The Experts below are selected from a list of 231 Experts worldwide ranked by ideXlab platform
Bud C. Tennant - One of the best experts on this subject based on the ideXlab platform.
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immunogenicity in mice and rabbits of dna vaccines expressing Woodchuck Hepatitis Virus antigens
Vaccine, 2008Co-Authors: Alain Luxembourg, Bud C. Tennant, Stephan Menne, Drew Hannaman, Ken Wills, Robert M Bernard, Paul J CoteAbstract:Abstract The licensed vaccine against Hepatitis B Virus (HBV) is an effective means to prevent infection, but is not an effective therapeutic strategy to treat established chronic infections when used alone. In an animal model of chronic HBV infection (the Woodchuck experimentally infected with Woodchuck Hepatitis Virus (WHV)), the combination of conventional vaccine and potent antiviral drugs has shown promise as a potential therapeutic intervention. This approach might be improved further through the application of newer vaccine technologies. In the present study, we evaluated electroporation (EP)-based intramuscular (i.m.) delivery of a codon-optimized DNA vaccine for the WHV surface antigen (WHsAg) in mice and rabbits. In mice, this immunization procedure compared favorably to vaccination by i.m. injection of the DNA vaccine or i.m. administration of a recombinant WHsAg–alum vaccine, exhibiting characteristics expected to be beneficial for a therapeutic vaccine strategy. These included dose efficiency, consistency, vigorous induction of antibody responses to WHsAg, as well as a Th1 bias. Following scale-up to rabbits, a species that approximates the size of the Woodchuck, the EP dosing regimen was markedly more effective than conventional i.m. injection of the DNA vaccine. Taken together, these results provide the foundation for studies of EP-based DNA immunization in the Woodchuck in order to further assess its potential as an immunotherapeutic approach for treatment of chronic HBV infection in humans.
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immunosuppression reactivates viral replication long after resolution of Woodchuck Hepatitis Virus infection
Hepatology, 2007Co-Authors: Stephan Menne, Paul J Cote, John L Gerin, Scott D Butler, Ilia Toshkov, Bud C. TennantAbstract:Resolution of Hepatitis B Virus (HBV) infection is characterized by coordinated humoral and cellular immune responses. Immunity is durable over decades, protecting the host from reinfection and potential activation of residual HBV. Woodchucks infected at birth with Woodchuck Hepatitis Virus (WHV) cleared viremia and developed antibodies to surface antigen (anti-WHs). Woodchucks became seronegative for anti-WHs 3-6 years later, but in some, WHV DNA was detected in serum, liver, and/or peripheral blood mononuclear cells (PBMCs). Those with WHV DNA had increased in vitro cellular immune responses to viral antigens, CD4 and CD8 markers, and Th1-type cytokines, suggesting active WHV-specific T lymphocytes. Immunosuppression for 12 weeks using cyclosporine A in such Woodchucks resulted in transient reactivation of WHV replication. Serum of 1 Woodchuck that became positive for WHV DNA during immunosuppression was inoculated into WHV-susceptible Woodchucks, and a productive infection was demonstrated. The results indicate that after infection durable cellular immunity to WHV is essential for the long-term control of viral replication and is probably maintained by continuous priming from residual Virus. Conclusion: These experimental observations demonstrate the potential of immunosuppression to reactivate HBV after resolution of infection. (HEPATOLOGY 2007;45:614–622.)
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decreased oxidative dna damage and accelerated cell turnover in Woodchuck Hepatitis Virus infected liver
Hepatology Research, 2003Co-Authors: Molly Ogorman, Bud C. Tennant, Ilia A Tochkov, Savitri Sharma, John D Groopman, Kathleen B SchwarzAbstract:Abstract Infection of newborn Woodchucks with Woodchuck Hepatitis Virus (WHV) results in hepatocellular carcinoma (HCC). Since oxidative damage may be carcinogenic, we investigated the relationship between WHV infection and oxidative damage to hepatic lipids and DNA. Eastern Woodchucks were infected with WHV. Hepatic lipid peroxidation was assessed in vitro in isolated hepatocytes by thiobarbituric acid reactive substances (TBARS). Oxidative DNA damage was assessed in vivo in snap-frozen livers by 8-hydroxy-2′-deoxyguanosine (8-OH-dG). The proliferation index (PI) and apoptotic index (AI) were also determined. WHV infection was associated with increased hepatic lipid peroxidation (0.51±0.04 nmols TBARS per mg protein for WHV+ hepatocytes vs. 0.38±0.04 for WHV negative controls, P 6 dG, P 20-fold. We conclude that WHV infection is associated with increased in vitro lipid peroxidation and decreased in vivo oxidative DNA damage. The increased PI and AI in the WHV+livers suggest that rapid cell turnover dilutes 8-OH-dG concentration.
