuck hepatitis virus (WHV) as an animal model for hepatocarcinogenesis and treatment of primary liver cancer or hepatocellular carcinoma (HCC) induced by the hepatitis B virus (HBV). Since laboratory animal models susceptible to HBV infection are limited, woodchucks experimentally infected with WHV, a hepatitis virus closely related to HBV, are increasingly used to enhance our understanding of virus-host interactions, immune response, and liver disease progression. A correlation of severe liver pathogenesis with high-level viral replication and deficient antiviral immunity has been established, which are present during chronic infection after WHV inoculation of neonatal woodchucks for modeling vertical HBV transmission in humans. HCC in chronic carrier woodchucks develops 17 to 36 mo after neonatal WHV infection and involves liver tumors that are comparable in size, morphology, and molecular gene signature to those of HBV-infected patients. Accordingly, woodchucks with WHV-induced liver tumors have been used for the improvement of imaging and ablation techniques of human HCC. In addition, drug efficacy studies in woodchucks with chronic WHV infection have revealed that prolonged treatment with nucleos(t)ide analogs, alone or in combination with other compounds, minimizes the risk of liver disease progression to HCC. More recently, woodchucks have been utilized in the delineation of mechanisms involved in innate and adaptive immune responses against WHV during acute, self-limited and chronic infections. Therapeutic interventions based on modulating the deficient host antiviral immunity have been explored in woodchucks for inducing functional cure in HBV-infected patients and for reducing or even delaying associated liver disease sequelae, including the onset of HCC. Therefore, woodchucks with chronic WHV infection constitute a well-characterized, fully immunocompetent animal model for HBV-induced liver cancer and for preclinical evaluation of the safety and efficacy of new modalities, which are based on chemo, gene, and immune therapy, for the prevention and treatment of HCC in patients for which current treatment options are dismal.

Liver-Targeted Toll-Like Receptor 7 Agonist Combined With Entecavir Promotes a Functional Cure in the Woodchuck Model of Hepatitis B Virus.

Current therapeutics for chronic infection with hepatitis B virus (HBV) rarely induce functional cure due to the immunotolerant status of patients. Small molecule agonists targeting toll‐like receptor 7 (TLR7) have been shown to elicit a functional cure in animal models of HBV but sometimes with poor tolerability due to immune‐related toxicities. In an effort to increase the therapeutic window of TLR7 agonists to treat chronic hepatitis B (CHB), we developed an oral TLR7 agonist, APR002, designed to act locally in the gastrointestinal tract and liver, thus minimizing systemic exposure and improving tolerability. Here, we describe the pharmacokinetic/pharmacodynamic (PK/PD) profile of APR002 in mice and uninfected woodchucks as well as the safety and antiviral efficacy in combination with entecavir (ETV) in woodchucks with CHB. Treatment of woodchucks chronically infected with woodchuck hepatitis virus (WHV) with weekly oral doses of APR002 was well‐tolerated. While APR002 and ETV single agents did not elicit sustained viral control, combination therapy resulted in durable immune‐mediated suppression of the chronic infection. These woodchucks also had detectable antibodies to viral antigens, enhanced interferon‐stimulated gene expression, and loss of WHV covalently closed circular DNA. Conclusion: APR002 is a novel TLR7 agonist exhibiting a distinct PK/PD profile that in combination with ETV can safely attain a functional cure in woodchucks with chronic WHV infection. Our results support further investigation of liver‐targeted TLR7 agonists in human CHB.

Prevention of liver tumor formation in woodchucks with established hepatocellular carcinoma by treatment with cationic liposome-DNA complexes

