Woodchuck Hepatitis Research Articles

Diverse Virus and Host-Dependent Mechanisms Influence the Systemic and Intrahepatic Immune Responses in the Woodchuck Model of Hepatitis B.

Woodchuck infected with woodchuck hepatitis virus (WHV) represents the pathogenically nearest model of hepatitis B and associated hepatocellular carcinoma (HCC). This naturally occurring animal model also is highly valuable for development and preclinical evaluation of new anti-HBV agents and immunotherapies against chronic hepatitis (CH) B and HCC. Studies in this system uncovered a number of molecular and immunological processes which contribute or likely contribute to the immunopathogenesis of liver disease and modulation of the systemic and intrahepatic innate and adaptive immune responses during hepadnaviral infection. Among them, inhibition of presentation of the class I major histocompatibility complex on chronically infected hepatocytes and a role of WHV envelope proteins in this process, as well as augmented hepatocyte cytotoxicity mediated by constitutively expressed components of CD95 (Fas) ligand- and perforin-dependent pathways, capable of eliminating cells brought to contact with hepatocyte surface, including activated T lymphocytes, were uncovered. Other findings pointed to a role of autoimmune response against hepatocyte asialoglycoprotein receptor in augmenting severity of liver damage in hepadnaviral CH. It was also documented that WHV in the first few hours activates intrahepatic innate immunity that transiently decreases hepatic virus load. However, this activation is not translated in a timely manner to induction of virus-specific T cell response which appears to be hindered by defective activation of antigen presenting cells and presentation of viral epitopes to T cells. The early WHV infection also induces generalized polyclonal activation of T cells that precedes emergence of virus-specific T lymphocyte reactivity. The combination of these mechanisms hinder recognition of virus allowing its dissemination in the initial, asymptomatic stages of infection before adaptive cellular response became apparent. This review will highlight a range of diverse mechanisms uncovered in the woodchuck model which affect effectiveness of the anti-viral systemic and intrahepatic immune responses, and modify liver disease outcomes. Further exploration of these and other mechanisms, either already discovered or yet unknown, and their interactions should bring more comprehensive understanding of HBV pathogenesis and help to identify novel targets for therapeutic and preventive interventions. The woodchuck model is uniquely positioned to further contribute to these advances.

Effects of viral promoters, the Woodchuck hepatitis post-transcriptional regulatory element, and weakened antibiotic resistance markers on transgene expression in Chinese hamster ovary cells

The product productivity of Chinese hamster ovary (CHO) cells need to be further increased and solved. Promoter and selection marker are essential elements for transgene expression. We investigated the effects of promoters, Woodchuck hepatitis post-transcriptional regulatory (WPRE) element, and selection marker on transgene expression in CHO cells. A weakened form of resistance gene neomycin phosphotransferase (NPT) and WPRE element were cloned into the expression vectors driven by the cytomegalovirus (CMV) and simian virus 40 (SV40) promoters, following by transfecting into CHO cells and screened under G418 pressure. qRT-PCR and Flow cytometry were used to detect the expression level of eGFP gene, and transgene copy number was determined using qPCR technology. Western blot and ELISA were utilized to analyze the expression levels of cetuximab. The weakened NPT gene enhanced eGFP expression both under the CMV or SV40 promoters. Moreover, WPRE, combined with the NPT gene considerably enhanced eGFP expression in CHO cells. Furthermore, the CMV promoter and WPRE combination resulted in higher cetuximab expression levels than CMV promoter alone. However, WPRE could not enhance the transgene expression under the circumstance of CMV promoter and weakened NPT gene combination, suggesting that the effects of the WPRE combined with selection markers were specificity.

Efficacy of an Inhibitor of Hepatitis B Virus Expression in Combination With Entecavir and Interferon-α in Woodchucks Chronically Infected With Woodchuck Hepatitis Virus.

