qPCR assay for detection of Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Elements from CAR-T and TCR-T cells in fresh and formalin-fixed tissue.

As adoptive cellular therapies become more commonplace in cancer care, there is a growing need to monitor site-specific localization of engineered cells-such as chimeric antigen receptor T (CAR-T) cells and T-cell receptor T (TCR-T) cells-in patients' tissues to understand treatment effectiveness as well as associated adverse events. Manufacturing CAR-T and TCR-T cells involves transduction with viral vectors commonly containing the WPRE gene sequence to enhance gene expression, providing a viable assay target unique to these engineered cells. Quantitative PCR (qPCR) is currently used clinically in fresh patient tissue samples and blood with target sequences specific to each immunotherapy product. Herein, we developed a WPRE-targeted qPCR assay that is broadly applicable for detection of engineered cell products in both fresh and archival formalin-fixed paraffin embedded (FFPE) tissues. Using both traditional PCR and SYBR Green PCR protocols, we demonstrate the use of this WPRE-targeted assay to successfully detect two CAR-T cell and two TCR-T cell products in FFPE tissue. Standard curve analysis reported a reproducible limit of detection at 100 WPRE copies per 20µL PCR reaction. This novel and inexpensive technique could provide better understanding of tissue abundance of engineered therapeutic T cells in both tumor and second-site toxicity tissues and provide quantitative assessment of immune effector cell trafficking in archival tissue.

Host cell-dependent late entry step as determinant of hepatitis B virus infection.

Hepatitis B virus (HBV) has a highly restricted host range and cell tropism. Other than the human sodium taurocholate cotransporting polypeptide (huNTCP), the HBV entry receptor, host determinants of HBV susceptibility are poorly understood. Woodchucks are naturally infected with woodchuck hepatitis virus (WHV), closely related to HBV, but not with HBV. Here, we investigated the capabilities of woodchuck hepatic and human non-hepatic cell lines to support HBV infection. DNA transfection assays indicated that all cells tested supported both HBV and WHV replication steps post entry, including the viral covalently closed circular DNA (cccDNA) formation, which is essential for establishing and sustaining infection. Ectopic expression of huNTCP rendered one, but not the other, woodchuck hepatic cell line and the non-hepatic human cell line competent to support productive HBV entry, defined here by cccDNA formation during de novo infection. All huNTCP-expressing cell lines tested became susceptible to infection with hepatitis D virus (HDV) that shares the same entry receptor and initial steps of entry with HBV, suggesting that a late entry/trafficking step(s) of HBV infection was defective in one of the two woodchuck cell lines. In addition, the non-susceptible woodchuck hepatic cell line became susceptible to HBV after fusion with human hepatic cells, suggesting the lack of a host cell-dependent factor(s) in these cells. Comparative transcriptomic analysis of the two woodchuck cell lines revealed widespread differences in gene expression in multiple biological processes that may contribute to HBV infection. In conclusion, other than huNTCP, neither human- nor hepatocyte-specific factors are essential for productive HBV entry. Furthermore, a late trafficking step(s) during HBV infection, following the shared entry steps with HDV and before cccDNA formation, is subject to host cell regulation and thus, a host determinant of HBV infection.

Agonistic Activation of Cytosolic DNA Sensing Receptors in Woodchuck Hepatocyte Cultures and Liver for Inducing Antiviral Effects.

Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-ß and interleukins 1ß and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB.

Combination Treatment with the Vimentin-Targeting Antibody hzVSF and Tenofovir Suppresses Woodchuck Hepatitis Virus Infection in Woodchucks.