BackgroundApproximately 250 million people worldwide are chronically infected with hepatitis B virus (HBV) and more than half of the hepatocellular carcinoma (HCC) cases are attributed to this infection. As HCC has a high mortality rate, and current treatment options are remarkably limited, the development of new therapeutic treatment strategies is warranted.MethodsIn this study, woodchucks infected with woodchuck hepatitis virus (WHV), and with pre-existing liver tumors, were used as a model to investigate if complexes of cationic liposomes and non-coding DNA (JVRS-100) were effective in treatment of HCC.ResultsIt was observed that the high serum viral load that is present in a typical chronic WHV infection (i.e., approximately 100-fold higher than human viral loads) results in immune suppression and resistance to treatment with JVRS-100. Treatment of woodchucks with lower serum viral load that more closely matched with the viral load usually seen in human HBV infection appears a better model for immunotherapeutic development based on the responsiveness to JVRS-100 treatment. In the latter case, marked declines in WHV DNA and WHV surface antigen were determined over the 12-week treatment period and WHV markers stayed suppressed during most time points of the 12-week follow-up period. Even more remarkably, the formation of new liver tumors was not observed in woodchucks treated with a well-tolerated dose of JVRS-100, as compared to several new tumors that developed in vehicle-treated control animals.ConclusionsAlthough there was little decrease in the volumes of the liver tumors existing at the time of treatment, it is generally accepted that preventing the spread and metastasis of almost always fatal cancers such as HCC and thus, reducing it to a chronic and treatable disease can also be a successful therapeutic approach. The results in woodchucks warrant the investigation of JVRS-100 as an intervention to prevent liver cancer in patients chronically infected with HBV and at high risk for HCC development.

Molecular cloning, characterization and expression analysis of Tim-3 and Galectin-9 in the woodchuck model.

In recent years, a critical role for T cell immunoglobulin mucin domain 3 (Tim-3) and its ligand Galectin-9 (Gal-9) has emerged in infectious disease, autoimmunity and cancer. Manipulating this immune checkpoint may have immunotherapeutic potential and could represent an alternative approach for improving immune responses to viral infections and cancer. The woodchuck (Marmot monax) infected by woodchuck hepatitis virus (WHV) represents an informative animal model to study HBV infection and HCC. In the current study, the cDNA sequences of woodchuck Tim-3 and Gal-9 were cloned, sequenced and characterized. The extracellular domain of Tim-3 cDNA sequence consisted of 576bp coding sequence (CDS) that encoded 192 amino acids. The 1076bp full-length Gal-9 cDNA sequence consisted of 1059bp coding sequence (CDS) that encoded 352 amino acids with a molecular weight of 39.7kDa. The phylogenetic tree analysis revealed that the woodchuck Tim-3 and Gal-9 had the closest genetic relationship with Ictidomys tridecemlineatus. The result of quantification PCR analysis showed that ubiquitous expression of Gal-9 but not Tim-3 in different tissues of naive woodchucks. Elevated liver Gal-9 expression was observed in woodchucks with chronic WHV infection. Moreover, a polyclonal antibody against the extracellular domain of woodchuck Tim-3 were generated and identified by flow cytometry. Our results serve as a foundation for further insight into the role of Tim-3/Galectin-9 signaling pathway in viral hepatitis and HCC in the woodchuck model.

Measurement of Antiviral Effect and Innate Immune Response During Treatment of Primary Woodchuck Hepatocytes.

An estimated 350 million people are chronically infected with hepatitis B virus (HBV), and over one million people die each year due to HBV-associated liver diseases, such as cirrhosis and liver cancer. Current therapeutics for chronic HBV infection are limited to nucleos(t)ide analogs and interferon. These anti-HBV drugs in general reduce viral load and improve the long-term outcome of infection but very rarely lead to a cure. Thus, new therapies for chronic HBV infection need to be developed by utilizing liver cell lines and primary cultures and small laboratory animals capable of replicating HBV or surrogate hepadnaviruses for antiviral testing. Natural infection with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to HBV, occurs in woodchucks. Chronic WHV infection has been established over decades as a suitable model for evaluating direct-acting antivirals as well as vaccines, vaccine adjuvants, and immunotherapeutics because animals are fully immunocompetent. Before HBV-specific compounds are applied to woodchucks, they are usually tested in primary woodchuck hepatocytes (PWHs) replicating WHV at high levels for confirming drug specificity against viral or host targets. Here we describe a protocol for the isolation of PWHs from liver of WHV-infected woodchucks, maintenance in culture, and use in assays for determining antiviral efficacy, safety, and associated host innate immune response of new experimental drugs. Exemplary assays were performed with the nucleoside analog, lamivudine, and the immunomodulator, interferon-alpha.

Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B.

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8–12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.

Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection

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.

389 ENHANCING VIRUS-SPECIFIC IMMUNITY IN VIVO BY COMBINING THERAPEUTIC VACCINATION AND PD1/PD-L1 BLOCKADE IN CHRONIC HEPADNAVIRAL INFECTION

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 PDL1 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. Citation: Liu J, Zhang E, Ma Z, Wu W, Kosinska A, et al. (2014) Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection. PLoS Pathog 10(1): e1003856. doi:10.1371/journal.ppat.1003856 Editor: Charles M. Rice, The Rockefeller University, United States of America Received April 17, 2013; Accepted November 13, 2013; Published January 2, 2014 Copyright: 2014 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Deutsche Forschungsgemeinschaft (TRR60 and GK 1045/2), National Major Science and Technology Project for Infectious Diseases of China (2008ZX10002-011, 2012ZX10004503), the National Natural Science Foundation of China (No. 30271170, 30571646, 81101248), and the International Science & Technology Cooperation Program of China (2011DFA31030). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: michael.roggendorf@uni-due.de

Transcriptomic analysis of the woodchuck model of chronic hepatitis B

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 (1) a limited intrahepatic type I interferon response; (2) intrahepatic induction of markers associated with T cell exhaustion; (3) elevated levels of suppressor of cytokine signaling 3 (SOCS3) in the liver; and (4) 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 insight into immune pathways which may play a role either in the persistence of HBV infection or the sequelae of CHB.

Molecular characterization of woodchuck interleukin-10 receptor and enhanced function of specific T cells from chronically infected woodchucks following its blockade

Interleukin 10 (IL-10) is a pleiotropic cytokine acting on a variety of immune cells through the cell surface receptor (IL-10R). It has been suggested to resuscitate antiviral immunity by interfering with IL-10/IL-10R pathway. The woodchuck model infected by woodchuck hepatitis virus (WHV) represents an informative animal model to study hepatitis B virus (HBV) infection. In this study, the woodchuck IL-10R (wIL-10R) was molecularly cloned and characterized, showing high similarity of its nucleotide and amino acid sequences to that of other mammalian species. The expression level of wIL-10R mRNA in woodchuck peripheral blood mononuclear cells was significantly increased in acute WHV infection but down-regulated during chronic WHV infection. Specific rabbit antibodies against wIL-10R were prepared and showed the ability to enhance the proliferation and degranulation of specific T-cells from chronically WHV-infected woodchucks in vitro. The present work on wIL-10R provided a good basis for future preclinical studies on therapeutic approaches for chronic HBV infection.

Modulation of regulatory T-cell activity in combination with interleukin-12 increases hepatic tolerogenicity in woodchucks with chronic hepatitis B

Regulatory T cells (Treg) play a critical role in the modulation of immune responses to viral antigens in chronic viral hepatitis. Woodchucks (Marmota monax) infected with the woodchuck hepatitis virus (WHV) represent the best animal model for chronic hepatitis B virus (HBV) infection. Examination of intrahepatic and peripheral Treg in uninfected and WHV chronically infected woodchucks showed a significant increase of intrahepatic Treg numbers in chronically infected animals, whereas no differences were found in peripheral blood. In agreement with these data, higher expression levels of Forkhead box P3 (Foxp3), interleukin (IL)-10, transforming growth factor beta (TGF-β) were detected in the liver of chronic WHV carriers in comparison to uninfected animals. Furthermore, treatment of WHV-infected animals with an adenovirus encoding IL-12 failed to reduce viral load, a finding that was associated with lymphocyte unresponsiveness to IL-12 stimulation in vitro. We observed that TGF-β and Treg play a major role in the lack of lymphocyte response to IL-12 stimulation, as TGF-β inhibition and Treg depletion allowed recovery of T-cell responsiveness to this cytokine. Based on these results, woodchucks were treated with IL-12 in combination with a TGF-β inhibitory peptide or Treg depletion. However, no antiviral effect was achieved and, instead, an enhancement of the intrahepatic tolerogenic environment was observed. Our data show that TGF-β inhibition or Treg depletion had no added benefit over IL-12 therapy in chronic WHV infection. IL-12 immunostimulation induces a strong immunosuppressive reaction in the liver of chronic WHV carriers that counteracts the antiviral effect of the treatment.