RG7834 is a small‐molecule inhibitor of hepatitis B virus (HBV) gene expression that significantly reduces the levels of hepatitis B surface antigen (HBsAg) and HBV DNA in a humanized liver HBV mouse model. In the current study, we evaluated the potency of RG7834 in the woodchuck model of chronic HBV infection, alone and in combination with entecavir (ETV) and/or woodchuck interferon‐α (wIFN‐α). RG7834 reduced woodchuck hepatitis virus (WHV) surface antigen (WHsAg) by a mean of 2.57 log10 from baseline and WHV DNA by a mean of 1.71 log10. ETV + wIFN‐α reduced WHsAg and WHV DNA by means of 2.40 log10 and 6.70 log10, respectively. The combination of RG7834, ETV, and wIFN‐α profoundly reduced WHsAg and WHV DNA levels by 5.00 log10 and 7.46 log10, respectively. However, both viral parameters rebounded to baseline after treatment was stopped and no antibody response against WHsAg was observed. Effects on viral RNAs were mainly seen with the triple combination treatment, reducing both pregenomic RNA (pgRNA) and WHsAg RNA, whereas RG7834 mainly reduced WHsAg RNA and ETV mainly affected pgRNA. When WHsAg was reduced by the triple combination, peripheral blood mononuclear cells (PBMCs) proliferated significantly in response to viral antigens, but the cellular response was diminished after WHsAg returned to baseline levels during the off‐treatment period. Consistent with this, Pearson correlation revealed a strong negative correlation between WHsAg levels and PBMC proliferation in response to peptides covering the entire WHsAg and WHV nucleocapsid antigen. Conclusion: A fast and robust reduction of WHsAg by combination therapy reduced WHV‐specific immune dysfunction in the periphery. However, the magnitude and/or duration of the induced cellular response were not sufficient to achieve a sustained antiviral response.

Nanoparticle Uptake in a Spontaneous and Immunocompetent Woodchuck Liver Cancer Model.

There is a tremendous focus on the application of nanomaterials for the treatment of cancer. Nonprimate models are conventionally used to assess the biomedical utility of nanomaterials. However, these animals often lack an intact immunological background, and the tumors in these animals do not develop spontaneously. We introduce a preclinical woodchuck hepatitis virus-induced liver cancer model as a platform for nanoparticle (NP)-based in vivo experiments. Liver cancer development in these out-bred animals occurs as a result of persistent viral infection, mimicking human hepatitis B virus-induced HCC development. We highlight how this model addresses key gaps associated with other commonly used tumor models. We employed this model to (1) track organ biodistribution of gold NPs after intravenous administration, (2) examine their subcellular localization in the liver, (3) determine clearance kinetics, and (4) characterize the identity of hepatic macrophages that take up NPs using RNA-sequencing (RNA-seq). We found that the liver and spleen were the primary sites of NP accumulation. Subcellular analyses revealed accumulation of NPs in the lysosomes of CD14+ cells. Through RNA-seq, we uncovered that immunosuppressive macrophages within the woodchuck liver are the major cell type that take up injected NPs. The woodchuck-HCC model has the potential to be an invaluable tool to examine NP-based immune modifiers that promote host anti-tumor immunity.

Liver-specific 3D sectioning molds for correlating in vivo CT and MRI with tumor histopathology in woodchucks(Marmota monax).

PurposeTo evaluate the spatial registration and correlation of liver and tumor histopathology sections with corresponding in vivo CT and MRI using 3D, liver-specific cutting molds in a woodchuck (Marmota monax) hepatic tumor model.MethodsFive woodchucks chronically infected with woodchuck hepatitis virus following inoculation at birth and with confirmed hepatic tumors were imaged by contrast enhanced CT or MRI. Virtual 3D liver or tumor models were generated by segmentation of in vivo CT or MR imaging. A specimen-specific cavity was created inside a block containing cutting slots aligned with an imaging plane using computer-aided design software, and the final cutting molds were fabricated using a 3D printer. Livers were resected two days after initial imaging, fixed with formalin or left unfixed, inserted into the 3D molds, and cut into parallel pieces by passing a sharp blade through the parallel slots in the mold. Histopathology sections were acquired and their spatial overlap with in vivo image slices was quantified using the Dice similarity coefficient (DSC).ResultsImaging of the woodchucks revealed heterogeneous hepatic tumors of varying size, number, and location. Specimen-specific 3D molds provided accurate co-localization of histopathology of whole livers, liver lobes, and pedunculated tumors with in vivo CT and MR imaging, with or without tissue fixation. Visual inspection of histopathology sections and corresponding in vivo image slices revealed spatial registration of analogous pathologic features. The mean DSC for all specimens was 0.83+/-0.05.ConclusionUse of specimen-specific 3D molds for en bloc liver dissection provided strong spatial overlap and feature correspondence between in vivo image slices and histopathology sections.