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immunogenic effects of Woodchuck Hepatitis Virus surface antigen vaccine in combination with antiviral therapy breaking of humoral and cellular immune tolerance in chronic Woodchuck Hepatitis Virus infection
Intervirology, 2002Co-Authors: Stephan Menne, Bud C. Tennant, Brent E Korba, John L Gerin, Carol A Roneker, Paul J CoteAbstract:Objective: A rational treatment strategy for chronic Hepatitis B Virus (HBV) infection might involve the modulation of immunity after the reduction of viremia and antigenemia. This strategy was tested in Woodchucks chronically infected with the Woodchuck Hepatitis Virus (WHV) by combining antiviral treatment with 1-(2-fluoro-5-methyl-β-L-arabinofuranosyl)-uracil (L-FMAU) and therapeutic vaccination with WHV surface antigen (WHsAg). Methods: Chronic WHV carriers were treated with L-FMAU or placebo for 32 weeks. Half the Woodchucks in each group then received four injections of a conventional WHsAg vaccine during the next 16 weeks. Results: Vaccination alone elicited low-level antibody to WHsAg (anti-WHs) in most carriers but did not affect serum WHV DNA, WHsAg or liver enzyme responses. Carriers treated first with L-FMAU to reduce WHV DNA and WHsAg and then vaccinated developed similar low-level anti-WHs and normalized liver enzymes. Following vaccinations, WHsAg-specific cell-mediated immunity (CMI) was demonstrated in both groups, but was significantly enhanced in carriers treated with L-FMAU, and was broadened to include WHV core antigen (WHcAg) and selected peptide epitopes of WHcAg and WHsAg. Anti-WHs and associated CMI to WHcAg and WHsAg were observed after drug discontinuation in half of the carriers that received L-FMAU alone. Conclusions: Vaccination with WHsAg following treatment with L-FMAU disrupted Virus-specific humoral and cell-mediated immune tolerance in chronic WHV infection and enhanced the immune response profiles beyond those seen with monotherapies alone. The combination therapy resulted in immune response profiles that resembled those observed during resolution of WHV infection. The results in Woodchucks demonstrate the feasibility of using such a combination therapy for the control of chronic HBV infection in humans.
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antiviral activity of clevudine l fmau 1 2 fluoro 5 methyl β l arabinofuranosyl uracil against Woodchuck Hepatitis Virus replication and gene expression in chronically infected Woodchucks marmota monax
Hepatology, 2001Co-Authors: Simon Peek, William E. Hornbuckle, Bud C. Tennant, Frances V Wells, Paul J Cote, John L Gerin, James R Jacob, Betty H Baldwin, Ilia Toshkov, Brent E KorbaAbstract:L-FMAU [1-(2-fluoro-5-methyl-β,L-arabinofuranosyl) uracil] has been shown to be an effective inhibitor of Hepatitis B Virus (HBV) and duck Hepatitis B Virus replication in cell culture and duck Hepatitis B Virus replication in acutely infected Peking ducks. The Woodchuck Hepatitis Virus (WHV) and its natural host, the Eastern Woodchuck (Marmota monax), have been established as a predictive model for the evaluation of antiviral therapies against chronic HBV infection. In this report, the antiviral activity of l-FMAU against WHV replication in chronically infected Woodchucks is described. Four weeks of once-daily oral administration of L-FMAU significantly reduced viremia, antigenemia, intrahepatic WHV replication, and intrahepatic expression of Woodchuck Hepatitis Virus core antigen (WHcAg) in a dose-dependent manner. At the highest dose administered (10 mg/kg/d), significant reductions of intrahepatic WHV RNA and covalently closed circular (ccc)WHV-DNA levels also were observed. The reduction in viremia was remarkably rapid at the higher doses of L-FMAU, with greater than 1,000-fold reductions in WHV-DNA serum levels observed after as little as 2 to 3 days of therapy. Following the withdrawal of therapy, a dose-related delay in viremia rebound was observed. At the highest doses used, viremia remained significantly suppressed in at least one half of the treated animals for 10 to 12 weeks' posttreatment. No evidence of drug-related toxicity was observed in the treated animals. L-FMAU is an exceptionally potent antihepadnaviral agent in vitro and in vivo, and is a suitable candidate for antiviral therapy of chronic HBV infection. (HEPATOLOGY 2001;33:254-266)
Paul J Cote - One of the best experts on this subject based on the ideXlab platform.
