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Song J, Sun X, Zhou Y, Li S, Wu J, Yang L, Zhou D, Yang Y, Liu A, Lu M, Michael R, Qin L, Yang D. Early application of IFNγ mediated the persistence of HBV in an HBV mouse model. Antiviral Res 2024; 225:105872. [PMID: 38556058 DOI: 10.1016/j.antiviral.2024.105872] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2024] [Revised: 03/20/2024] [Accepted: 03/23/2024] [Indexed: 04/02/2024]
Abstract
The antiviral activity of interferon gamma (IFNγ) against hepatitis B virus (HBV) was demonstrated both in vivo and in vitro in a previous study. IFNγ can suppress HBV replication by accelerating the decay of replication-competent nucleocapsids of HBV. However, in this study, we found that the direct application of the mouse IFNγ (mIFNγ) expression plasmid to the liver of an HBV hydrodynamic injection (HI) mouse model led to the persistence of HBV, as indicated by sustained HBsAg and HBeAg levels in the serum as well as an increased percentage of the HBsAg positive mice, whereas the level of HBV DNA in the serum and the expression of HBcAg in the liver were inhibited at the early stage after HI. Meanwhile, we found that the productions of both HBcAb and HBsAb were suppressed after the application of mIFNγ. In addition, we found that HBV could be effectively inhibited in mice immunized with HBsAg expression plasmid before the application of mIFNγ. Furthermore, mIFNγ showed antiviral effect and promoted the production of HBsAb when the mice subjected to the core-null HBV plasmid. These results indicate that the application of mIFNγ in the HBV HI mouse model, the mice showed defective HBcAg-specific immunity that impeded the production of HBcAb and HBsAb, finally allowing the persistence of the virus. Moreover, IFNγ-induced negative immune regulatory factors also play an important role in virus persistence.
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Affiliation(s)
- Jingjiao Song
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Xiliang Sun
- Clinical Laboratory, Qingdao West Coast New District People's Hospital, Shandong, PR China.
| | - Yun Zhou
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Key Laboratory of Receptors-mediated Gene Regulation and Drug Discovery, School of Medicine, Henan University, Kaifeng, PR China.
| | - Sheng Li
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Jun Wu
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Lu Yang
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Di Zhou
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Yan Yang
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Anding Liu
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Mengji Lu
- Institute of Virology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany.
| | | | - Li Qin
- Department of Dermatology, Traditional Chinese and Western Medicine Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, Hubei, PR China.
| | - Dongliang Yang
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
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Corkum CP, Wiede LL, Ruble CLA, Qiu J, Mulrooney-Cousins PM, Steeves MA, Watson DE, Michalak TI. Identification of antibodies cross-reactive with woodchuck immune cells and activation of virus-specific and global cytotoxic T cell responses by anti-PD-1 and anti-PD-L1 in experimental chronic hepatitis B and persistent occult hepadnaviral infection. Front Microbiol 2022; 13:1011070. [PMID: 36560951 PMCID: PMC9764628 DOI: 10.3389/fmicb.2022.1011070] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Accepted: 11/14/2022] [Indexed: 12/12/2022] Open
Abstract
Woodchuck (Marmota monax) infected with woodchuck hepatitis virus (WHV) is the most pathogenically compatible naturally occurring model of human hepatitis B virus (HBV) infection, chronic hepatitis B, and HBV-induced hepatocellular carcinoma. This system plays a crucial role in discovery and preclinical evaluation of anti-HBV therapies. Its utilization remains tempered by the relatively narrow range of validated immunologic and molecular tools. We evaluated commercial antibodies against immune cell phenotypic markers and T cell molecules for cross-reactivity with woodchuck antigenic equivalents. The confirmed antibodies against programed cell death protein-1 (PD-1) and its ligand (PD-L1) were examined for ex vivo ability to activate WHV-specific, global and bystander cytotoxic T cells (CTLs) in chronic hepatitis and asymptomatic infection persisting after self-resolved acute hepatitis. Examination of 65 antibodies led to identification or confirmation of 23 recognizing woodchuck T, regulatory T, B and natural killer cells, T cell-associated PD-1, PD-L1, CTLA-4 and TIM-3 molecules, CD25 and CD69 markers of T cell activation, and interferon gamma (IFNγ). Antibodies against woodchuck PD-1 and PD-L1 triggered in vitro highly individualized WHV-specific and global activation of CTLs in both chronic hepatitis and persistent occult infection. WHV-specific CTLs were more robustly augmented by anti-PD-1 than by anti-PD-L1 in chronic hepatitis, while global IFNγ-positive CTL response was significantly suppressed in chronic hepatitis compared to persistent occult infection. Anti-PD-1 and anti-PD-L1 also occasionally activated CTLs to specificities other than those tested suggesting their potency to trigger side effects. This was particularly apparent when T cells from chronic hepatitis were treated with anti-PD-L1. The current findings indicate that inhibition of the PD-1/PD-L1 pathway could reactivate virus-specific and global T cell responses in both chronic hepatitis and asymptomatic persistent infection. They suggest a mechanism of potential reactivation of clinically silent infection during anti-PD-1/PD-L1 treatment and indicate that this therapy may also subdue occult HBV infection.
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Affiliation(s)
- Christopher P. Corkum
- Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John’s, NL, Canada
| | - Louisa L. Wiede
- Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John’s, NL, Canada
| | - Cara L.-A. Ruble
- Lilly Research Laboratories, Elli Lilly and Company, Indianapolis, IN, United States
| | - Jiabin Qiu
- Lilly Research Laboratories, Elli Lilly and Company, Indianapolis, IN, United States
| | - Patricia M. Mulrooney-Cousins
- Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John’s, NL, Canada
| | - Meredith A. Steeves
- Non-Clinical Safety Assessment, Toxicology, Elli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, United States
| | - David E. Watson
- Lilly Research Laboratories, Elli Lilly and Company, Indianapolis, IN, United States
| | - Tomasz I. Michalak
- Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John’s, NL, Canada,*Correspondence: Tomasz I. Michalak,
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Suresh M, Menne S. Application of the woodchuck animal model for the treatment of hepatitis B virus-induced liver cancer. World J Gastrointest Oncol 2021; 13:509-535. [PMID: 34163570 PMCID: PMC8204361 DOI: 10.4251/wjgo.v13.i6.509] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/21/2021] [Revised: 05/02/2021] [Accepted: 05/15/2021] [Indexed: 02/06/2023] Open
Abstract
This review describes woodchucks chronically infected with the woodchuck hepatitis virus (WHV) as an animal model for hepatocarcinogenesis and treatment of primary liver cancer or hepatocellular carcinoma (HCC) induced by the hepatitis B virus (HBV). Since laboratory animal models susceptible to HBV infection are limited, woodchucks experimentally infected with WHV, a hepatitis virus closely related to HBV, are increasingly used to enhance our understanding of virus-host interactions, immune response, and liver disease progression. A correlation of severe liver pathogenesis with high-level viral replication and deficient antiviral immunity has been established, which are present during chronic infection after WHV inoculation of neonatal woodchucks for modeling vertical HBV transmission in humans. HCC in chronic carrier woodchucks develops 17 to 36 mo after neonatal WHV infection and involves liver tumors that are comparable in size, morphology, and molecular gene signature to those of HBV-infected patients. Accordingly, woodchucks with WHV-induced liver tumors have been used for the improvement of imaging and ablation techniques of human HCC. In addition, drug efficacy studies in woodchucks with chronic WHV infection have revealed that prolonged treatment with nucleos(t)ide analogs, alone or in combination with other compounds, minimizes the risk of liver disease progression to HCC. More recently, woodchucks have been utilized in the delineation of mechanisms involved in innate and adaptive immune responses against WHV during acute, self-limited and chronic infections. Therapeutic interventions based on modulating the deficient host antiviral immunity have been explored in woodchucks for inducing functional cure in HBV-infected patients and for reducing or even delaying associated liver disease sequelae, including the onset of HCC. Therefore, woodchucks with chronic WHV infection constitute a well-characterized, fully immunocompetent animal model for HBV-induced liver cancer and for preclinical evaluation of the safety and efficacy of new modalities, which are based on chemo, gene, and immune therapy, for the prevention and treatment of HCC in patients for which current treatment options are dismal.
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Affiliation(s)
- Manasa Suresh
- Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, United States
| | - Stephan Menne
- Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, United States
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Michalak TI. Diverse Virus and Host-Dependent Mechanisms Influence the Systemic and Intrahepatic Immune Responses in the Woodchuck Model of Hepatitis B. Front Immunol 2020; 11:853. [PMID: 32536912 PMCID: PMC7267019 DOI: 10.3389/fimmu.2020.00853] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2019] [Accepted: 04/14/2020] [Indexed: 12/15/2022] Open
Abstract
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.