Current treatment options for patients infected with hepatitis B virus (HBV) are suboptimal, because the approved drugs rarely induce cure due to the persistence of the viral DNA genome in the nucleus of infected hepatocytes, and are associated with either severe side effects (pegylated interferon-alpha) or require life-long administration (nucleos(t)ide analogs). We report here the evaluation of the safety and therapeutic efficacy of a novel, humanized antibody (hzVSF) in the woodchuck model of HBV infection. hzVSF has been shown to act as a viral entry inhibitor, most likely by suppressing vimentin-mediated endocytosis of virions. Targeting the increased vimentin expression on liver cells by hzVSF after infection with HBV or woodchuck hepatitis virus (WHV) was demonstrated initially. Thereafter, hzVSF safety was assessed in eight woodchucks naïve for WHV infection. Antiviral efficacy of hzVSF was evaluated subsequently in 24 chronic WHV carrier woodchucks by monotreatment with three ascending doses and in combination with tenofovir alafenamide fumarate (TAF). Consistent with the proposed blocking of WHV reinfection, intravenous hzVSF administration for 12 weeks resulted in a modest but transient reduction of viral replication and associated liver inflammation. In combination with oral TAF dosing, the antiviral effect of hzVSF was enhanced and sustained in half of the woodchucks with an antibody response to viral proteins. Thus, hzVSF safely but modestly alters chronic WHV infection in woodchucks; however, as a combination partner to TAF, its antiviral efficacy is markedly increased. The results of this preclinical study support future evaluation of this novel anti-HBV drug in patients.

Woodchuck Hepatitis Virus Post-Transcriptional Regulation Element (WPRE) Promotes Anti-CD19 BiTE Expression in Expi293 Cells.

Background: Bispecific antibodies represent an important class of monoclonal antibodies (mAbs), with great therapeutic potentials due to their ability to target simultaneously two distinct epitopes. The generation of functional bispecific antibodies with the highest possible yields is particularly critical for the production of these compounds on industrial scales. Anti-CD3 × CD19 bispecific antibody (bsAb) is a bispecific T-cell engager currently used for treating ALL. Herein, we have tried to optimize the expression level of this antibody in mammalian hosts. Methods: Woodchuck hepatitis virus post-transcriptional regulation (WPRE) sequence was incorporated at the 3' end of the expression cassette. This modification resulted in a notable about two-fold increase in the expression of the bsAb in the Expi293 cell line. Results & Conclusion: Follow-up flow cytometry analysis demonstrated the binding properties of the produced antibody at acceptable levels, and in vitro bioactivity assays showed that this product is potent enough for targeting and destroying CD19-positive cells. Our findings show that WPRE enhances the expression of this type of bispecific mAbs in human embryonic kidney-293 family cell lines. This approach can be used in biopharma industry for the mass production of anti-CD3 × CD19 bispecific antibody.

Characterization and Application of Precore/Core-Related Antigens in Animal Models of Hepatitis B Virus Infection.

BACKGROUND AND AIMS: The hepatitis B core-related antigen (HBcrAg), a composite antigen of precore/core gene including classical hepatitis B core protein (HBc) and HBeAg and, additionally, the precore-related antigen PreC, retaining the N-terminal signal peptide, has emerged as a surrogate marker to monitor the intrahepatic HBV covalently closed circular DNA (cccDNA) and to define meaningful treatment endpoints. APPROACH AND RESULTS: Here, we found that the woodchuck hepatitis virus (WHV) precore/core gene products (i.e., WHV core-related antigen [WHcrAg]) include the WHV core protein and WHV e antigen (WHeAg) as well as the WHV PreC protein (WPreC) in infected woodchucks. Unlike in HBV infection, WHeAg and WPreC proteins were N-glycosylated, and no significant amounts of WHV empty virions were detected in WHV-infected woodchuck serum. WHeAg was the predominant form of WHcrAg, and a positive correlation was found between the serum WHeAg and intrahepatic cccDNA. Both WHeAg and WPreC antigens displayed heterogeneous proteolytic processing at their C-termini, resulting in multiple species. Analysis of the kinetics of each component of the precore/core-related antigen, along with serum viral DNA and surface antigens, in HBV-infected chimpanzees and WHV-infected woodchucks revealed multiple distinct phases of viral decline during natural resolution and in response to antiviral treatments. A positive correlation was found between HBc and intrahepatic cccDNA but not between HBeAg or HBcrAg and cccDNA in HBV-infected chimpanzees, suggesting that HBc can be a better marker for intrahepatic cccDNA. CONCLUSIONS: In conclusion, careful monitoring of each component of HBcrAg along with other classical markers will help understand intrahepatic viral activities to elucidate natural resolution mechanisms as well as guide antiviral development.

Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry.

Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.

Ovarian teratoma in a woodchuck (Marmota monax) with hepatocellular carcinoma: radiologic and pathologic features.

BACKGROUND: Teratomas are germ cell neoplasms composed of a wide variety of tissues. In the woodchuck, only one testicular teratoma has been described in the literature. The objective of this report was to describe the radiologic and pathologic findings in a female woodchuck (Marmota monax) with an ovarian teratoma consisting of mature tissues originating from all three germ layers. CASE PRESENTATION: A 2-year-old female woodchuck that had been infected at birth with woodchuck hepatitis virus and subsequently developed hepatocellular carcinoma was incidentally discovered to have a mobile 6.6 × 4.8 × 4.7 cm abdominal mass on computed tomography (CT) imaging. The tumor was predominantly solid and heterogenous on CT with soft tissue, fat, and areas of dense calcification. The teratoma did not enhance with intravenous contrast administration. On ultrasound, the tumor was solid with heterogeneous echogenicity, reflecting the fat content and areas of calcification. Sonolucent areas were present that may have represented cysts. There was heterogeneously increased signal on T1-weighted magnetic resonance imaging (MRI) and heterogeneous hyperintensity in T2-weighted imaging. Fat was evident within the tumor. At necropsy, the tumor was attached to the distal end of the right uterine horn. Histopathology showed mature tissue types representing all three germ layers. CONCLUSIONS: Ovarian teratoma should be considered in the differential diagnosis of ovarian or abdominal masses in woodchucks. The tumor displayed mature tissue derived from all three germ layers. CT, ultrasound, and MRI findings were presented in detail and matched the typical imaging appearance of teratomas.

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.

Transarterial Chemoembolization in a Woodchuck Model of Hepatocellular Carcinoma.

PURPOSE: To assess the feasibility of transarterial chemoembolization with drug-eluting embolic (DEE) microspheres in a woodchuck model of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Nine 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. RESULTS: The 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. CONCLUSIONS: Woodchuck 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.

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.

Intrahepatic hepatitis B virus large surface antigen induces hepatocyte hyperploidy via failure of cytokinesis.

Hepatitis B virus (HBV) is an aetiological factor for liver cirrhosis and hepatocellular carcinoma (HCC). Despite current antiviral therapies that successfully reduce the viral load in patients with chronic hepatitis B, persistent hepatitis B surface antigen (HBsAg) remains a risk factor for HCC. To explore whether intrahepatic viral antigens contribute directly to hepatocarcinogenesis, we monitored the mitotic progression of HBV-positive cells. Cytokinesis failure was increased in HBV-positive HepG2.2.15 and 1.3ES2 cells, as well as in HuH-7 cells transfected with a wild-type or X-deficient HBV construct, but not in cells transfected with an HBsAg-deficient construct. We show that expression of viral large surface antigen (LHBS) was sufficient to induce cytokinesis failure of immortalized hepatocytes. Premitotic defects with DNA damage and G2 /M checkpoint attenuation preceded cytokinesis in LHBS-positive cells, and ultimately resulted in hyperploidy. Inhibition of polo-like kinase-1 (Plk1) not only restored the G2 /M checkpoint in these cells, but also suppressed LHBS-mediated in vivo tumourigenesis. Finally, a positive correlation between intrahepatic LHBS expression and hepatocyte hyperploidy was detected in >70% of patients with chronic hepatitis B. We conclude that HBV LHBS provokes hyperploidy by inducing DNA damage and upregulation of Plk1; the former results in atypical chromatin structures, and the latter attenuates the function of the G2 /M DNA damage checkpoint. Our data uncover a mechanism by which genomic integrity of hepatocytes is disrupted by viral LHBS. These findings highlight the role of intrahepatic surface antigen as an oncogenic risk factor in the development of HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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

BACKGROUND: Approximately 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. METHODS: In 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. RESULTS: It 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. CONCLUSIONS: Although 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.