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immunogenicity in mice and rabbits of dna vaccines expressing Woodchuck Hepatitis Virus antigens
Vaccine, 2008Co-Authors: Alain Luxembourg, Bud C. Tennant, Stephan Menne, Drew Hannaman, Ken Wills, Robert M Bernard, Paul J CoteAbstract:Abstract The licensed vaccine against Hepatitis B Virus (HBV) is an effective means to prevent infection, but is not an effective therapeutic strategy to treat established chronic infections when used alone. In an animal model of chronic HBV infection (the Woodchuck experimentally infected with Woodchuck Hepatitis Virus (WHV)), the combination of conventional vaccine and potent antiviral drugs has shown promise as a potential therapeutic intervention. This approach might be improved further through the application of newer vaccine technologies. In the present study, we evaluated electroporation (EP)-based intramuscular (i.m.) delivery of a codon-optimized DNA vaccine for the WHV surface antigen (WHsAg) in mice and rabbits. In mice, this immunization procedure compared favorably to vaccination by i.m. injection of the DNA vaccine or i.m. administration of a recombinant WHsAg–alum vaccine, exhibiting characteristics expected to be beneficial for a therapeutic vaccine strategy. These included dose efficiency, consistency, vigorous induction of antibody responses to WHsAg, as well as a Th1 bias. Following scale-up to rabbits, a species that approximates the size of the Woodchuck, the EP dosing regimen was markedly more effective than conventional i.m. injection of the DNA vaccine. Taken together, these results provide the foundation for studies of EP-based DNA immunization in the Woodchuck in order to further assess its potential as an immunotherapeutic approach for treatment of chronic HBV infection in humans.
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Serologic Survey of Woodchuck Hepatitis Virus in North Carolina Woodchucks (Marmota monax)
Journal of Zoo and Wildlife Medicine, 2008Co-Authors: John M. Cullen, D. Lindsey-pegram, Paul J CoteAbstract:Abstract The prevalence of Woodchuck Hepatitis Virus (WHV) in wild populations of Woodchucks is understudied and therefore unclear. Although infection is common in the southeastern region of Pennsylvania and surrounding states, it is virtually absent in New York and New England. Sera were collected from wild Woodchucks from Orange County, North Carolina and tested for the presence of markers of current or previous infection with WHV. Of the 24 Woodchucks tested, there were three animals (12.5%) with WHV surface antigen as well as antibodies to Woodchuck Hepatitis core antigen in their serum, indicative of active infection. There were four (17%) animals with antibodies to WHV core antigen but no Woodchuck Hepatitis surface antigen, indicative of prior infections. The remaining 17 animals had no detectable markers of WHV infection. These data indicate that WHV is present in central North Carolina at rates approaching those seen in endemic areas, such as the mid-Atlantic region of the United States.
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immunosuppression reactivates viral replication long after resolution of Woodchuck Hepatitis Virus infection
Hepatology, 2007Co-Authors: Stephan Menne, Paul J Cote, John L Gerin, Scott D Butler, Ilia Toshkov, Bud C. TennantAbstract:Resolution of Hepatitis B Virus (HBV) infection is characterized by coordinated humoral and cellular immune responses. Immunity is durable over decades, protecting the host from reinfection and potential activation of residual HBV. Woodchucks infected at birth with Woodchuck Hepatitis Virus (WHV) cleared viremia and developed antibodies to surface antigen (anti-WHs). Woodchucks became seronegative for anti-WHs 3-6 years later, but in some, WHV DNA was detected in serum, liver, and/or peripheral blood mononuclear cells (PBMCs). Those with WHV DNA had increased in vitro cellular immune responses to viral antigens, CD4 and CD8 markers, and Th1-type cytokines, suggesting active WHV-specific T lymphocytes. Immunosuppression for 12 weeks using cyclosporine A in such Woodchucks resulted in transient reactivation of WHV replication. Serum of 1 Woodchuck that became positive for WHV DNA during immunosuppression was inoculated into WHV-susceptible Woodchucks, and a productive infection was demonstrated. The results indicate that after infection durable cellular immunity to WHV is essential for the long-term control of viral replication and is probably maintained by continuous priming from residual Virus. Conclusion: These experimental observations demonstrate the potential of immunosuppression to reactivate HBV after resolution of infection. (HEPATOLOGY 2007;45:614–622.)