Transarterial Chemoembolization in a Woodchuck Model of Hepatocellular Carcinoma

PurposeTo assess the feasibility of transarterial chemoembolization with drug-eluting embolic (DEE) microspheres in a woodchuck model of hepatocellular carcinoma (HCC). Materials and MethodsNine woodchucks were studied: 4 normal animals and 5 animals infected with woodchuck hepatitis virus in which HCC had developed. Three animals with HCC underwent multidetector CT. A 3-F sheath was introduced into the femoral artery, and the hepatic arteries were selectively catheterized with 2.0–2.4-F microcatheters. Normal animals underwent diagnostic angiography and bland embolization. Animals with HCC underwent DEE transarterial chemoembolization with 70–150-μm radiopaque microspheres loaded with 37.5 mg doxorubicin per milliliter. Cone-beam CT and multidetector CT were performed. Following euthanasia, explanted livers underwent micro-CT, histopathologic examination, and fluorescence imaging of doxorubicin. ResultsThe tumors were hypervascular and supplied by large-caliber tortuous vessels, with arteriovenous shunts present in 2 animals. There was heterogeneous enhancement on multidetector CT with areas of necrosis. Six tumors were identified. The most common location was the right medial lobe (n = 3). Mean tumor volume was 30.7 cm3 ± 12.3. DEE chemoembolization of tumors was achieved. Excluding the 2 animals with arteriovenous shunts, the mean volume of DEE microspheres injected was 0.49 mL ± 0.17. Fluorescence imaging showed diffusion of doxorubicin from the DEE microspheres into the tumor. ConclusionsWoodchuck HCC shares imaging appearances and biologic characteristics with human HCC. Selective catheterization and DEE chemoembolization may similarly be performed. Woodchucks may be used to model interventional therapies and possibly characterize radiologic–pathologic correlations.

Kinetics of DNA damage repair response accompanying initial hepadnavirus-host genomic integration in woodchuck hepatitis virus infection of hepatocyte

Mechanism of initial hepatitis B virus (HBV) integrations and kinetics of DNA repair immediately after infection remain essentially unknown impairing understanding of hepadnaviral oncogenesis. WCM260 hepatocytes susceptible to HBV-compatible woodchuck hepatitis virus (WHV) were examined from 15 min to 72 h post-infection (p.i.). WHV strongly induced reactive oxygen species (ROS), transiently inducible nitric oxide (iNOS) and DNA damage from 15 min p.i. All initial WHV-host fusions had the head-to-tail format indicating their formation by non-homologous end joining (NHEJ). Transcription of poly(ADP-ribose) polymerase 1 (PARP1) and X-ray repair cross-complementing protein 1 (XRCC1), the PARP1 binding partner, were induced in 30 min p.i. and that of 8-oxyguanine DNA glycosylse (OGG1) responding to oxidative DNA damage at 12 h p.i. Nicotinamide adenine dinucleotide (NAD+), a marker of PARP1 activation, and heme oxygenase-1 (HO1), an indicator of pro-oxidative stress, were significantly augmented from 15-30 min p.i. Additionally, PARP1 cleavage activity was evident from 30 min p.i. confirming that PARP1-mediated DNA repair became operational almost instatly after hepatocyte contact with virus. By applying complementary approaches, the study showed that initial WHV integration was due to virus-induced oxidative DNA damage and implied that the NHEJ PARP1-dependent repair pathway determined format of the first virus-host DNA junctions.

Innate and adaptive immunity associated with resolution of acute woodchuck hepatitis virus infection in adult woodchucks.