Abstract 4546: Variability analysis of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of hepatocellular carcinoma (HCC)

Abstract INTRODUCTION: HCC is a highly vascular, rapidly fatal cancer and antiangiogenic drugs have shown clinical benefit despite lack of radiographic tumor shrinkage. We standardized imaging methods to assess HCC in the woodchuck animal model (Marmota monax). This is a large unique animal model of spontaneous HCC with several similarities to human Hepatitis b(HBV) related HCC and such studies would allow utilization of this model to assess the functional changes with currently used targeted therapies. METHODS: Fourteen woodchucks infected at birth with known titers of sera from a chronic infectious pool of chronic woodchuck hepatitis virus (WHV), which is similar in structure and replicative function to HBV. Serial measurement of WHV antigen titers was conducted. Chronic WHV infection occurred in greater than 60% of woodchucks by 12 months. The WHV carrier woodchucks were screened with serial ultrasound and ten animals which developed HCC at a median age of 24- 36 months were included. Mediports were placed in the femoral vein under aseptic precautions for intravenous (IV) contrast administration. DCE-MRI methods for assessment of woodchuck HCC blood flow and size were optimized in a 1.5T magnet human head coil and IV gadolinium injected at 5cc per second using a rapid injector. Four variables - the median Ktrans, median AUCBN90, necrotic volume (NV) and total volume (TV) were analyzed by Virtualscopics using published methods. A total of 44 images were obtained, 32 were included in the study. Death due to disease progression or missing paired data resulted in exclusion of 12 images. Statistical analysis: We performed a variability analysis by calculating the concordance correlation coefficient (CCC). This coefficient was calculated for all four variables and compared at different time points. RESULTS: The CCC calculated for images obtained on day 1 and day 8 images was 0.85 for Ktrans, 0.59 for AUCBN90, 0.93 for NV and 0.99 for TV. Strong concordance was seen in 3 of the 4 DCE-MRI variables recorded a week apart. This lack of change over an 8-day period is expected with this cancer. We also compared CCC for day 8 and day 14 with Ktrans 0.76, AUCBN 0.6, NV 0.76, TV 0.89 showing less agreement than day 1 and 8. When compared to day 14 and day 30 CCC for different variables were Ktrans 0.59, AUCBN90 0.38, NV 0.82, TV 0.85, suggesting a downward trend with increasing time. Our data using this technique is able to identify clinically relevant changes in tumor size with little variability in short interval scanning. CONCLUSION: This is the first study validating the accuracy of DCE-MRI in a translational animal model for HCC and has potential to aid development of newer antiangiogenic therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4546. doi:1538-7445.AM2012-4546

The Expression of PD-1 Ligands and Their Involvement in Regulation of T Cell Functions in Acute and Chronic Woodchuck Hepatitis Virus Infection