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immunogenic effects of Woodchuck Hepatitis Virus surface antigen vaccine in combination with antiviral therapy breaking of humoral and cellular immune tolerance in chronic Woodchuck Hepatitis Virus infection
Intervirology, 2002Co-Authors: Stephan Menne, Bud C. Tennant, Brent E Korba, John L Gerin, Carol A Roneker, Paul J CoteAbstract:Objective: A rational treatment strategy for chronic Hepatitis B Virus (HBV) infection might involve the modulation of immunity after the reduction of viremia and antigenemia. This strategy was tested in Woodchucks chronically infected with the Woodchuck Hepatitis Virus (WHV) by combining antiviral treatment with 1-(2-fluoro-5-methyl-β-L-arabinofuranosyl)-uracil (L-FMAU) and therapeutic vaccination with WHV surface antigen (WHsAg). Methods: Chronic WHV carriers were treated with L-FMAU or placebo for 32 weeks. Half the Woodchucks in each group then received four injections of a conventional WHsAg vaccine during the next 16 weeks. Results: Vaccination alone elicited low-level antibody to WHsAg (anti-WHs) in most carriers but did not affect serum WHV DNA, WHsAg or liver enzyme responses. Carriers treated first with L-FMAU to reduce WHV DNA and WHsAg and then vaccinated developed similar low-level anti-WHs and normalized liver enzymes. Following vaccinations, WHsAg-specific cell-mediated immunity (CMI) was demonstrated in both groups, but was significantly enhanced in carriers treated with L-FMAU, and was broadened to include WHV core antigen (WHcAg) and selected peptide epitopes of WHcAg and WHsAg. Anti-WHs and associated CMI to WHcAg and WHsAg were observed after drug discontinuation in half of the carriers that received L-FMAU alone. Conclusions: Vaccination with WHsAg following treatment with L-FMAU disrupted Virus-specific humoral and cell-mediated immune tolerance in chronic WHV infection and enhanced the immune response profiles beyond those seen with monotherapies alone. The combination therapy resulted in immune response profiles that resembled those observed during resolution of WHV infection. The results in Woodchucks demonstrate the feasibility of using such a combination therapy for the control of chronic HBV infection in humans.
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antiviral activity of clevudine l fmau 1 2 fluoro 5 methyl β l arabinofuranosyl uracil against Woodchuck Hepatitis Virus replication and gene expression in chronically infected Woodchucks marmota monax
Hepatology, 2001Co-Authors: Simon Peek, William E. Hornbuckle, Bud C. Tennant, Frances V Wells, Paul J Cote, John L Gerin, James R Jacob, Betty H Baldwin, Ilia Toshkov, Brent E KorbaAbstract:L-FMAU [1-(2-fluoro-5-methyl-β,L-arabinofuranosyl) uracil] has been shown to be an effective inhibitor of Hepatitis B Virus (HBV) and duck Hepatitis B Virus replication in cell culture and duck Hepatitis B Virus replication in acutely infected Peking ducks. The Woodchuck Hepatitis Virus (WHV) and its natural host, the Eastern Woodchuck (Marmota monax), have been established as a predictive model for the evaluation of antiviral therapies against chronic HBV infection. In this report, the antiviral activity of l-FMAU against WHV replication in chronically infected Woodchucks is described. Four weeks of once-daily oral administration of L-FMAU significantly reduced viremia, antigenemia, intrahepatic WHV replication, and intrahepatic expression of Woodchuck Hepatitis Virus core antigen (WHcAg) in a dose-dependent manner. At the highest dose administered (10 mg/kg/d), significant reductions of intrahepatic WHV RNA and covalently closed circular (ccc)WHV-DNA levels also were observed. The reduction in viremia was remarkably rapid at the higher doses of L-FMAU, with greater than 1,000-fold reductions in WHV-DNA serum levels observed after as little as 2 to 3 days of therapy. Following the withdrawal of therapy, a dose-related delay in viremia rebound was observed. At the highest doses used, viremia remained significantly suppressed in at least one half of the treated animals for 10 to 12 weeks' posttreatment. No evidence of drug-related toxicity was observed in the treated animals. L-FMAU is an exceptionally potent antihepadnaviral agent in vitro and in vivo, and is a suitable candidate for antiviral therapy of chronic HBV infection. (HEPATOLOGY 2001;33:254-266)
Thomas J Hope - One of the best experts on this subject based on the ideXlab platform.
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enhanced expression of transgenes from adeno associated Virus vectors with the Woodchuck Hepatitis Virus posttranscriptional regulatory element implications for gene therapy
Human Gene Therapy, 1999Co-Authors: Jonathan E Loeb, Wendy S Cordier, Matthew E Harris, Matthew D Weitzman, Thomas J HopeAbstract:The Woodchuck Hepatitis Virus posttranscriptional regulatory element (WPRE) evolved to stimulate the expression of intronless viral messages. To determine whether this ability to enhance expression could be useful in nonviral and heterologous viral gene delivery systems, we analyzed the ability of the WPRE to elevate the expression of a cDNA encoding the green fluorescent protein (GFP) in these contexts. We find that the WPRE can stimulate the expression of GFP when the gene is delivered by transfection or transduction with recombinant adeno-associated Virus (AAV). Enhancement occurred both during transient expression and when the gene is stably incorporated into the genome of target cells. This enhancement required that the WPRE be located in cis within the GFP message, and was observed in both transformed cell lines and primary human fibroblasts. These results demonstrate that the WPRE will be an effective tool for increasing the long-term expression of transgenes in gene therapy.