Viral and/or host factors that are directly responsible for the acute versus chronic outcome of hepatitis B virus (HBV) infection have not been identified yet. Information on immune response during the early stages of HBV infection in humans is mainly derived from blood samples of patients with acute hepatitis B (AHB), which are usually obtained after the onset of clinical symptoms. Features of intrahepatic immune response in these patients are less studied due to the difficulty of obtaining multiple liver biopsies. Woodchuck hepatitis virus (WHV) infection in woodchucks is a model for HBV infection in humans. In the present study, five adult woodchucks were experimentally infected with WHV and then followed for 18 weeks. Blood and liver tissues were frequently collected for assaying markers of WHV replication and innate and adaptive immune responses. Liver tissues were further analyzed for pathological changes and stained for important immune cell subsets and cytokines. The increase and subsequent decline of viral replication markers in serum and liver, the elicitation of antibodies against viral proteins, and the induction of virus-specific T-cell responses indicated eventual resolution of acute WHV infection in all animals. Intrahepatic innate immune makers stayed unchanged immediately after the infection, but increased markedly during resolution, as determined by changes in transcript levels. The presence of interferon-gamma and expression of natural killer (NK) cell markers suggested that a non-cytolytic response mechanism is involved in the initial viral control in liver. This was followed by the expression of T-cell markers and cytolytic effector molecules, indicating the induction of a cytolytic response mechanism. Parallel increases in regulatory T-cell markers suggested that this cell subset participates in the overall immune cell infiltration in liver and/or has a role in regulating AHB induced by the cytolytic response mechanism. Since the transcript levels of immune cell markers in blood, when detectable, were lower than in liver, and the kinetics, except for NK-cells and interferon-gamma, did not correlate well with their intrahepatic expression, this further indicated enrichment of immune cells within liver. Conclusion: The coordinated interplay of innate and adaptive immunity mediates viral clearance in the woodchuck animal model of HBV infection. The initial presence of NK-cell associated interferon-gamma response points to an important role of this cytokine in HBV resolution.

Designing a therapeutic hepatitis B vaccine to circumvent immune tolerance

ABSTRACTAn effective prophylactic hepatitis B virus (HBV) vaccine has long been available but is ineffective for chronic infection. The primary cause of chronic hepatitis B (CHB) and greatest impediment for a therapeutic vaccine is the direct and indirect effects of immune tolerance to HBV antigens. The resulting defective CD4+/CD8+ T cell response, poor cytokine production, insufficient neutralizing antibody (nAb) and poor response to HBsAg vaccination characterize CHB infection. The objective of this study was to develop virus-like-particles (VLPs) that elicit nAb to prevent viral spread and prime CD4+/CD8+ T cells to eradicate intracellular HBV. Eight neutralizing B cell epitopes from the envelope PreS1 region were consolidated onto a species-variant of the HBV core protein, the woodchuck hepatitis core antigen (WHcAg). PreS1-specific B cell epitopes were chosen because of preferential expression on HBV virions. Because WHcAg and HBcAg are not crossreactive at the B cell level and only partially cross-reactive at the CD4+/CD8+ T cell level, CD4+ T cells specific for WHcAg-unique T cell sites can provide cognate T-B cell help for anti-PreS1 Ab production that is not curtailed by immune tolerance. Immunization of immune tolerant HBV transgenic (Tg) mice with PreS1-WHc VLPs elicited levels of high titer anti-PreS1 nAbs equivalent to wildtype mice. Passive transfer of PreS1 nAbs into human-liver chimeric mice prevented acute infection and cleared serum HBV from mice previously infected with HBV in a model of CHB. At the T cell level, PreS1-WHc VLPs and hybrid WHcAg/HBcAg DNA immunogens elicited HBcAg-specific CD4+ Th and CD8+ CTL responses.

TLR2 Stimulation Increases Cellular Metabolism in CD8+ T Cells and Thereby Enhances CD8+ T Cell Activation, Function, and Antiviral Activity.