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Affiliation(s)
- Tomasz I Michalak
- Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's, NL, Canada
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Suresh M, Czerwinski S, Murreddu MG, Kallakury BV, Ramesh A, Gudima SO, Menne S. Innate and adaptive immunity associated with resolution of acute woodchuck hepatitis virus infection in adult woodchucks. PLoS Pathog 2019; 15:e1008248. [PMID: 31869393 PMCID: PMC6946171 DOI: 10.1371/journal.ppat.1008248] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2019] [Revised: 01/07/2020] [Accepted: 11/29/2019] [Indexed: 01/03/2023] Open
Abstract
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.
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Affiliation(s)
- Manasa Suresh
- Department of Microbiology & Immunology, Georgetown University Medical Center, Washington, DC, United States of America
| | - Stefanie Czerwinski
- Department of Microbiology & Immunology, Georgetown University Medical Center, Washington, DC, United States of America
| | - Marta G. Murreddu
- Department of Microbiology & Immunology, Georgetown University Medical Center, Washington, DC, United States of America
| | - Bhaskar V. Kallakury
- Department of Pathology, Georgetown University Medical Center, Washington, DC, United States of America
| | - Ashika Ramesh
- Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC, United States of America
| | - Severin O. Gudima
- Department of Microbiology, Molecular Genetics & Immunology, University of Kansas Medical Center, Kansas City, KS, United States of America
| | - Stephan Menne
- Department of Microbiology & Immunology, Georgetown University Medical Center, Washington, DC, United States of America
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Williams JB, Hüppner A, Mulrooney-Cousins PM, Michalak TI. Differential Expression of Woodchuck Toll-Like Receptors 1-10 in Distinct Forms of Infection and Stages of Hepatitis in Experimental Hepatitis B Virus Infection. Front Microbiol 2018; 9:3007. [PMID: 30581424 PMCID: PMC6292964 DOI: 10.3389/fmicb.2018.03007] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2018] [Accepted: 11/20/2018] [Indexed: 12/17/2022] Open
Abstract
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.
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Affiliation(s)
- John Bradley Williams
- Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's, NL, Canada
| | - Alena Hüppner
- Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's, NL, Canada
| | - Patricia M Mulrooney-Cousins
- Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's, NL, Canada
| | - Tomasz I Michalak
- Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's, NL, Canada
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Mulrooney-Cousins PM, Michalak TI. Asymptomatic Hepadnaviral Persistence and Its Consequences in the Woodchuck Model of Occult Hepatitis B Virus Infection. J Clin Transl Hepatol 2015; 3:211-9. [PMID: 26623268 PMCID: PMC4663203 DOI: 10.14218/jcth.2015.00020] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/02/2015] [Revised: 07/20/2015] [Accepted: 07/21/2015] [Indexed: 02/06/2023] Open
Abstract
Woodchuck hepatitis virus (WHV) is molecularly and pathogenically closely related to hepatitis B virus (HBV). Both viruses display tropism towards hepatocytes and cells of the immune system and cause similar liver pathology, where acute hepatitis can progress to chronic hepatitis and to hepatocellular carcinoma (HCC). Two forms of occult hepadnaviral persistence were identified in the woodchuck-WHV model: secondary occult infection (SOI) and primary occult infection (POI). SOI occurs after resolution of a serologically apparent infection with hepatitis or after subclinical serologically evident virus exposure. POI is caused by small amounts of virus and progresses without serological infection markers, but the virus genome and its replication are detectable in the immune system and with time in the liver. SOI can be accompanied by minimal hepatitis, while the hallmark of POI is normal liver morphology. Nonetheless, HCC develops in about 20% of animals with SOI or POI within 3 to 5 years. The virus persists throughout the lifespan in both SOI and POI at serum levels rarely greater than 100 copies/mL, causes hepatitis and HCC when concentrated and administered to virus-naïve woodchucks. SOI is accompanied by virus-specific T and B cell immune responses, while only virus-specific T cells are detected in POI. SOI coincides with protection against reinfection, while POI does not and hepatitis develops after challenge with liver pathogenic doses >1000 virions. Both SOI and POI are associated with virus DNA integration into the liver and the immune system genomes. Overall, SOI and POI are two distinct forms of silent hepadnaviral persistence that share common characteristics. Here, we review findings from the woodchuck model and discuss the relevant observations made in human occult HBV infection (OBI).
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Affiliation(s)
| | - Tomasz I. Michalak
- Correspondence to: Tomasz I. Michalak, Molecular Virology and Hepatology Research Group, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John’s, NL A1B 3V6, Canada. Tel: +1-709-777-7301, Fax: +1-709-777-8279, E-mail:
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Repeated exposure to trace amounts of woodchuck hepadnavirus induces molecularly evident infection and virus-specific T cell response in the absence of serological infection markers and hepatitis. J Virol 2012; 87:1035-48. [PMID: 23135718 DOI: 10.1128/jvi.01363-12] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Exposure to multiple small doses of hepatitis B virus (HBV) is a frequent occurrence in high-risk groups, including close relatives of infected individuals, primary care givers, and intravenous drug users. It remains uncertain whether such repeated contact may culminate in a symptomatic infection coinciding with hepatitis in individuals not immunoprotected. In this study, we evaluated consequences of multiple exposures to small, liver-nonpathogenic amounts of infectious hepadnavirus in the woodchuck model of hepatitis B. Virus-naïve animals were intravenously injected with 6 weekly doses of 110 DNase digestion-protected virions of woodchuck hepatitis virus (WHV), injected again with 6 weekly 110-virion doses after 7.5 months, and then challenged or not with a liver-pathogenic dose of 1.1 × 10(6) virions of the same inoculum. The data revealed that two rounds of such repeated exposure did not result in serologically evident infection or hepatitis. However, a low-level WHV DNA-positive infection accompanied by a WHV-specific T cell response in the absence of antiviral antibody reactivity was established. The kinetics of the virus-specific and mitogen-induced (generalized) T cell responses and the inability to induce immunoprotection against challenge with a large, liver-pathogenic virus dose were closely comparable to those previously reported for occult infection initiated by a single liver-nonpathogenic dose of WHV. Thus, repeated exposures to small quantities of hepadnavirus induce molecularly evident but serologically silent infection that does not culminate in hepatitis or generate immune protection. The findings imply that the HBV-specific T cell response encountered in the absence of serological markers of infection likely reflects ongoing occult infection.
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Khawaja G, Buronfosse T, Jamard C, Abdul F, Guerret S, Zoulim F, Luxembourg A, Hannaman D, Evans CF, Hartmann D, Cova L. In vivo electroporation improves therapeutic potency of a DNA vaccine targeting hepadnaviral proteins. Virology 2012; 433:192-202. [PMID: 22921316 DOI: 10.1016/j.virol.2012.07.014] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2012] [Revised: 06/01/2012] [Accepted: 07/16/2012] [Indexed: 12/15/2022]
Abstract
This preclinical study investigated the therapeutic efficacy of electroporation (EP)-based delivery of plasmid DNA (pDNA) encoding viral proteins (envelope, core) and IFN-γ in the duck model of chronic hepatitis B virus (DHBV) infection. Importantly, only DNA EP-therapy resulted in a significant decrease in mean viremia titers and in intrahepatic covalently closed circular DNA (cccDNA) levels in chronic DHBV-carrier animals, compared with standard needle pDNA injection (SI). In addition, DNA EP-therapy stimulated in all virus-carriers a humoral response to DHBV preS protein, recognizing a broader range of major antigenic regions, including neutralizing epitopes, compared with SI. DNA EP-therapy led also to significant higher intrahepatic IFN-γ RNA levels in DHBV-carriers compared to other groups, in the absence of adverse effects. We provide the first evidence on DNA EP-therapy benefit in terms of hepadnaviral infection clearance and break of immune tolerance in virus-carriers, supporting its clinical application for chronic hepatitis B.