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Woodchuck Hepatitis Virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors
Journal of Virology, 1999Co-Authors: Romain Zufferey, John Edward Donello, Didier Trono, Thomas J HopeAbstract:The expression of genes delivered by retroviral vectors is often inefficient, a potential obstacle for their widespread use in human gene therapy. Here, we explored the possibility that the posttranscriptional regulatory element of Woodchuck Hepatitis Virus (WPRE) might help resolve this problem. Insertion of the WPRE in the 3′ untranslated region of coding sequences carried by either oncoretroviral or lentiviral vectors substantially increased their levels of expression in a transgene-, promoter- and vector-independent manner. The WPRE thus increased either luciferase or green fluorescent protein production five- to eightfold, and effects of a comparable magnitude were observed with either the immediate-early cytomegaloVirus or the herpesVirus thymidine kinase promoter and with both human immunodeficiency Virus- and murine leukemia Virus-based vectors. The WPRE exerted this influence only when placed in the sense orientation, consistent with its predicted posttranscriptional mechanism of action. These results demonstrate that the WPRE significantly improves the performance of retroviral vectors and emphasize that posttranscriptional regulation of gene expression should be taken into account in the design of gene delivery systems.
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Woodchuck Hepatitis Virus contains a tripartite posttranscriptional regulatory element
Journal of Virology, 1998Co-Authors: John Edward Donello, Jonathan E Loeb, Thomas J HopeAbstract:The Hepatitis B Virus posttranscriptional regulatory element (HBVPRE) is a cis-acting RNA element that partially overlaps with enhancer I and is required for the cytoplasmic accumulation of HBV surface RNAs. We find that the closely related Woodchuck Hepatitis Virus (WHV), which has been shown to lack a functional enhancer I, also contains a posttranscriptional regulatory element (WPRE). Deletion analysis suggests that the WPRE consists of three independent subelements. Comparison of the bipartite HBVPRE and tripartite WPRE activities reveals that the tripartite WPRE is two to three times more active than the bipartite HBVPRE. Mutation of a single WPRE subelement decreases WPRE activity to the level of the HBVPRE. Bipartite and tripartite chimeras of the WPRE and HBVPRE possess activities which suggest that elements containing three subelements are posttranscriptionally stronger than those containing two. These data demonstrate that the posttranscriptional regulatory element is conserved within the mammalian hepadnaViruses and that its strength is determined by the number of subelements within the RNA.
Michael Roggendorf - One of the best experts on this subject based on the ideXlab platform.
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lipopolysaccharide induced innate immune responses in primary hepatocytes downregulates Woodchuck Hepatitis Virus replication via interferon independent pathways
Cellular Microbiology, 2009Co-Authors: Xiaoyong Zhang, Michael Roggendorf, Dongliang Yang, Zhongji Meng, Yang Xu, J F Schlaak, Mengji LuAbstract:Summary Our previous studies have shown that Toll-like receptor (TLR) ligands, Poly I:C and lipopolysaccharide (LPS), are able to activate non-parenchymal liver cells and trigger the production of interferon (IFN) to inhibit Hepatitis B Virus replication in vivo and in vitro. However, little is known about TLR-mediated cellular responses in primary hepatocytes. By the model of Woodchuck Hepatitis Virus (WHV) infected primary Woodchuck hepatocytes (PWHs), Poly I:C and LPS stimulation resulted in upregulation of cellular antiviral genes and relevant TLRs mRNA expression respectively. LPS stimulation led to a pronounced reduction of WHV replicative intermediates without a significant IFN induction. Poly I:C transfection resulted in the production of IFN and a highly increased expression of antiviral genes in PWHs and slight inhibitory effect on WHV replication. LPS could activate nuclear factor kappa B, MAPK and PI-3k/Akt pathways in PWHs. Further, inhibitors of MAPK-ERK and PI-3k/Akt pathways, but not that of IFN signalling pathway, were able to block the antiviral effect of LPS. These results indicate that IFN- independent pathways which activated by LPS are able to downregulate hepadnaviral replication in hepatocytes.