TLR2 serves as a costimulatory molecule on activated T cells. However, it is unknown how the functionality and antiviral activity of CD8+ T cells are modulated by direct TLR2 signaling. In this study, we looked at the TLR2-mediated enhancement of TCR-driven CD8+ T cell activation in vitro and in woodchuck hepatitis virus transgenic mice. In vitro stimulation of CD8+ T cells purified from C57BL/6 mice showed that TLR2 agonist Pam3CSK4 directly enhanced the TCR-dependent CD8+ T cell activation. Transcriptome analysis revealed that TLR2 signaling increased expression of bioenergy metabolism-related genes in CD8+ T cells, such as IRF4, leading to improved glycolysis and glutaminolysis. This was associated with the upregulation of genes related to immune regulation and functions such as T-bet and IFN-γ. Glycolysis and glutaminolysis were in turn essential for the TLR2-mediated enhancement of T cell activation. Administration of TLR2 agonist Pam3CSK4 promoted the expansion and functionality of vaccine-primed, Ag-specific CD8+ T cells in both wild type and transgenic mice and improved viral suppression. Thus, TLR2 could promote CD8+ T cell immunity through regulating the energy metabolism.

The Genome Sequence of the Eastern Woodchuck (Marmota monax) – A Preclinical Animal Model for Chronic Hepatitis B

The Eastern woodchuck (Marmota monax) has been extensively used in research of chronic hepatitis B and liver cancer because its infection with the woodchuck hepatitis virus closely resembles a human hepatitis B virus infection. Development of novel immunotherapeutic approaches requires genetic information on immune pathway genes in this animal model. The woodchuck genome was assembled with a combination of high-coverage whole-genome shotgun sequencing of Illumina paired-end, mate-pair libraries and fosmid pool sequencing. The result is a 2.63 Gigabase (Gb) assembly with a contig N50 of 74.5 kilobases (kb), scaffold N50 of 892 kb, and genome completeness of 99.2%. RNA sequencing (RNA-seq) from seven different tissues aided in the annotation of 30,873 protein-coding genes, which in turn encode 41,826 unique protein products. More than 90% of the genes have been functionally annotated, with 82% of them containing open reading frames. This genome sequence and its annotation will enable further research in chronic hepatitis B and hepatocellular carcinoma and contribute to the understanding of immunological responses in the woodchuck.

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.

Structural Differences between the Woodchuck Hepatitis Virus Core Protein in the Dimer and Capsid States Are Consistent with Entropic and Conformational Regulation of Assembly.

Hepadnaviruses are hepatotropic enveloped DNA viruses with an icosahedral capsid. Hepatitis B virus (HBV) causes chronic infection in an estimated 240 million people; woodchuck hepatitis virus (WHV), an HBV homologue, has been an important model system for drug development. The dimeric capsid protein (Cp) has multiple functions during the viral life cycle and thus has become an important target for a new generation of antivirals. Purified HBV and WHV Cp spontaneously assemble into 120-dimer capsids. Though they have 65% identity, WHV Cp has error-prone assembly with stronger protein-protein association. We have taken advantage of the differences in assemblies to investigate the basis of assembly regulation. We determined the structures of the WHV capsid to 4.5-Å resolution by cryo-electron microscopy (cryo-EM) and of the WHV Cp dimer to 2.9-Å resolution by crystallography and examined the biophysical properties of the dimer. We found, in dimer, that the subdomain that makes protein-protein interactions is partially disordered and rotated 21° from its position in capsid. This subdomain is susceptible to proteolysis, consistent with local disorder. WHV assembly shows similar susceptibility to HBV antiviral molecules, suggesting that HBV assembly follows similar transitions. These data show that there is an entropic cost for assembly that is compensated for by the energetic gain of burying hydrophobic interprotein contacts. We propose a series of stages in assembly that incorporate a disorder-to-order transition and structural shifts. We suggest that a cascade of structural changes may be a common mechanism for regulating high-fidelity capsid assembly in HBV and other viruses.IMPORTANCE Virus capsids assemble spontaneously with surprisingly high fidelity. This requires strict geometry and a narrow range of association energies for these protein-protein interactions. It was hypothesized that requiring subunits to undergo a conformational change to become assembly active could regulate assembly by creating an energetic barrier and attenuating association. We found that woodchuck hepatitis virus capsid protein undergoes structural transitions between its dimeric and its 120-dimer capsid states. It is likely that the closely related hepatitis B virus capsid protein undergoes similar structural changes, which has implications for drug design. Regulation of assembly by structural transition may be a common mechanism for many viruses.