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MESH Headings
- Animals
- Antibodies, Viral/biosynthesis
- Antibodies, Viral/immunology
- Chronic Disease
- DNA, Circular/genetics
- DNA, Circular/immunology
- Disease Models, Animal
- Ducks
- Electroporation
- Epitopes
- Hepadnaviridae Infections/immunology
- Hepadnaviridae Infections/prevention & control
- Hepadnaviridae Infections/veterinary
- Hepadnaviridae Infections/virology
- Hepatitis B Vaccines/administration & dosage
- Hepatitis B Vaccines/immunology
- Hepatitis B Virus, Duck/immunology
- Hepatitis, Viral, Animal/immunology
- Hepatitis, Viral, Animal/prevention & control
- Hepatitis, Viral, Animal/virology
- Immune Tolerance
- Immunity, Humoral
- Interferon-gamma/biosynthesis
- Interferon-gamma/immunology
- Plasmids
- Vaccines, DNA/administration & dosage
- Vaccines, DNA/immunology
- Viral Core Proteins/genetics
- Viral Core Proteins/immunology
- Viral Envelope Proteins/genetics
- Viral Envelope Proteins/immunology
- Viremia/immunology
- Viremia/prevention & control
- Viremia/veterinary
- Viremia/virology
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Specific expression of human interferon-gamma controls hepatitis B virus replication in vitro in secreting hepatitis B surface antigen hepatocytes. J Virol Methods 2012; 180:84-90. [DOI: 10.1016/j.jviromet.2011.12.016] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2011] [Revised: 11/04/2011] [Accepted: 12/30/2011] [Indexed: 12/31/2022]
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von Freyend MJ, Untergasser A, Arzberger S, Oberwinkler H, Drebber U, Schirmacher P, Protzer U. Sequential control of hepatitis B virus in a mouse model of acute, self-resolving hepatitis B. J Viral Hepat 2011; 18:216-26. [PMID: 20367794 DOI: 10.1111/j.1365-2893.2010.01302.x] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
The determinants of an immune response to the human hepatitis B virus (HBV) are poorly understood. As studies in man and chimpanzees are limited, we aimed at developing a model of self-limiting hepatitis B in mice that helps to dissect the control of HBV by humoral and cellular immune responses. Adenoviral vectors containing 1.3-fold HBV genomes allowed an efficient and reproducible transfer of HBV genomes into mouse livers and initiated HBV replication in mice. HBV transcripts were detected in mouse livers for more than 3 months. HBsAg and HBeAg peaked around day 6 and slowly declined thereafter. A two-phase mild to moderate liver inflammation with elevated serum alanine transaminase activities was observed around day 7 and around day 70 when the vast majority of HBV-specific T cells were detected in the liver. HBV was initially controlled when specific and nonspecific T cells infiltrated the liver and intrahepatic interferon γ levels peaked around day 7, but replicated again from day 10 to day 24 and persisted at low levels thereafter despite the presence of HBV-specific T cells. Finally, HBV replication was terminated after a sufficient B-cell response had been mounted- indicated by anti-HBs seroconversion around day 35. HBV-specific T cells infiltrated the liver a second time around day 70 postinfection. This demonstrates that the established mouse model allows studying the onset and termination of HBV infection and will help to dissect the determinants of HBV control and clearance by the immune response.
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Affiliation(s)
- M John von Freyend
- Institute for Medical Microbiology, Immunology and Hygiene and Center for Molecular Medicine Cologne, University of Cologne Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
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Gujar SA, Jenkins AKM, Macparland SA, Michalak TI. Pre-acute hepadnaviral infection is associated with activation-induced apoptotic death of lymphocytes in the woodchuck (Marmota monax) model of hepatitis B. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2010; 34:999-1008. [PMID: 20451550 DOI: 10.1016/j.dci.2010.05.001] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/06/2010] [Revised: 04/30/2010] [Accepted: 05/01/2010] [Indexed: 05/29/2023]
Abstract
Woodchucks (Marmota monax) infected with woodchuck hepatitis virus (WHV) represent a highly valuable immunopathogenic model of hepatitis B virus (HBV) infection. Both WHV and HBV are noncytopathic hepadnaviruses which induce a strong but delayed virus-specific cellular immune response believed to be a cause of hepatitis. The reason behind this postponement is not well understood and its dissection in the woodchuck model has been hampered by the lack of appropriate research tools. In this study, we applied an assay for the simultaneous detection of cell apoptosis and proliferation to determine the fate of T lymphocytes after WHV infection leading to acute hepatitis. The results revealed that pre-acute WHV infection is associated with the significantly heightened susceptibility of T lymphocytes to activation-induced apoptotic death. This suggests that T lymphocyte function is compromised very early in the course of hepadnaviral infection and this may directly contribute to the postponement of virus-specific T cell response.
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Affiliation(s)
- Shashi A Gujar
- Molecular Virology and Hepatology Research Group, Division of BioMedical Science, Faculty of Medicine, Health Sciences Center, Memorial University, St. John's, Newfoundland, Canada
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13
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Guy CS, Wang J, Michalak TI. Hepadnaviral infection augments hepatocyte cytotoxicity mediated by both CD95 ligand and perforin pathways. Liver Int 2010; 30:396-405. [PMID: 19912529 DOI: 10.1111/j.1478-3231.2009.02168.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
Abstract
BACKGROUND/AIM Recently, we documented that hepatocytes can eliminate contacted cells via the CD95 ligand (CD95L)-CD95 pathway and that they are also equipped in perforin and granzyme B and can eradicate other cells via the granule exocytosis mechanism. The aim of this study was to assess whether hepadnaviral infection modifies hepatocyte-mediated cell killing. METHODS Primary hepatocytes from woodchucks with progressing or resolved hepadnaviral hepatitis and hepatocyte lines transfected with woodchuck hepatitis virus (WHV) genes were examined for cytotoxic effector activity against cell targets susceptible to CD95L and/or perforin-dependent killing. Hepatocytes from healthy animals served as controls. RESULTS Actively progressing and resolved hepadnaviral hepatitis is associated with a significantly greater capacity of hepatocytes to kill contacted cells. Both hepatocyte CD95L- and perforin-dependent cytotoxicity were augmented. Hepatocytes transfected with WHV X gene, but not those with complete WHV genome or virus envelope or core gene, transcribed significantly more CD95L and perforin and killed cell targets more efficiently. Exposure to interferon-gamma profoundly enhanced hepatocyte cell killing. CONCLUSIONS Hepatocyte cytotoxic potential is significantly augmented during and following resolution of active hepadnaviral hepatitis. Hepatocyte cytotoxic activity may contribute to both liver physiological functions and the pathogenesis and progression of liver disease, including viral hepatitis.
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Affiliation(s)
- Clifford S Guy
- Molecular Virology and Hepatology Research Group, Health Sciences Centre, Division of Biomedical Sciences, Faculty of Medicine, Memorial University, St John's, NF, Canada
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14
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Gujar SA, Michalak TI. Flow Cytometric Quantification of T Cell Proliferation and Division Kinetics in Woodchuck Model of Hepatitis B. Immunol Invest 2009. [DOI: 10.1081/imm-55834] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
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15
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Primary occult hepadnavirus infection induces virus-specific T-cell and aberrant cytokine responses in the absence of antiviral antibody reactivity in the Woodchuck model of hepatitis B virus infection. J Virol 2009; 83:3861-76. [PMID: 19193791 DOI: 10.1128/jvi.02521-08] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Although the virological features of serologically silent hepadnaviral primary occult infection (POI) have been relatively well recognized in the woodchuck model of hepatitis B virus infection, the characteristics of accompanying immune responses remain unknown. In this study, the kinetics of woodchuck hepatitis virus (WHV)-specific and generalized (mitogen-induced) T-cell proliferative responses and cytokine expression profiles in circulating lymphoid cells and the liver, along with WHV-specific antibody responses, were investigated during experimentally induced POI and subsequent challenge with a liver-pathogenic dose (>10(3) virions) or liver-nonpathogenic dose (50 virions) of the same virus. The data revealed that POI, which does not prompt WHV surface antigenemia, antiviral antibody response, and hepatitis or protect from challenge with a liver-pathogenic virus dose, was accompanied by the appearance of a strong WHV-specific T-cell response directed against multiple viral epitopes that intermittently persisted at low levels for up to 10-months during follow-up. Furthermore, immediately after exposure to a liver-nonpathogenic dose of WHV, lymphocytes acquired a heightened capacity to proliferate in response to mitogenic stimuli and displayed augmented expression of alpha interferon, interleukin-12 (IL-12), and IL-2, but not tumor necrosis factor alpha. Overall, the kinetics of WHV-specific and mitogen-induced T-cell proliferative and cytokine responses in POI were closely comparable to those seen in infection induced by liver-pathogenic viral doses. The data demonstrated that virus-specific T-cell proliferative reactivity is a very sensitive indicator of exposure to hepadnavirus, even to small amounts inducing serologically mute infection. They also showed that hepadnaviral POI is not only a molecularly but also an immunologically identifiable and distinctive entity.