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inhibition of Woodchuck Hepatitis Virus gene expression in primary hepatocytes by sirna enhances the cellular gene expression
Virology, 2009Co-Authors: Zhongji Meng, Michael Roggendorf, Dongliang Yang, Xiaoyong Zhang, Jun Wu, Thomas Schreiter, Yang Xu, J F Schlaak, Mengji LuAbstract:Small interfering RNA (siRNA) has been shown to be active to inhibit the Hepatitis B Virus gene expression and replication in transient and stable transfection systems. Here in primary hepatocytes prepared from naturally Woodchuck Hepatitis Virus (WHV)-infected Woodchucks, four siRNAs targeting the WHV preS1, S, C, and X region led to a depletion of WHV transcripts and replicative intermediates with different kinetics and a decreased production of viral particles. Two siRNAs targeting WHV S and X region had the highest efficacy to deplete 70% of WHV transcripts and replicative intermediates. In addition, siRNA-mediated suppression of WHV enhanced the expression of cellular genes like MxA and MHC I. Specific siRNAs are able to inhibit the hepadnaviral replication and enhance the expression of cellular genes relevant for antiviral actions. Thus, siRNAs might be useful as novel antiviral agents for the treatment of chronic HBV infection.
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The interferon-α gene family of Marmota himalayana, a Chinese marmot species with susceptibility to Woodchuck Hepatitis Virus infection
Developmental and Comparative Immunology, 2007Co-Authors: Yinping Lu, Michael Roggendorf, Yongjun Tian, Mengji Lu, Baoju Wang, Hong-ping Huang, Ji-hua Dong, Dongliang YangAbstract:Abstract The interferon- α (IFN- α ) gene family is an important part of the immune system. Recombinant interferon- α is widely used to treat viral Hepatitis and malignant diseases. Marmota himalayana has been found to be susceptible to Woodchuck Hepatitis Virus, a Virus genetically related to Hepatitis B Virus (HBV), and is suitable as an animal model for studies on HBV infection. Here, the IFN- α gene family of M. himalayana (cwIFN- α ) was characterized. Sequence data indicate that the cwIFN- α family consists of at least 8 functional sequences and 6 pseudogenes with high homology within the family and to IFN- α of Marmota monax , a related species and well-established animal model. The recombinant cwIFN- α subtypes were expressed and tested to be active in viral protection assay and to induce expression of MxA in a species-specific manner. This work provides essential information for future work on testing new therapeutic approaches of HBV infection based on IFN- α in M. himalayana .
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Identification of a glycosylation site in the Woodchuck Hepatitis Virus preS2 protein and its role in protein trafficking.
Journal of General Virology, 2004Co-Authors: Oliver Schildgen, Michael Roggendorf, M. LuAbstract:The middle surface antigen (M-sAg) of hepadnaViruses is one of three envelope proteins that share a common C-terminal S domain. M-sAg contains the preS2 domain in addition to the S region. The preS2 region of Woodchuck Hepatitis Virus (WHV) contains a potential glycosylation site Asn-Gln-Thr at amino acid (aa) positions 3–5. In this study, we mutated this site by site-directed mutagenesis and confirmed that glycosylation occurs here. In in vitro translation assays, the mutation Thr to Asn at aa 5 significantly impaired glycosylation of M-sAg. The mutated M-sAg formed abnormal clustered structures in transfected cells as determined by immunofluorescent staining. Confocal microscopic analysis showed that an enrichment of this glycosylation-deficient protein in the Golgi apparatus occurred, which is not typical for the wild-type protein. These results are consistent with earlier findings that incorrect glycosylation of viral proteins may interfere with Virus assembly.
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Immunization with recombinant Woodchuck Hepatitis Virus nucleocapsid antigen or Hepatitis B Virus nucleocapsid antigen protects Woodchucks from Woodchuck Hepatitis Virus infection.
Vaccine, 2002Co-Authors: Florian Schodel, Klaus Fuchs, Darrell L Peterson, Georg Neckermann, Steve Fuller, Hans Will, Michael RoggendorfAbstract:Woodchucks were immunized with recombinant Woodchuck Hepatitis Virus (WHV) nucleocapsid antigen (WHcAg) or Hepatitis B Virus (HBV) nucleocapsid antigen (HBcAg) and challenged with 106 WHV ID50. Six out of six Woodchucks immunized with WHcAg and four out of six immunized with HBcAg were protected from WHV infection. Woodchucks immunized with WHcAg or HBcAg developed high serum antibody titres against WHcAg or HBcAg. Antibodies against WHc and HBc displayed little cross-reactivity (
John L Gerin - One of the best experts on this subject based on the ideXlab platform.