Down-regulation of hepatitis delta virus super-infection in the woodchuck model

Mechanisms mediating clearance of hepatitis delta virus (HDV) are poorly understood. This study analyzed in detail profound down-regulation of HDV infection in the woodchuck model. Super-infection with HDV of woodchucks chronically infected with HBV-related woodchuck hepatitis virus produced two patterns. In the first, HDV viremia had a sharp peak followed by a considerable decline, and initial rise of HDV virions' infectivity followed by abrupt infectivity loss. In the second, HDV titer rose and later displayed plateau-like profile with high HDV levels; and HDV infectivity became persistently high when HDV titer reached the plateau. The infectivity loss was not due to defects in the virions' envelope, binding to anti-envelope antibodies, or mutations in HDV genome, but it correlated with profound reduction of the replication capacity of virion-associated HDV genomes. Subsequent finding that in virions with reduced infectivity most HDV RNAs were not full-length genomes suggests possible HDV clearance via RNA fragmentation.

Abstract No. 610 Immunologic characterization of woodchuck hepatitis virus–induced hepatic tumors

To characterize immune patterns in woodchuck viral-induced hepatic tumors, including hepatocellular carcinoma (HCC). All studies were conducted under a research protocol approved by the Institutional Animal Care and Use Committee. Woodchucks (N=5) chronically infected with woodchuck hepatitis virus (WHV) developed hepatic tumors which are similar to human tumors. US, CT, and MRI in vivo imaging were performed under anesthesia. After euthanasia, whole blood was collected, peripheral blood mononuclear cells (PBMCs) were isolated by density gradient and PD-1 expression was assessed by flow cytometry. Tissues were sectioned using 3D-printed molds based on in vivo imaging. Correlative histology and immunohistochemistry analyses of the tissues were performed. For immune characterization of tumors, CD3 (T cells), CD4 (T helpers), TLR-7 (innate immunity activator), PDL-1 (inhibitory checkpoint protein) and alpha-fetoprotein (AFP) staining were performed. Tissue histological examination confirmed hepatic tumors including hyperplasia, hepatocellular adenoma and HCC. Histopathology of tissue adjacent to HCC typically showed inflammation consistent with viral-induced hepatitis. Staining quantification using an H-score revealed that there were no significant differences between normal and tumor tissues for CD3, CD4, TLR-7, and PDL-1 markers. Distribution analysis of the stained slides suggested preferential accumulation of immune cells in the tumor periphery. AFP expression was significantly greater in tumors (P=0.01) compared to normal tissues. Flow cytometry analysis showed low expression of PD-1 on PBMCs. Woodchucks chronically infected with WHV develop hepatic tumors with histopathology and the serum biomarker, AFP, that are comparable to human HCC. Defining the immunologic characteristics in untreated tumors serves as a baseline for using this model for the assessment of local and regional interventional oncology therapies. Such clarity may facilitate the evaluation of the myriad of scientific and mechanistic questions regarding the potential for interventional oncology tools to augment immune pharmacotherapies for HCC.

Differential Expression of Woodchuck Toll-Like Receptors 1-10 in Distinct Forms of Infection and Stages of Hepatitis in Experimental Hepatitis B Virus Infection.