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16
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Glebe D, Lorenz H, Gerlich WH, Butler SD, Tochkov IA, Tennant BC, Cote P, Menne S. Correlation of virus and host response markers with circulating immune complexes during acute and chronic woodchuck hepatitis virus infection. J Virol 2009; 83:1579-91. [PMID: 19052077 PMCID: PMC2643793 DOI: 10.1128/jvi.01934-08] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2008] [Accepted: 11/25/2008] [Indexed: 12/27/2022] Open
Abstract
Woodchuck hepatitis virus (WHV) is an established model for human hepatitis B virus. The kinetics of virus and host responses in serum and liver during acute, self-limited WHV infection in adult woodchucks were studied. Serum WHV DNA and surface antigen (WHsAg) were detected as early as 1 to 3 weeks following experimental infection and peaked between 1 and 5 weeks postinfection. Thereafter, serum WHsAg levels declined rapidly and became undetectable, while WHV DNA levels became undetectable much later, between 4 and 20 weeks postinfection. Decreasing viremia correlated with transient liver injury marked by an increase in serum sorbitol dehydrogenase (SDH) levels. Clearance of WHV DNA from serum was associated with the normalization of serum SDH. Circulating immune complexes (CICs) of WHsAg and antibodies against WHsAg (anti-WHs) that correlated temporarily with the peaks in serum viremia and WHs antigenemia were detected. CICs were no longer detected in serum once free anti-WHs became detectable. The detection of CICs around the peak in serum viremia and WHs antigenemia in resolving woodchucks suggests a critical role for the humoral immune response against WHsAg in the early elimination of viral and subviral particles from the peripheral blood. Individual kinetic variation during WHV infections in resolving woodchucks infected with the same WHV inoculum and dose is likely due to the outbred nature of the animals, indicating that the onset and magnitude of the individual immune response determine the intensity of virus inhibition and the timing of virus elimination from serum.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology, Justus Liebig University Giessen, Germany.
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17
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Guy CS, Mulrooney-Cousins PM, Churchill ND, Michalak TI. Intrahepatic expression of genes affiliated with innate and adaptive immune responses immediately after invasion and during acute infection with woodchuck hepadnavirus. J Virol 2008; 82:8579-91. [PMID: 18596101 PMCID: PMC2519695 DOI: 10.1128/jvi.01022-08] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2008] [Accepted: 06/23/2008] [Indexed: 02/07/2023] Open
Abstract
The importance of effective immune responses in recovery from acute hepadnaviral hepatitis has been demonstrated. However, there is no conclusive delineation of virological and immunological events occurring in the liver immediately after hepadnavirus invasion and during the preacute phase of infection. These very early events might be of primary importance in determining the recovery or progression to chronic hepatitis and the intrinsic hepadnaviral propensity to persist. In this study, applying the woodchuck model of acute hepatitis B, the hepatic kinetics of hepadnavirus replication and activation of genes encoding cytokines, cytotoxicity effectors, and immune cell markers were quantified in sequential liver biopsies collected from 1 h postinoculation onward by sensitive real-time cDNA amplification assays. The results revealed that hepadnavirus replication is established in the liver as early as 1 hour after infection. In 3 to 6 h, significantly augmented intrahepatic transcription of gamma interferon and interleukin-12 were evident, suggesting activation of antigen-presenting cells. In 48 to 72 h, NK and NKT cells were activated and virus replication was transiently but significantly reduced, implying that this early innate response is at least partially successful in limiting virus propagation. Nonetheless, T cells were activated 4 to 5 weeks later when hepatitis became histologically evident. Collectively, our data demonstrate that virus replication is initiated and the innate response activated in the liver soon after exposure to a liver-pathogenic dose of hepadnavirus. Nevertheless, this response is unable to prompt a timely adaptive T-cell response, in contrast to infections caused by other viral pathogens.
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Affiliation(s)
- Clifford S Guy
- Division of BioMedical Science, Molecular Virology and Hepatology Research Group, Discipline of Laboratory Medicine, Faculty of Medicine, Health Science Centre, Memorial University, St John's, Newfoundland, Canada
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18
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Aberrant lymphocyte activation precedes delayed virus-specific T-cell response after both primary infection and secondary exposure to hepadnavirus in the woodchuck model of hepatitis B virus infection. J Virol 2008; 82:6992-7008. [PMID: 18480439 DOI: 10.1128/jvi.00661-08] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The contribution of virus-specific T lymphocytes to the outcome of acute hepadnaviral hepatitis is well recognized, but a reason behind the consistent postponement of this response remains unknown. Also, the characteristics of T-cell reactivity following reexposure to hepadnavirus are not thoroughly recognized. To investigate these issues, healthy woodchucks (Marmota monax) were infected with liver-pathogenic doses of woodchuck hepatitis virus (WHV) and investigated unchallenged or after challenge with the same virus. As expected, the WHV-specific T-cell response appeared late, 6 to 7 weeks postinfection, remained high during acute disease, and then declined but remained detectable long after the resolution of hepatitis. Interestingly, almost immediately after infection, lymphocytes acquired a heightened capacity to proliferate in response to mitogenic (nonspecific) stimuli. This reactivity subsided before the WHV-specific T-cell response appeared, and its decline coincided with the cells' augmented susceptibility to activation-induced death. The analysis of cytokine expression profiles confirmed early in vivo activation of immune cells and revealed their impairment of transcription of tumor necrosis factor alpha and gamma interferon. Strikingly, reexposure of the immune animals to WHV swiftly induced hyperresponsiveness to nonspecific stimuli, followed again by the delayed virus-specific response. Our data show that both primary and secondary exposures to hepadnavirus induce aberrant activation of lymphocytes preceding the virus-specific T-cell response. They suggest that this activation and the augmented death of the cells activated, accompanied by a defective expression of cytokines pivotal for effective T-cell priming, postpone the adaptive T-cell response. These impairments likely hamper the initial recognition and clearance of hepadnavirus, permitting its dissemination in the early phase of infection.
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19
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Guy CS, Rankin SL, Wang J, Michalak TI. Hepatocytes can induce death of contacted cells via perforin-dependent mechanism. Hepatology 2008; 47:1691-701. [PMID: 18393317 DOI: 10.1002/hep.22228] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
UNLABELLED The liver displays unique immunological properties including the ability to remove aberrant cells and pathogens and to induce peripheral immunotolerance. We have previously demonstrated that hepatocytes can cause cell death by a CD95 ligand-mediated mechanism. Here, we provide evidence that hepatocytes can kill other cells via a perforin-dependent pathway. Using cultured woodchuck hepatocytes and human liver cells as well as freshly isolated woodchuck, mouse, and human hepatocytes, we show that hepatocyte-mediated death of CD95-deficient target cells requires microtubule polymerization, a feature of the granule exocytosis-mediated cytotoxicity. Neutralizing anti-perforin antibodies and short-hairpin RNA directed against perforin messenger RNA confirmed the involvement of perforin in hepatocyte-mediated cell killing. CONCLUSION This study shows that hepatocytes express biologically competent perforin capable of killing susceptible cells and emphasizes the role of hepatocytes as cytotoxic effectors. This also is the first demonstration of perforin in a non-lymphoid cell type.
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Affiliation(s)
- Clifford S Guy
- Molecular Virology and Hepatology Research, Division of Biomedical Sciences, Memorial University, St. John's, Newfoundland, Canada
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20
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Ribeiro CSS, Visentainer JEL, Moliterno RA. Association of cytokine genetic polymorphism with hepatitis B infection evolution in adult patients. Mem Inst Oswaldo Cruz 2008; 102:435-40. [PMID: 17612762 DOI: 10.1590/s0074-02762007005000043] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2006] [Accepted: 03/07/2007] [Indexed: 12/12/2022] Open
Abstract
The infection by the hepatitis B virus (HBV) has different forms of evolution, ranging from self-limited infection to chronic hepatic disease. The objective of this study was to evaluate the influence of cytokine genetic polymorphisms in the disease evolution. The patients were divided into two groups, one with chronic HBV (n = 30), and the other with self-limited infection (n = 41). The genotyping for TNF (-308), TGFB1 (+869, +915), IL-10 (1082, -819, and -592), IL-6 (-174), and IFNG (+874) was accomplished by the PCR-SSP (polymerase chain reaction with sequence specific primers technique using the One Lambda kit. Although no statistically significant differences were found between the groups, the combination of TNF -308GG and IFNG +874TA was found in a lower frequency in chronic patients than in individuals with self-limited infection (26.7 versus 46.3%; P = 0.079; OR = 0.40; IC95% = 0.14-1.11). In chronic patients with histological alterations it was not observed the genotype TGFB1+869 C/C, against 24.4% in the self limited infection group (100 versus 75.6%; P = 0.096; OR = 7.67; IC95% = 0.42-141.63). Further studies in other populations, and evaluation of a greater number of individuals could contribute for a better understanding of the cytokine genetic polymorphism influence in HBV infection evolution.
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21
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Mulrooney-Cousins PM, Michalak TI. Persistent occult hepatitis B virus infection: Experimental findings and clinical implications. World J Gastroenterol 2007; 13:5682-6. [PMID: 17963292 PMCID: PMC4171252 DOI: 10.3748/wjg.v13.i43.5682] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) is a highly pathogenic virus that causes chronic liver diseases in millions of people globally. In addition to a symptomatic, serologically evident infection, occult persistent HBV carriage has been identified since nucleic acid amplification assays of enhanced sensitivity became introduced for detection of hepadnaviral genomes and their replicative intermediates. Current evidence indicates that occult HBV infection is a common and long-term consequence of resolution of acute hepatitis B. This form of residual infection is termed as secondary occult infection (SOI). The data from the woodchuck model of HBV infection indicate that exposure to small amounts of hepadnavirus can also cause primary occult infection (POI) where virus genome, but no serological makers of exposure to virus, are detectable, and the liver may not be involved. However, virus replicates at low levels in the lymphatic system in both these forms. We briefly summarize the current understanding of the nature and characteristics of occult hepadnaviral persistence as well as of its documented and expected pathological consequences.