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immunosuppression reactivates viral replication long after resolution of Woodchuck Hepatitis Virus infection
Hepatology, 2007Co-Authors: Stephan Menne, Paul J Cote, John L Gerin, Scott D Butler, Ilia Toshkov, Bud C. TennantAbstract:Resolution of Hepatitis B Virus (HBV) infection is characterized by coordinated humoral and cellular immune responses. Immunity is durable over decades, protecting the host from reinfection and potential activation of residual HBV. Woodchucks infected at birth with Woodchuck Hepatitis Virus (WHV) cleared viremia and developed antibodies to surface antigen (anti-WHs). Woodchucks became seronegative for anti-WHs 3-6 years later, but in some, WHV DNA was detected in serum, liver, and/or peripheral blood mononuclear cells (PBMCs). Those with WHV DNA had increased in vitro cellular immune responses to viral antigens, CD4 and CD8 markers, and Th1-type cytokines, suggesting active WHV-specific T lymphocytes. Immunosuppression for 12 weeks using cyclosporine A in such Woodchucks resulted in transient reactivation of WHV replication. Serum of 1 Woodchuck that became positive for WHV DNA during immunosuppression was inoculated into WHV-susceptible Woodchucks, and a productive infection was demonstrated. The results indicate that after infection durable cellular immunity to WHV is essential for the long-term control of viral replication and is probably maintained by continuous priming from residual Virus. Conclusion: These experimental observations demonstrate the potential of immunosuppression to reactivate HBV after resolution of infection. (HEPATOLOGY 2007;45:614–622.)
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immunogenic effects of Woodchuck Hepatitis Virus surface antigen vaccine in combination with antiviral therapy breaking of humoral and cellular immune tolerance in chronic Woodchuck Hepatitis Virus infection
Intervirology, 2002Co-Authors: Stephan Menne, Bud C. Tennant, Brent E Korba, John L Gerin, Carol A Roneker, Paul J CoteAbstract:Objective: A rational treatment strategy for chronic Hepatitis B Virus (HBV) infection might involve the modulation of immunity after the reduction of viremia and antigenemia. This strategy was tested in Woodchucks chronically infected with the Woodchuck Hepatitis Virus (WHV) by combining antiviral treatment with 1-(2-fluoro-5-methyl-β-L-arabinofuranosyl)-uracil (L-FMAU) and therapeutic vaccination with WHV surface antigen (WHsAg). Methods: Chronic WHV carriers were treated with L-FMAU or placebo for 32 weeks. Half the Woodchucks in each group then received four injections of a conventional WHsAg vaccine during the next 16 weeks. Results: Vaccination alone elicited low-level antibody to WHsAg (anti-WHs) in most carriers but did not affect serum WHV DNA, WHsAg or liver enzyme responses. Carriers treated first with L-FMAU to reduce WHV DNA and WHsAg and then vaccinated developed similar low-level anti-WHs and normalized liver enzymes. Following vaccinations, WHsAg-specific cell-mediated immunity (CMI) was demonstrated in both groups, but was significantly enhanced in carriers treated with L-FMAU, and was broadened to include WHV core antigen (WHcAg) and selected peptide epitopes of WHcAg and WHsAg. Anti-WHs and associated CMI to WHcAg and WHsAg were observed after drug discontinuation in half of the carriers that received L-FMAU alone. Conclusions: Vaccination with WHsAg following treatment with L-FMAU disrupted Virus-specific humoral and cell-mediated immune tolerance in chronic WHV infection and enhanced the immune response profiles beyond those seen with monotherapies alone. The combination therapy resulted in immune response profiles that resembled those observed during resolution of WHV infection. The results in Woodchucks demonstrate the feasibility of using such a combination therapy for the control of chronic HBV infection in humans.