Woodchucks infected with woodchuck hepatitis virus (WHV) represent a highly valuable model of human hepatitis B virus (HBV) infection, chronic hepatitis (CH), and virus induced-primary liver cancer. Toll-like receptors (TLRs) are important mediators of immune responses playing pivotal roles in the pathogenesis of viral diseases; however, their expression profiles in different forms of infection and stages of hepatitis, and in healthy animals remain essentially unknown. In this study, woodchuck TLRs 1–10 exon fragments were identified and TLR genes transcription quantified in livers, primary hepatocytes, peripheral blood mononuclear cells (PBMC), and in selected organs during experimental WHV infection. Among others, liver biopsies from acute hepatitis (AH) and CH showed significantly augmented expression of the majority of TLRs when compared to healthy and woodchucks prior to AH, with resolved AH or primary occult infection. In contrast to the liver tissue, significant upregulation of TLR3, TLR4, and TLR10, but downregulation of TLR7, characterized hepatocytes derived from livers of animals with resolved AH accompanied by secondary occult infection. Hepatocytes from CH showed significantly lower expression or a trend toward suppression of several TLRs when compared to hepatocytes from healthy animals and woodchucks with other forms of infection or hepatitis, suggesting that hepatocyte innate immune response is downregulated during CH. Contrastingly, upregulated transcription of some TLRs characterized PBMC throughout CH. Our study uncovered that TLR expression significantly varies between different forms of hepadnaviral infection and whether infection is accompanied or not by hepatitis. The results showed that the profiles of TLRs’ expression in circulating lymphomononuclear cells do not mirror accurately those of livers and hepatocytes from infected animals. These findings are of importance to the understanding of immune process operating at different sites targeted by virus in the course of hepadnaviral infection and evaluation of future therapies modifying antiviral innate responses in the woodchuck model.

In Vitro Anti-hepatitis B Virus Activity of 2',3'-Dideoxyguanosine.

2',3'-dideoxyguanosine (DoG) has been demonstrated to inhibit duck hepatitis B virus (DHBV) replication in vivo in a duck model of HBV infection. In the current study, the in vitro antiviral effects of DoG on human and animal hepadnaviruses were investigated. Our results showed that DoG effectively inhibited HBV, DHBV, and woodchuck hepatitis virus (WHV) replication in hepatocyte-derived cells in a dose-dependent manner, with 50% effective concentrations (EC50) of 0.3 ± 0.05, 6.82 ± 0.25, and 23.0 ± 1.5μmol/L, respectively. Similar to other hepadnaviral DNA polymerase inhibitors, DoG did not alter the levels of intracellular viral RNA but induced the accumulation of a less-than-full-length viral RNA species, which was recently demonstrated to be generated by RNase H cleavage of pgRNA. Furthermore, using a transient transfection assay, DoG showed similar antiviral activity against HBV wild-type, 3TC-resistant rtA181V, and adefovir-resistant rtN236T mutants. Our results suggest that DoG has potential as a nucleoside analogue drug with anti-HBV activity.

Chinese woodchucks with different susceptibility to WHV infection differ in their genetic background exemplified by cytochrome B and MHC-DRB molecules

BackgroundChinese woodchucks (M. himalayana) were recently found to be susceptible to woodchuck hepatitis virus (WHV) infection. In this study, we aimed to determine the susceptibility to WHV infection of M. himalayana from different areas and their association with the animal genetic background exemplified by cytochrome B and MHC-DRB molecules.MethodsAnimals from four different areas in Qinghai province were inoculated with WHV59 strains. The virological markers including WHV surface antigen (WHsAg), WHV core antibody (WHcAb), and WHV DNA in serum were measured by ELISA and Real-time PCR, respectively. The sequences of cytochrome B gene and MHC-DRB molecules were obtained and sorted with Clustalx software. The nucleotide variation sites were identified using MEGA5 software.ResultsThe animals from four different areas had different susceptibility to WHV infection. Animals from TR and TD areas had a high level of long-lasting viremia, while those from GD and WL areas had a low level of transient viremia after WHV inoculation. All of the animals belong to the same subspecies M. himalayana robusta identified by cytochrome B gene sequences. Based on their nucleotide variation pattern, 8 alleles of cytochrome B gene were identified, and 7 MHC-DRB alleles were identified. Allele A of cytochrome B and Allele Mamo-DRB1*02 of MHC-DRB was found to be frequent in animals from TR and TD areas, while Allele H of cytochrome B and Allele Mamo-DRB1*07 of MHC-DRB was predominant in animals from GD and WL areas.ConclusionChinese woodchucks from different areas differed in their susceptibility to WHV infection, though they belong to the same subspecies M. himalayana robusta. The genetic background exemplified by cytochrome B and MHC-DRB differed in Chinese woodchucks with different susceptibility to WHV infection.