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22
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Michalak TI, Pham TNQ, Mulrooney-Cousins PM. Molecular diagnosis of occult HCV and HBV infections. Future Virol 2007. [DOI: 10.2217/17460794.2.5.451] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
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23
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Puro R, Schneider RJ. Tumor necrosis factor activates a conserved innate antiviral response to hepatitis B virus that destabilizes nucleocapsids and reduces nuclear viral DNA. J Virol 2007; 81:7351-62. [PMID: 17475655 PMCID: PMC1933346 DOI: 10.1128/jvi.00554-07] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Tumor necrosis factor (TNF) is critical for the control of hepatitis B virus (HBV) in the clinical setting and in model systems. TNF induces noncytopathic suppression and clearance of HBV in animal models, possibly through reduction of viral nucleocapsids, but the mechanism is not well described. Here, we demonstrate the molecular mechanism and broad host range for TNF action against HBV. We show that TNF rapidly blocks HBV replication by promoting destabilization of preexisting cytoplasmic viral nucleocapsids containing viral RNA and DNA, as well as empty nucleocapsids. TNF destabilized human HBV nucleocapsids in a variety of human hepatocytic cell lines and in primary rat hepatocytes and also destabilized duck HBV (DHBV) nucleocapsids in chicken hepatocytic cells. Lysates from TNF-treated uninfected cells also destabilized HBV nucleocapsids in vitro. Moreover, inhibition of DHBV DNA replication by TNF blocks nuclear accumulation of the viral transcription template, maintenance of which is essential for the establishment and maintenance of chronic infection. We show that TNF destabilization of HBV nucleocapsids does not involve ubiquitination or methylation of the viral core protein and is not mediated by the nitric oxide free radical arm of the TNF pathway. These results define a novel antiviral mechanism mediated by TNF against multiple types of HBVs in different species.
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Affiliation(s)
- Robyn Puro
- Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA
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24
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You J, Zhuang L, Ma YL, Tang BZ. Research advances in the imbalance of helper T lymphocyte subpopulations and cytokine network in patients with chronic hepatitis B. Shijie Huaren Xiaohua Zazhi 2007; 15:791-799. [DOI: 10.11569/wcjd.v15.i8.791] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Helper T (Th) lymphocytes, important immune regulating cells of organism, could be divided into four functional subsets, Th0, Th1, Th2, Th3, on the basis of the immunoregulatory cytokines that these clones produced. Most of the current work in this field is exploratory and focuses on Th1 and Th2 subsets. Th1 cells secrete interferon-γ (IFN-γ), interkeukin-2 (IL-2) and tumor necrosis factor-β and are principally involved in cell-mediated immunity. They play an important role in the protection against intracellular pathogens, including a variety of viruses. Th2 cells secret interleukin-4 (IL-4), IL-5, IL-6 and IL-10 and regulate the humoral immune response. Th0 cells are naive Th cells, secreting Th1/Th2 phenotype cytokines at low levels. However, Th3 cells, which secrete active transforming growth factor-β, exert a negative immunoregulatory action on the immune response. The cross-regulatory properties of Th1 and Th2 subset cells and relevant cytokines network are very important to maintain normal immunity of organisms. HBV can destroy the balance of Th lymphocytes and cytokines network. The imbalance of pro-inflammatory Th1 and anti-inflammatory Th2 cytokine production play an important role in the immunopathogenesis of hepatitis B virus infection and alter chronic liver disease development, progression and outcome.
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25
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Menne S, Cote PJ. The woodchuck as an animal model for pathogenesis and therapy of chronic hepatitis B virus infection. World J Gastroenterol 2007; 13:104-24. [PMID: 17206759 PMCID: PMC4065868 DOI: 10.3748/wjg.v13.i1.104] [Citation(s) in RCA: 130] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/28/2006] [Revised: 08/25/2006] [Accepted: 10/04/2006] [Indexed: 02/06/2023] Open
Abstract
This review describes the woodchuck and the woodchuck hepatitis virus (WHV) as an animal model for pathogenesis and therapy of chronic hepatitis B virus (HBV) infection and disease in humans. The establishment of woodchuck breeding colonies, and use of laboratory-reared woodchucks infected with defined WHV inocula, have enhanced our understanding of the virology and immunology of HBV infection and disease pathogenesis, including major sequelae like chronic hepatitis and hepatocellular carcinoma. The role of persistent WHV infection and of viral load on the natural history of infection and disease progression has been firmly established along the way. More recently, the model has shed new light on the role of host immune responses in these natural processes, and on how the immune system of the chronic carrier can be manipulated therapeutically to reduce or delay serious disease sequelae through induction of the recovery phenotype. The woodchuck is an outbred species and is not well defined immunologically due to a limitation of available host markers. However, the recent development of several key host response assays for woodchucks provides experimental opportunities for further mechanistic studies of outcome predictors in neonatal- and adult-acquired infections. Understanding the virological and immunological mechanisms responsible for resolution of self-limited infection, and for the onset and maintenance of chronic infection, will greatly facilitate the development of successful strategies for the therapeutic eradication of established chronic HBV infection. Likewise, the results of drug efficacy and toxicity studies in the chronic carrier woodchucks are predictive for responses of patients chronically infected with HBV. Therefore, chronic WHV carrier woodchucks provide a well-characterized mammalian model for preclinical evaluation of the safety and efficacy of drug candidates, experimental therapeutic vaccines, and immunomodulators for the treatment and prevention of HBV disease sequelae.
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Affiliation(s)
- Stephan Menne
- Department of Clinical Sciences, College of Veterinary Medicine, Veterinary Medical Center, Cornell University, Ithaca, NY 14853, USA.
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26
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MacParland SA, Pham TNQ, Gujar SA, Michalak TI. De novo infection and propagation of wild-type Hepatitis C virus in human T lymphocytes in vitro. J Gen Virol 2006; 87:3577-3586. [PMID: 17098973 DOI: 10.1099/vir.0.81868-0] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
While exploring previous findings that ex vivo treatment of lymphoid cells from Hepatitis C virus (HCV)-infected individuals with T cell-stimulating mitogens augments detection of the residing virus, an in vitro HCV replication system was established, in which mitogen-induced T cell-enriched cultures served as HCV targets and the derived T cells multiplied virus during repeated serial passage. HCV replication was ascertained by detecting HCV RNA positive and negative strands, HCV NS5a and E2 proteins, release of HCV virions and nucleocapsids (confirmed by immunoelectron microscopy) and de novo infection of mitogen-induced T cells prepared from healthy donors. Further, affinity-purified normal human T lymphocytes were also susceptible to HCV infection in vitro and HCV replication was detected in pure T cells isolated from a patient with chronic hepatitis C. These results document that T cells can support propagation of HCV both in vivo and in vitro. The infection system established offers a valuable tool for in vitro studies on the entire cycle of HCV replication, virus cytopathogenicity and evaluation of antiviral agents against wild-type HCV in the natural host-cell milieu.
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Affiliation(s)
- Sonya A MacParland
- Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St John's, NL A1B 3V6, Canada
| | - Tram N Q Pham
- Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St John's, NL A1B 3V6, Canada
| | - Shashi A Gujar
- Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St John's, NL A1B 3V6, Canada
| | - Tomasz I Michalak
- Discipline of Laboratory Medicine, Faculty of Medicine, Health Sciences Centre, Memorial University, St John's, NL A1B 3V6, Canada
- Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St John's, NL A1B 3V6, Canada
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27
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Wang J, Gujar SA, Cova L, Michalak TI. Bicistronic woodchuck hepatitis virus core and gamma interferon DNA vaccine can protect from hepatitis but does not elicit sterilizing antiviral immunity. J Virol 2006; 81:903-16. [PMID: 17079319 PMCID: PMC1797430 DOI: 10.1128/jvi.01537-06] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The immunity elicited against nucleocapsid of hepatitis B virus (HBV) and closely related woodchuck hepatitis virus (WHV) has been shown to be important in resolution of hepatitis and protection from infection. Further, activity of gamma interferon (IFN-gamma), which may directly inhibit hepadnavirus replication, promotes antiviral defense and favors T helper cell type 1 (Th1) response, which is seemingly a prerequisite of HBV clearance. In this study, to enhance induction of protective immunity against hepadnavirus, healthy woodchucks were immunized with a bicistronic DNA vaccine carrying WHV core (WHc) and woodchuck IFN-gamma (wIFN-gamma) gene sequences. Three groups, each group containing three animals, were injected once or twice with 0.5 mg, 0.9 mg, or 1.5 mg per dose of this vaccine. In addition, four animals received two injections of 0.6 mg or 1 mg WHc DNA alone. All animals were challenged with WHV. The results showed that four of nine animals injected with the bicistronic vaccine and one of four immunized with WHc DNA became protected from serologically evident infection and hepatitis. This protection was not linked to induction of WHc antigen-specific antibodies or T-cell proliferative response and was not associated with enhanced transcription of Th1 cytokines or 2',5'-oligoadenylate synthetase. Strikingly, all animals protected from hepatitis became reactive for WHV DNA and carried low levels of replicating virus in hepatic and lymphoid tissues after challenge with WHV. This study shows that the bicistronic DNA vaccine encoding both hepadnavirus core antigen and IFN-gamma was more effective in preventing hepatitis than that encoding virus core alone, but neither of them could mount sterile immunity against the virus or prevent establishment of occult infection.