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antiviral activity of clevudine l fmau 1 2 fluoro 5 methyl β l arabinofuranosyl uracil against Woodchuck Hepatitis Virus replication and gene expression in chronically infected Woodchucks marmota monax
Hepatology, 2001Co-Authors: Simon Peek, William E. Hornbuckle, Bud C. Tennant, Frances V Wells, Paul J Cote, John L Gerin, James R Jacob, Betty H Baldwin, Ilia Toshkov, Brent E KorbaAbstract:L-FMAU [1-(2-fluoro-5-methyl-β,L-arabinofuranosyl) uracil] has been shown to be an effective inhibitor of Hepatitis B Virus (HBV) and duck Hepatitis B Virus replication in cell culture and duck Hepatitis B Virus replication in acutely infected Peking ducks. The Woodchuck Hepatitis Virus (WHV) and its natural host, the Eastern Woodchuck (Marmota monax), have been established as a predictive model for the evaluation of antiviral therapies against chronic HBV infection. In this report, the antiviral activity of l-FMAU against WHV replication in chronically infected Woodchucks is described. Four weeks of once-daily oral administration of L-FMAU significantly reduced viremia, antigenemia, intrahepatic WHV replication, and intrahepatic expression of Woodchuck Hepatitis Virus core antigen (WHcAg) in a dose-dependent manner. At the highest dose administered (10 mg/kg/d), significant reductions of intrahepatic WHV RNA and covalently closed circular (ccc)WHV-DNA levels also were observed. The reduction in viremia was remarkably rapid at the higher doses of L-FMAU, with greater than 1,000-fold reductions in WHV-DNA serum levels observed after as little as 2 to 3 days of therapy. Following the withdrawal of therapy, a dose-related delay in viremia rebound was observed. At the highest doses used, viremia remained significantly suppressed in at least one half of the treated animals for 10 to 12 weeks' posttreatment. No evidence of drug-related toxicity was observed in the treated animals. L-FMAU is an exceptionally potent antihepadnaviral agent in vitro and in vivo, and is a suitable candidate for antiviral therapy of chronic HBV infection. (HEPATOLOGY 2001;33:254-266)
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antiviral activities of oral 1 o hexadecylpropanediol 3 phosphoacyclovir and acyclovir in Woodchucks with chronic Woodchuck Hepatitis Virus infection
Antimicrobial Agents and Chemotherapy, 2000Co-Authors: Karl Y Hostetler, William E. Hornbuckle, Brent E Korba, Paul J Cote, John L Gerin, James R Beadle, Christine A Bellezza, Ilia A Tochkov, Bud C. TennantAbstract:Acyclovir triphosphate is a potent inhibitor of Hepatitis B Virus DNA polymerase, but acyclovir treatment provides no benefit in patients with Hepatitis B Virus infection. This is due in part to the fact that Hepatitis B Virus, unlike herpes simplex Virus, does not code for a viral thymidine kinase which catalyzes the initial phosphorylation of acyclovir. We synthesized 1-O-octadecyl-sn-glycero-3-phospho (3-P)-acyclovir and found that it was highly active in reducing Hepatitis B Virus replication in 2.2.15 cells, while acyclovir was inactive. The greater antiviral activity of 1-O-octadecyl-sn-glycero-3-P-acyclovir appeared to be due to liver cell metabolism of the compound to acyclovir monophosphate (K. Y. Hostetler et al., Biochem. Pharmacol. 53:1815–1822, 1997). However, a closely related compound without a hydroxyl group at the sn-2 position of glycerol, 1-O-hexadecylpropanediol-3-P-acyclovir, was more active and selective in 2.2.15 cells in vitro. In this study, we treated Woodchucks chronically infected with Woodchuck Hepatitis Virus with increasing oral doses of 1-O-hexadecylpropanediol-3-P-acyclovir and assessed the response to therapy versus acyclovir or a placebo. At a dosage of 10 mg/kg of body weight twice a day, the test compound significantly inhibited viral replication in vivo, as indicated by a 95% reduction in serum Woodchuck Hepatitis Virus DNA levels and by a 54% reduction in levels of Woodchuck Hepatitis Virus replicative intermediates in the liver. Higher doses were somewhat less effective. In contrast, 20 mg of acyclovir/kg twice daily, a 5.3-fold-higher molar dosage, had no demonstrable activity against Woodchuck Hepatitis Virus. Oral 1-O-hexadecylpropanediol-3-P-acyclovir appeared to be safe and effective in chronic Woodchuck Hepatitis Virus infection.
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titration of recombinant Woodchuck Hepatitis Virus dna in adult Woodchucks
Journal of Medical Virology, 1998Co-Authors: Hongshu Chen, William E. Hornbuckle, Bud C. Tennant, Paul J Cote, John L Gerin, Roger H Miller, Robert H PurcellAbstract:In vivo transfection of Eastern Woodchucks (Marmota monax) with recombinant Woodchuck Hepatitis Virus (WHV) DNA is effective in inducing Virus infection for the study of replication, pathogenicity, and oncogenicity of wild-type and mutated WHV. The one drawback to this procedure is the need for preparation of large amounts of WHV DNA. Reduction of the amount of WHV DNA in the transfection protocol necessary to induce infection would save considerable time and resources. Therefore, we conducted a titration of WHV DNA, ranging from 50 μg to 50 pg of DNA, in adult Woodchucks to determine the minimum infectious dose of recombinant WHV DNA. As little as 50 ng of transfected WHV DNA induced productive infection in adult Woodchucks. Thus, transfection with large amounts of recombinant WHV DNA appears to be unnecessary. J. Med. Virol. 54:92–94, 1998. © 1998 Wiley-Liss,Inc.