4:03 PM Abstract No. 319 Development and use of the common woodchuck as a model for treatment of hepatocellular carcinoma

Define the utility of woodchucks as a model for development and evaluation of image-guided interventional therapies for the treatment of primary hepatocellular carcinoma (HCC). All studies were conducted under a research protocol approved by the Institutional Animal Care and Use Committee. Woodchuck chronically infected with woodchuck hepatitis virus (WHV) develop neoplasms similar to human HCCs associated with hepatitis B. Woodchucks with and without HCC underwent preoperative CT and US. A 3F introducer sheath was placed in the femoral artery and diagnostic angiography performed via 1.7-2.8F microcatheters. Following superselective catheterization, embolization of arteries supplying HCC tumors was performed to stasis with radiopaque beads (70-150um, Biocompatibles UK LTD) that had been loaded with doxorubicin at 37.5 mg/mL wet bead volume. Following embolization, CBCT, post mortem microCT, and specimen radiography were performed. Tissue was prepared for histopathology and visualization of doxorubicin distribution. Preoperative CT demonstrated single or multifocal hypervascular tumors with varied areas of necrosis, confirmed by superselective catheterization and angiography. Tumors were successfully embolized with post-mortem demonstration of vascular filling with radiopaque beads on CBCT, microCT and specimen radiography. Histology showed beads present within the vasculature. These data may help guide future studies of HCC therapies. Additional research is needed to refine the methods and validate the model; however, a more predictive HCC model than previously described would be valuable for translational medical research.

Safety and efficacy of anti-PD-L1 therapy in the woodchuck model of HBV infection.

Immune clearance of Hepatitis B virus (HBV) is characterized by broad and robust antiviral T cell responses, while virus-specific T cells in chronic hepatitis B (CHB) are rare and exhibit immune exhaustion that includes programmed-death-1 (PD-1) expression on virus-specific T cells. Thus, an immunotherapy able to expand and activate virus-specific T cells may have therapeutic benefit for CHB patients. Like HBV-infected patients, woodchucks infected with woodchuck hepatitis virus (WHV) can have increased hepatic expression of PD-1-ligand-1 (PD-L1), increased PD-1 on CD8+ T cells, and a limited number of virus-specific T cells with substantial individual variation in these parameters. We used woodchucks infected with WHV to assess the safety and efficacy of anti-PD-L1 monoclonal antibody therapy (αPD-L1) in a variety of WHV infection states. Experimentally-infected animals lacked PD-1 or PD-L1 upregulation compared to uninfected controls, and accordingly, αPD-L1 treatment in lab-infected animals had limited antiviral effects. In contrast, animals with naturally acquired WHV infections displayed elevated PD-1 and PD-L1. In these same animals, combination therapy with αPD-L1 and entecavir (ETV) improved control of viremia and antigenemia compared to ETV treatment alone, but with efficacy restricted to a minority of animals. Pre-treatment WHV surface antigen (sAg) level was identified as a statistically significant predictor of treatment response, while PD-1 expression on peripheral CD8+ T cells, T cell production of interferon gamma (IFN-γ) upon in vitro antigen stimulation (WHV ELISPOT), and circulating levels of liver enzymes were not. To further assess the safety of this strategy, αPD-L1 was tested in acute WHV infection to model the risk of liver damage when the extent of hepatic infection and antiviral immune responses were expected to be the greatest. No significant increase in serum markers of hepatic injury was observed over those in infected, untreated control animals. These data support a positive benefit/risk assessment for blockade of the PD-1:PD-L1 pathway in CHB patients and may help to identify patient groups most likely to benefit from treatment. Furthermore, the efficacy of αPD-L1 in only a minority of animals, as observed here, suggests that additional agents may be needed to achieve a more robust and consistent response leading to full sAg loss and durable responses through anti-sAg antibody seroconversion.