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Affiliation(s)
- Jinguo Wang
- Molecular Virology and Hepatology Research, Division of Basic Medical Science, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John's, Newfoundland, Canada
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28
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Narayan R, Buronfosse T, Schultz U, Chevallier-Gueyron P, Guerret S, Chevallier M, Saade F, Ndeboko B, Trepo C, Zoulim F, Cova L. Rise in gamma interferon expression during resolution of duck hepatitis B virus infection. J Gen Virol 2006; 87:3225-3232. [PMID: 17030856 DOI: 10.1099/vir.0.82170-0] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Gamma interferon (IFN-γ) expression plays a crucial role in the control of mammalian hepatitis B virus (HBV) infection. However, the role of duck INF-γ (DuIFN-γ) in the outcome of duck HBV (DHBV) infection, a reference model for hepadnavirus replication studies, has not yet been investigated. This work explored the dynamics of DuIFN-γ expression in liver and peripheral blood mononuclear cells (PBMCs) during resolution of DHBV infection in adolescent ducks in relation to serum and liver markers of virus replication, histological changes and humoral response induction. DHBV infection of 3-week-old ducks resulted in transient expression of intrahepatic preS protein (days 3–14) and mild histological changes. Low-level viraemia was detected only during the first 10 days of infection and was accompanied by early anti-preS antibody response induction. Importantly, a strong increase in intrahepatic DuIFN-γ RNA was detected by real-time RT-PCR at days 6–14, which coincided with a sharp decrease in both viral DNA and preS protein in the liver. Interestingly, liver DuIFN-γ expression remained augmented to the end of the follow-up period (day 66) and correlated with portal lymphocyte infiltration and persistence of trace quantities of intrahepatic DHBV DNA in animals that had apparently completely resolved the infection. Moreover, in infected ducks, a moderate increase was detected in the levels of DuIFN-γ in PBMCs (days 12–14), which coincided with the peak in liver DuIFN-γ RNA levels. These data reveal that increased DuIFN-γ expression in liver and PBMCs is concomitant with viral clearance, characterizing the resolution of infection, and provide new insights into the host–virus interactions that control DHBV infection.
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MESH Headings
- Animals
- DNA, Viral/analysis
- DNA, Viral/genetics
- Ducks
- Hepadnaviridae Infections/blood
- Hepadnaviridae Infections/metabolism
- Hepadnaviridae Infections/veterinary
- Hepadnaviridae Infections/virology
- Hepatitis B Antibodies/blood
- Hepatitis B Surface Antigens/immunology
- Hepatitis B Surface Antigens/metabolism
- Hepatitis B Virus, Duck/isolation & purification
- Hepatitis, Viral, Animal/blood
- Hepatitis, Viral, Animal/metabolism
- Hepatitis, Viral, Animal/virology
- Interferon-gamma/genetics
- Interferon-gamma/metabolism
- Leukocytes, Mononuclear/metabolism
- Liver/metabolism
- Liver/virology
- Polymerase Chain Reaction
- RNA, Viral/analysis
- RNA, Viral/genetics
- Time Factors
- Viremia
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Affiliation(s)
| | - Thierry Buronfosse
- Ecole Nationale Vétérinaire, Marcy l'Etoile, France
- INSERM Unit 271, 151 Cours Albert Thomas, 69003 Lyon, France
| | - Ursula Schultz
- Department of Internal Medicine II/Molecular Biology, University Hospital, Freiburg, Germany
| | | | | | | | - Fadi Saade
- INSERM Unit 271, 151 Cours Albert Thomas, 69003 Lyon, France
| | | | - Christian Trepo
- INSERM Unit 271, 151 Cours Albert Thomas, 69003 Lyon, France
| | - Fabien Zoulim
- INSERM Unit 271, 151 Cours Albert Thomas, 69003 Lyon, France
| | - Lucyna Cova
- INSERM Unit 271, 151 Cours Albert Thomas, 69003 Lyon, France
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29
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Lutsiak MEC, Kwon GS, Samuel J. Biodegradable nanoparticle delivery of a Th2-biased peptide for induction of Th1 immune responses. J Pharm Pharmacol 2006; 58:739-47. [PMID: 16734975 DOI: 10.1211/jpp.58.6.0004] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The type of immune response developed against the hepatitis B virus (HBV) is crucial in determining the outcome of the disease. The protective effects of vaccine-induced antibody responses against subsequent exposure to HBV are well-established. After the establishment of chronic HBV infection, cell-mediated immune response is curative while humoral response is detrimental. A therapeutic vaccine that could switch the type of response could lead to disease resolution. Hepatitis B core antigen (HBcAg)(129-140) has been identified as a Th2-biased peptide in H-2(b) mice when it is administered along with complete Freund's adjuvant (CFA). We formulated HBcAg(129-140) along with monophosphoryl lipid A in poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles. Naive mice immunized with the nanoparticle formulation developed a strong Th1-type response while mice immunized with the control formulation of CFA and peptide did not. We then primed mice with CFA and peptide to establish a Th2-type immune response before administering the nanoparticle formulation. Mice receiving the nanoparticle formulation being primed with CFA still developed a strong Th1-type response, while mice that received incomplete Freund's adjuvant and peptide instead of nanoparticles did not. The ability of PLGA nanoparticles to alter the type of immune response elicited by a peptide, even in the context of an ongoing immune response, makes PLGA nanoparticles a strong candidate for the formulation of therapeutic vaccines.
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Affiliation(s)
- M E Christine Lutsiak
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
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30
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Gujar SA, Michalak TI. Characterization of bioactive recombinant woodchuck interleukin-2 amplified by RLM-RACE and produced in eukaryotic expression system. Vet Immunol Immunopathol 2006; 112:183-98. [PMID: 16631932 DOI: 10.1016/j.vetimm.2006.02.011] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2006] [Revised: 02/14/2006] [Accepted: 02/15/2006] [Indexed: 10/24/2022]
Abstract
Woodchucks (Marmota monax) infected with woodchuck hepatitis virus (WHV) represent a highly valuable laboratory model of hepatitis B virus (HBV) infection, in which molecular, immunological and pathological events occurring in infected humans are adequately reflected. To advance studies on T cell immune responses and propagation of hepadnavirus in T lymphocytes in this animal model, we determined the complete sequence of woodchuck interleukin-2 (wIL-2) cDNA by utilizing RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) reaction. The wIL-2 sequence revealed a single open reading frame encoding for the predicted precursor protein comprised of a signal peptide and a 134 amino acid-long mature protein. The mature wIL-2 protein produced in the Escherichia coli expression system, designated as ec-rwIL-2, was found to be immunogenic but not biologically active. In contrast, precursor wIL-2 protein cloned into baculovirus transfer vector and expressed in Sf9 cells, designated as bac-rwIL-2, demonstrated functional competence. Further, bac-rwIL-2 was able to stimulate proliferation and to induce multiple daughter cell generations in woodchuck T cells, as well as facilitated the survival of standard IL-2-dependent mouse CTLL-2 cells in culture. Western blot analysis of bac-rwIL-2 using antibodies generated against ec-rwIL-2 revealed a single protein band of 15.5kDa. The availability of biologically active recombinant wIL-2 should facilitate ex vivo studies on functional competence of woodchuck T lymphocytes derived from different stages of hepadnaviral hepatitis and assist in recognizing their contribution to the pathogenesis of liver injury in the woodchuck model of hepatitis B.
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Affiliation(s)
- Shashi A Gujar
- Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John's, Nfld A1B 3V6, Canada
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31
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Abstract
Hepatitis B virus (HBV) is a major cause of chronic liver inflammation worldwide. Recent knowledge of the virological and immunological events secondary to HBV infection has increased our understanding of the mechanisms involved in viral clearance and persistence. In this review, how the early virological and immunological events might influence the development of a coordinate activation of adaptive immunity necessary to control HBV infection is analysed. The mechanism(s) by which high levels of viral antigens, liver immunological features, regulatory cells and dendritic cell defects might maintain the HBV-specific immunological collapse, typical of chronic hepatitis B patients, is also examined.