BackgroundThe programmed cell death 1 (PD-1)/programmed death-1 ligand 1 (PD-L1) system may play a role in the negative regulation of T cell functions in hepatitis B virus (HBV) infection. Thus, it is important to study its role in the widely used animal model for HBV infection of woodchucks with woodchuck hepatitis virus (WHV).MethodsWoodchuck PD-L1 (wPD-L1) and -L2 (wPD-L2) were cloned and characterized. The levels of wPD-L1 expression in primary woodchuck hepatocytes (PWH), peripheral blood mononuclear cells (PBMCs), and liver tissue of naive and WHV-infected woodchucks were examined by real time reverse transcription (RT)-PCR and flow cytometry. Using antibodies against wPD-L1 and -L2, the effect of blocking PD-1/PD-L1/PD-L2 interaction on the proliferation and degranulation of woodchuck PBMCs was examined.Principal FindingsBoth wPD-L1 and -L2 showed a high homology to their counterparts of other mammalian species and humans. WPD-L1 expression in PWH and PBMCs of naive animals was low but could be stimulated by Toll-like receptor (TLR) ligands and interferons (IFN). WPD-L1 expression in liver tissue was significantly higher than that measured in PWHs and was slightly elevated during acute and chronic WHV infection. However, wPD-1 and wPD-L1 expression on PBMCs was strongly up-regulated during acute and chronic infection. In vitro blockade with antibodies against wPD-L1 and -L2 partially enhanced proliferation and degranulation of PBMCs from WHV-infected woodchucks.ConclusionsOur results demonstrated that wPD-1/wPD-L1 expression in hepatocytes and PBMCs can be induced by different inflammatory stimuli and is up-regulated mainly on PBMCs during WHV infection. A blockade of the woodchuck PD-1/PD-L pathway could partially enhance T cell functions in WHV infection.

Profound Antiviral Effect of Oral Administration of MIV-210 on Chronic Hepadnaviral Infection in a Woodchuck Model of Hepatitis B

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.

Correlation of Virus and Host Response Markers with Circulating Immune Complexes during Acute and Chronic Woodchuck Hepatitis Virus Infection

Woodchuck hepatitis virus (WHV) is an established model for human hepatitis B virus. The kinetics of virus and host responses in serum and liver during acute, self-limited WHV infection in adult woodchucks were studied. Serum WHV DNA and surface antigen (WHsAg) were detected as early as 1 to 3 weeks following experimental infection and peaked between 1 and 5 weeks postinfection. Thereafter, serum WHsAg levels declined rapidly and became undetectable, while WHV DNA levels became undetectable much later, between 4 and 20 weeks postinfection. Decreasing viremia correlated with transient liver injury marked by an increase in serum sorbitol dehydrogenase (SDH) levels. Clearance of WHV DNA from serum was associated with the normalization of serum SDH. Circulating immune complexes (CICs) of WHsAg and antibodies against WHsAg (anti-WHs) that correlated temporarily with the peaks in serum viremia and WHs antigenemia were detected. CICs were no longer detected in serum once free anti-WHs became detectable. The detection of CICs around the peak in serum viremia and WHs antigenemia in resolving woodchucks suggests a critical role for the humoral immune response against WHsAg in the early elimination of viral and subviral particles from the peripheral blood. Individual kinetic variation during WHV infections in resolving woodchucks infected with the same WHV inoculum and dose is likely due to the outbred nature of the animals, indicating that the onset and magnitude of the individual immune response determine the intensity of virus inhibition and the timing of virus elimination from serum.

Antiviral Effects of Lamivudine, Emtricitabine, Adefovir Dipivoxil, and Tenofovir Disoproxil Fumarate Administered Orally Alone and in Combination to Woodchucks with Chronic Woodchuck Hepatitis Virus Infection

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 log(10) and 6.1 log(10) genome equivalents/ml serum, respectively, followed by TDF plus 3TC (5.6 log(10) genome equivalents/ml), ADV alone (4.8 log(10) genome equivalents/ml), ADV plus FTC (one survivor) (4.4 log(10) genome equivalents/ml), TDF alone (2.9 log(10) genome equivalents/ml), 3TC alone (2.7 log(10) genome equivalents/ml), and FTC alone (2.0 log(10) 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.

Molecular characterization of woodchuck type I interferons and their expression by woodchuck peripheral blood lymphocytes