Transduction Profile of the Marmoset Central Nervous System Using Adeno-Associated Virus Serotype 9 Vectors.

The common marmoset is a small New World primate that has attracted remarkable attention as a potential experimental animal link between rodents and humans. Adeno-associated virus (AAV) vector-mediated expression of a disease-causing gene or a potential therapeutic gene in the brain may allow the construction of a marmoset model of a brain disorder or an exploration of the possibility of gene therapy. To gain more insights into AAV vector-mediated transduction profiles in the marmoset central nervous system (CNS), we delivered AAV serotype 9 (AAV9) vectors expressing GFP to the cisterna magna or the cerebellar cortex. Intracisternally injected AAV9 vectors expanded in the CNS according to the cerebrospinal fluid (CSF) flow, by retrograde transport through neuronal axons or via intermediary transcytosis, resulting in diffuse and global transduction within the CNS. In contrast, cerebellar parenchymal injection intensely transduced a more limited area, including the cerebellar cortex and cerebellar afferents, such as neurons of the pontine nuclei, vestibular nucleus and inferior olivary nucleus. In the spinal cord, both administration routes resulted in labeling of the dorsal column and spinocerebellar tracts, presumably by retrograde transport from the medulla oblongata and cerebellum, respectively. Motor neurons and dorsal root ganglia were also transduced, possibly by diffusion of the vector down the subarachnoid space along the cord. Thus, these two administration routes led to distinct transduction patterns in the marmoset CNS, which could be utilized to generate different disease animal models and to deliver therapeutic genes for the treatment of diseases affecting distinct brain areas.

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.

Immunogenicity Evaluation of Modified Adenovirus Vaccines Expressing Porcine Circovirus Type 2 Capsid Protein in Pigs.

Porcine circovirus type 2 (PCV2) adenovirus vaccine has been reported, but strong immune responses induced by adenovirus vector can decrease vaccine efficacy. To reduce the immunogenicity of adenovirus proteins, in previous study, we constructed the PCV2 adenovirus vaccine either modified with human cytomegalovirus first intron (Intron A) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to increase the expression of Cap, or coexpressed porcine tumor necrosis factor-related activate protein (CD40L) and granulocyte macrophage colony-stimulating factor (GMCSF) to improve the immunogenicity of PCV2 Cap adenovirus vaccine. All these vaccines were evaluated in mice. In the present study, the protective immune responses of Intron A/WPRE-modified recombinant adenovirus Ad-A-C-W and CD40L/GMCSF-modified recombinant adenovirus Ad-CD40L-Cap-GMCSF were evaluated in pigs. Enzyme-linked immunosorbent assay and virus neutralization assay showed that both Ad-A-C-W and Ad-CD40L-Cap-GMCSF could induce a higher specific antibody and neutralizing antibody than Ad-Cap (p < 0.05). Lymphocyte proliferation assay and cytokine release assay showed that Ad-A-C-W and Ad-CD40L-Cap-GMCSF induced a stronger cellular immune response than Ad-Cap. The PCV2 challenge experiment showed that viral loads of Ad-A-C-W-vaccinated group and Ad-CD40L-Cap-GMCSF-vaccinated group were lower than Ad-Cap vaccinated group (p < 0.05) after pigs were oronasally challenged with 5 × 105 TCID50 PCV2. Autopsy and histopathological examination showed that no obvious clinical and microscopic lesions were observed in groups Ad-Cap, Ad-A-C-W, and Ad-CD40L-Cap-GMCSF. Taken together, the results demonstrated that two modified recombinant adenovirus vaccines (Ad-A-C-W and Ad-CD40L-Cap-GMCSF) induced stronger humoral and cellular immune responses and provided better protection than unmodified adenovirus Ad-Cap. Therefore, Ad-A-C-W and Ad-CD40L-Cap-GMCSF would be used as potential vaccines for prevention and control of PCV2 infection.

Molecular cloning, characterization and expression analysis of woodchuck retinoic acid-inducible gene I.

Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-ß (IFN-ß). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-ß transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.