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Affiliation(s)
- Antonio Bertoletti
- The UCL Institute of Hepatology, University College of London, 69-75 Chenies Mews, London WC1E 6HX, UK
| | - Adam J Gehring
- The UCL Institute of Hepatology, University College of London, 69-75 Chenies Mews, London WC1E 6HX, UK
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32
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Guy CS, Wang J, Michalak TI. Hepatocytes as cytotoxic effector cells can induce cell death by CD95 ligand-mediated pathway. Hepatology 2006; 43:1231-40. [PMID: 16729304 DOI: 10.1002/hep.21201] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The liver plays an increasingly recognized role in the host's immune responses. The direct contribution of hepatocytes as effector cells to local immunity, pathogen containment, and liver disease is not determined. This in vitro study examined whether hepatocytes can eliminate other cells via a CD95 ligand (CD95L or FasL)/CD95 (Fas)-mediated mechanism and whether this cytotoxic activity can be modulated by cytokines such as interferon gamma (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha). We have found that normal woodchuck and human hepatocytes, both cultured and primary freshly isolated, as well as human HepG2 cells, intrinsically transcribe not only CD95 but also CD95L when examined by reverse transcription-polymerase chain reaction (RT-PCR) assays. The functional competence of CD95L, which was detectable in hepatocytes and HepG2 cells by Western blotting, was confirmed in bioassays by induction of apoptosis of CD95-bearing P815 and LS102.9 cell targets and validated by inhibition of the cell killing with CD95 antagonistic antibody or with a general caspase inhibitor. Furthermore, exposure of cultured hepatocytes to IFN-gamma or their stable transfection with IFN-gamma cDNA or TNF-alpha cDNA increased hepatocyte CD95L/CD95-mediated cell killing. In conclusion, hepatocytes express both CD95L and CD95 and they can induce death of other cells by a CD95L-dependent mechanism. IFN-gamma and, to a lesser extent, TNF-alpha can enhance hepatocyte CD95L-mediated cytotoxicity. This suggests that the local cytokine environment may modulate the hepatocyte contribution to liver immunity.
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Affiliation(s)
- Clifford S Guy
- Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Memorial University, St. John's, Newfoundland A1B 3V6, Canada
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33
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Schildgen O, Fiedler M, Dahmen U, Li J, Lohrengel B, Lu M, Roggendorf M. Fluctuation of the cytokine expression in the liver during the chronic woodchuck hepatitis virus (WHV) infection is not related to viral load. Immunol Lett 2005; 102:31-7. [PMID: 16046239 DOI: 10.1016/j.imlet.2005.06.007] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2005] [Revised: 06/20/2005] [Accepted: 06/21/2005] [Indexed: 11/28/2022]
Abstract
The woodchuck together with the woodchuck hepatitis virus (WHV) is an excellent model to study the pathogenesis of hepadnaviral infections. Chronic WHV infection causes severe liver disease and hepatocellular carcinoma in woodchucks. The mechanism of viral clearance is not fully understood, interferons seem to play a major role in down-regulating viral replication prior to elimination of infected hepatocytes. We investigated on the pattern of cytokine and T-cell-marker expression in livers of woodchucks chronically infected with WHV. RNase-protection-assay (RPA) was used to determine mRNA of woodchuck specific genes (TNF-alpha, IFN-gamma, IL-15, CD3, CD4, CD8). Serial liver biopsies were performed daily or weekly in eight chronic WHV-carrier woodchucks. Cytokine/T-cell-marker expression differed significantly between the time points up to +/-50% within each woodchuck. The different expression patterns of cytokines or T-cell-markers did not correlate to the (weak) fluctuations in the viremia but may explain the observed fluctuations in the WHV/HBV-load in chronically infected individuals. Furthermore, we observed associations between cytokine and T-cell-marker expression. The marginal fluctuations in viremia during the chronic infection may indicate, that, once the chronic hepadnaviral infection is established, cytokines/interferons expressed endogenously (i.e. not vector-borne or injected) play only a minor role.
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Affiliation(s)
- O Schildgen
- University Hospital Essen, Institute of Virology, Hufelandstrasse 55, D-45122 Essen, Germany.
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34
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Wang J, Michalak TI. Comparison of biological activity of recombinant woodchuck interferon gamma and tumor necrosis factor alpha produced in baculovirus and Escherichia coli expression systems. Cytokine 2005; 30:22-34. [PMID: 15784409 DOI: 10.1016/j.cyto.2004.11.006] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2004] [Revised: 10/20/2004] [Accepted: 11/03/2004] [Indexed: 01/12/2023]
Abstract
The full-length cDNAs of recombinant woodchuck interferon gamma (rwIFN gamma) and woodchuck tumor necrosis factor alpha (rwTNF alpha) were cloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The recombinant proteins secreted by the insect cells, bac-rwIFN gamma and bac-rwTNF alpha, were found to be functionally competent. Their biological activities were compared to those of rwIFN gamma and rwTNF alpha produced in the Escherichia coli (E. coli) expression system. The bac-rwIFN gamma demonstrated a 4.5-fold greater protective activity against encephalomyocarditis virus-induced cytolysis of woodchuck hepatocytes and that of class I MHC antigen presentation on the hepatocytes than rwIFN gamma derived from E. coli. The bac-rwTNF alpha was cytotoxic towards murine fibroblasts and able to upregulate class I MHC antigen display and these effects were about 18-fold greater than those triggered by rwTNF alpha from E. coli at a comparable protein level. In addition, the antiviral activity of bac-rwIFN gamma was inhibited by anti-wIFN gamma antibodies and the cytotoxicity of bac-rwTNF alpha neutralized by cross-reactive antibodies to murine TNF alpha. The study showed that the expression of rwIFN gamma and rwTNF alpha in the baculovirus system generated biologically active cytokines whose potency was considerably greater than those produced in E. coli.
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Affiliation(s)
- J Wang
- Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Health Sciences Centre, Memorial University of Newfoundland, St. John's, NL A1B 3V6, Canada
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35
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Li DH, Kumanogoh A, Cao TM, Parnes JR, Cullen JM. Woodchuck interleukin-6 gene: structure, characterization, and biologic activity. Gene 2005; 342:157-64. [PMID: 15527975 DOI: 10.1016/j.gene.2004.07.034] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2004] [Revised: 07/20/2004] [Accepted: 07/29/2004] [Indexed: 01/08/2023]
Abstract
Woodchuck is an important animal model for studying human hepatitis B virus (HBV) infection. Within the cytokine network, interleukin-6 (IL-6) plays an important role in immune responses that may lead to viral clearance. To further understand woodchuck IL-6 biology, we cloned and characterized the IL-6 gene from white blood cells. The complete woodchuck IL-6 gene is about 7 kb and consists of five exons and four introns. The IL-6 gene organization of the woodchuck is similar to those of the human, rat, and mouse. Also several elements are highly conserved in the 300 bp promoter region of the IL-6 gene, including a nuclear factor kappa B (NF-kappaB) binding site. The woodchuck IL-6 gene encodes a polypeptide of 207 amino acids in a precursor form and 189 amino acids in the mature form. The expressed protein was 23 kDa according to SDS-PAGE. To demonstrate biologic activity, we expressed woodchuck IL-6 and showed that the purified recombinant protein induced terminal differentiation, as reflected by upregulation of Fcgamma receptor expression, and substantially inhibited proliferation of M1 cells, a murine myeloid leukemia cell line. The inhibitory effect of woodchuck IL-6 on M1 cells was blocked by an anti-gp130 monoclonal antibody, suggesting that woodchuck IL-6 activity is specifically mediated by signaling through the IL-6 receptor complex. Cloning of the woodchuck IL-6 gene and demonstrating biologic activity of the gene product will facilitate studies of human hepatitis B virus using the woodchuck model.
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Affiliation(s)
- Daniel H Li
- Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, 269 W. Campus Drive, CCSR Building 2230, Stanford, CA 94305-5166, USA.