Interferon (IFN)-α and -β are important antiviral mediators. IFN-α is widely used for the treatment of chronic hepatitis B. In our previous studies, a subtype of woodchuck IFN-α (wIFN-α) was characterized and has been shown to be active in suppressing the replication of woodchuck hepatitis virus (WHV) in vitro and vivo. Here, we refined the analysis of the IFN-α/β system of the woodchuck and studied the expression of wIFN-α/β in peripheral blood lymphocytes (PBLs) from naïve and WHV-infected woodchucks. A number of wIFN-α genes were sequenced and could be classified into 10 subtypes and 3 pseudotypes. The biological activity of different subtypes of wIFN-α was demonstrated by their ability to protect woodchuck cells against encephalomyocarditis virus infection and to induce MxA expression in woodchuck cells. Additionally, a partial sequence of wIFN-β was characterized. A subtyping method for wIFN-α based on restriction length polymorphism analysis was developed. Further, the expression of wIFN by woodchuck PBLs after stimulation with polyI/C was investigated. The maximal production of wIFN by woodchuck PBLs occurred within the first 48 h after addition poly I/C. The wIFN-α subtypes 1, 4, and 5 were found to be produced by poly I/C-stimulated woodchuck PBLs, indicating a selective expression of wIFN-α subtypes. PBLs from chronically WHV-infected woodchucks showed a reduced ability to produce wIFN when stimulated with poly I/C. The results suggest that woodchucks with chronic WHV infection have impaired immunological responses to poly I/C.

Antiviral Effect of Orally Administered (−)-β-d-2-Aminopurine Dioxolane in Woodchucks with Chronic Woodchuck Hepatitis Virus Infection

(-)-beta-D-2-Aminopurine dioxolane (APD) is a nucleoside prodrug that is efficiently converted to 9-(beta-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 log(10) copies/ml, respectively. The 30-mg/kg/day dose resulted in a more pronounced, statistically significant decline in serum WHV viremia of 1.9 log(10) 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.

Molecular characterization of woodchuck interleukin 15 (wIL-15) and detection of its expression in liver samples of woodchucks infected with woodchuck hepatitis virus (WHV)

Interleukin 15 (IL-15) is a member of the four-helix bundle cytokine family and has T cell growth factor activity. IL-15 plays a unique role in both innate and adaptive immune cell homeostasis, particularly for the development of NK cells and CD8 + memory cells. It may be useful for stimulation of specific immune responses in chronic viral infection such as hepatitis B virus infection. The woodchuck model is an informative animal model for studies on hepadnavirus infection and therapeutic interventions. Here, the complete coding sequence of woodchuck IL-15 (wIL-15) was cloned and sequenced. wIL-15 shows a high homology (>70%) to its counterparts of other mammalian species. His-tagged recombinant wIL-15 protein was expressed and purified and showed the ability to promote the proliferation of activated mouse splenocytes and woodchuck peripheral blood lymphocytes. Further, examination of mRNA amounts in liver samples of woodchucks by semi-quantitative RT-PCR showed a slightly increased expression of wIL-15 in woodchuck livers during chronic woodchuck hepatitis virus infection. This available information will provide a basis for further studies on the function of IL-15 in the context of acute and chronic hepadnavirus infection and its potential therapeutic use for chronic hepatitis B virus infection in the woodchuck model.

Fluctuation of the cytokine expression in the liver during the chronic woodchuck hepatitis virus (WHV) infection is not related to viral load

The woodchuck together with the woodchuck hepatitis virus (WHV) is an excellent model to study the pathogenesis of hepadnaviral infections. Chronic WHV infection causes severe liver disease and hepatocellular carcinoma in woodchucks. The mechanism of viral clearance is not fully understood, interferons seem to play a major role in down-regulating viral replication prior to elimination of infected hepatocytes. We investigated on the pattern of cytokine and T-cell-marker expression in livers of woodchucks chronically infected with WHV. RNase-protection-assay (RPA) was used to determine mRNA of woodchuck specific genes (TNF-α, IFN-γ, IL-15, CD3, CD4, CD8). Serial liver biopsies were performed daily or weekly in eight chronic WHV-carrier woodchucks. Cytokine/T-cell-marker expression differed significantly between the time points up to ±50% within each woodchuck. The different expression patterns of cytokines or T-cell-markers did not correlate to the (weak) fluctuations in the viremia but may explain the observed fluctuations in the WHV/HBV-load in chronically infected individuals. Furthermore, we observed associations between cytokine and T-cell-marker expression. The marginal fluctuations in viremia during the chronic infection may indicate, that, once the chronic hepadnaviral infection is established, cytokines/interferons expressed endogenously (i.e. not vector-borne or injected) play only a minor role.