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36
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Coffin CS, Pham TNQ, Mulrooney PM, Churchill ND, Michalak TI. Persistence of isolated antibodies to woodchuck hepatitis virus core antigen is indicative of occult infection. Hepatology 2004; 40:1053-61. [PMID: 15382154 DOI: 10.1002/hep.20419] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Antibodies against virus nucleocapsid (anticore) normally accompany hepadnaviral hepatitis but they may also occur in the absence of symptoms and other serological indicators of the infection. This situation can be encountered following a clinically and serologically unapparent exposure to hepatitis B virus (HBV) or after recovery from hepatitis B. In this study, woodchucks inoculated with woodchuck hepatitis virus (WHV) were investigated to determine the relationship between anticore detection and the molecular status of virus replication in a primary WHV surface antigen (WHsAg)-negative infection or long-after resolution of WHV hepatitis. Serial, parallel samples of sera, peripheral blood mononuclear cells (PBMC) and liver tissue, collected for more than 5 years after inoculation with virus, were examined for WHV DNA by highly sensitive polymerase chain reaction (PCR)/nucleic acid hybridization assays. Sera were also tested for WHV DNA after DNase treatment and for WHV DNA and WHsAg after concentration in sucrose. Liver and PBMC were examined for WHV covalently closed circular DNA and viral RNA transcripts by PCR-based techniques to assess virus replication status. The study showed that anticore antibodies existing in the absence of other serological markers are a reliable indicator of occult WHV infection. This state can be accompanied by traces of circulating particles behaving as intact virions and by intermittent minimal-to-mild liver inflammation. In conclusion, the long-term presence of anticore antibodies alone is a consequence of sustained restimulation of the immune system by virus nucleocapsid produced during low-level hepadnaviral assembly.
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Affiliation(s)
- Carla S Coffin
- Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3V6, Canada
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37
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N/A. N/A. Shijie Huaren Xiaohua Zazhi 2004; 12:2459-2461. [DOI: 10.11569/wcjd.v12.i10.2459] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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38
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Buke AC, Buke M, Altuglu IE, Ciceklioglu M, Kamcioglu S, Karakartal G, Huseyinov A. Tumor necrosis factor alpha and interleukin 6 productions in response to platelet-activating factor in chronic hepatitis B virus infection. Med Princ Pract 2004; 13:273-6. [PMID: 15316260 DOI: 10.1159/000079526] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
OBJECTIVE The aim of this study was to determine tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) release in response to platelet-activating factor (PAF) induction in peripheral blood mononuclear cells (PBMCs) from chronic hepatitis B virus (HBV) carriers. METHODS Subjects were grouped into three subgroups. The mean age was 37 +/- 10 years. Group A (n = 15), group B (n = 10) and group C (n = 9) subjects were HBV serology-negative, had natural immunity after recovery from an acute HBV infection, and were chronic HBV carriers, respectively. RESULTS Compared with group A, PBMCs from naturally immune subjects and chronic HBV carriers produced significantly higher amounts of TNF-alpha and IL-6 in response to PAF. In chronic HBV carriers, TNF-alpha (1,633.3 +/- 793.7) and IL-6 (2,533.3 +/- 466.3) production was statistically lower than TNF-alpha (2,630.0 +/- 727.3) and IL-6 (3,870.0 +/- 728.4) obtained from naturally immune subjects to HBV. CONCLUSION Differences of TNF-alpha levels between chronic HBV carriers and naturally immune subjects suggest that TNF-alpha may be a critical mediator of HBV clearance.
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Affiliation(s)
- A Cagri Buke
- Department of Infectious Diseases and Clinical Microbiology, Ege University Medical Faculty, Bornova, Izmir, Turkey.
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39
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Wang Y, Jacob JR, Menne S, Bellezza CA, Tennant BC, Gerin JL, Cote PJ. Interferon-gamma-associated responses to woodchuck hepatitis virus infection in neonatal woodchucks and virus-infected hepatocytes. J Viral Hepat 2004; 11:404-17. [PMID: 15357645 DOI: 10.1111/j.1365-2893.2004.00529.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Acute hepatitis and recovery from woodchuck hepatitis virus (WHV) infection involves increased intrahepatic expression of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) mRNAs. In the present study, recovery correlated with increased intrahepatic expression of mRNAs for major histocompatibility complex class 1 (MHC1), beta(2)-microglobulin, 2'5'-oligoadenylate synthetase (2'5'-OAS), and indoleamine dioxygenase (IDO). By comparison, acute WHV infection progressing to chronicity was associated with diminished expression of these IFN-gamma-associated mRNAs in liver. Transfection of WHV-infected primary hepatocytes (WPH) from WHV carriers with an IFN-gamma-expressing plasmid (pIFN-gamma) resulted in dose-dependent accumulations of MHC1, TNF-alpha, 2'5'-OAS, and IDO mRNAs within 96 h. Markers of T cells and immune-mediated cytotoxicity that accumulate in recovering liver were not apparent in WPH based on the relative lack of CD3, CD4, Fas ligand, perforin, and granzyme B mRNAs. Expression of pIFN-gamma, and TNF-alpha-expressing plasmid (pTNF-alpha), did not affect total WHV RNA, or fully double-stranded WHV DNA in WPH, but each reduced some of the replicative intermediate (RI) species of WHV DNA synthesis. WPH treated with recombinant IFN-alpha protein had a higher fold induction of 2'5'-OAS mRNA associated with partial reductions in WHV RNAs and the major RI species. Thus, IFN-gamma expression in carrier WPH induced several host responses often observed in liver of recovering woodchucks, and impaired a stage of WHV DNA synthesis by a non-cytolytic mechanism mediated by TNF-alpha. Local enhancement of IFN-gamma-associated responses in chronic WHV-infected hepatocytes may promote therapeutic antiviral effects, but additional effector mechanisms evident during recovery appear necessary for more complete clearance of WHV infection.
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Affiliation(s)
- Y Wang
- Division of Molecular Virology and Immunology, Department of Microbiology and Immunology, Georgetown University Medical Center, Rockville, MD 20850, USA.
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40
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Wang Y, Menne S, Baldwin BH, Tennant BC, Gerin JL, Cote PJ. Kinetics of viremia and acute liver injury in relation to outcome of neonatal woodchuck hepatitis virus infection. J Med Virol 2004; 72:406-15. [PMID: 14748064 DOI: 10.1002/jmv.20019] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The kinetics of serum viral responses and acute liver injury were studied during neonatal woodchuck hepatitis virus (WHV) infection in relation to the chronic or resolved outcome. The mean concentrations of serum WHV DNA and surface antigen were significantly higher by week 10 post infection in chronic infections compared to resolving infections, and diverged even further by the time of peak viral load development in serum (week 12). After week 12, these viral markers were detected less frequently with time and at lower concentrations in the resolved outcome. In both outcomes, mean serum activities of hepatic enzymes became increased significantly above baseline by weeks 10-12, peaked at week 14, and normalized by weeks 20-22, thus indicating transient acute liver injury. The increasing liver injury responses were comparable between outcomes at week 12, when serum viral load was markedly higher in the developing chronic infections. This suggested a deficiency in early non-cytolytic control of infection in the chronic outcome. At week 14, liver injury was significantly greater in the resolved outcome and associated with higher mean Fas ligand (FasL) and perforin messenger RNAs (mRNAs) in liver compared to the chronic outcome. This indicated greater immune-mediated killing of infected hepatocytes during resolution. Thus, chronicity as an outcome of neonatal WHV infection develops relatively early during the acute phase of infection due to reduced immune-mediated clearance of infected hepatocytes by both cytolytic and non-cytolytic processes.
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MESH Headings
- Animals
- Animals, Newborn
- Antigens, Surface/blood
- Antigens, Viral/blood
- DNA, Viral/blood
- Disease Progression
- Enzymes/blood
- Fas Ligand Protein
- Gene Expression
- Hepatitis B/immunology
- Hepatitis B/pathology
- Hepatitis B/physiopathology
- Hepatitis B/virology
- Hepatitis B Virus, Woodchuck/growth & development
- Hepatitis B Virus, Woodchuck/immunology
- Hepatitis B Virus, Woodchuck/isolation & purification
- Hepatitis B, Chronic/immunology
- Hepatitis B, Chronic/pathology
- Hepatitis B, Chronic/physiopathology
- Hepatitis B, Chronic/virology
- Hepatitis, Viral, Animal/immunology
- Hepatitis, Viral, Animal/pathology
- Hepatitis, Viral, Animal/physiopathology
- Hepatitis, Viral, Animal/virology
- Kinetics
- Liver/pathology
- Marmota/virology
- Membrane Glycoproteins/analysis
- Membrane Glycoproteins/genetics
- Perforin
- Pore Forming Cytotoxic Proteins
- Viral Load
- Viremia/virology
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Affiliation(s)
- Yun Wang
- Division of Molecular Virology and Immunology, Department of Microbiology and Immunology, Georgetown University Medical Center, Rockville, Maryland 20852, USA
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41
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Michalak TI, Mulrooney PM, Coffin CS. Low doses of hepadnavirus induce infection of the lymphatic system that does not engage the liver. J Virol 2004; 78:1730-8. [PMID: 14747538 PMCID: PMC369489 DOI: 10.1128/jvi.78.4.1730-1738.2004] [Citation(s) in RCA: 80] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Woodchuck hepatitis virus (WHV), which is closely related to human hepatitis B virus and is considered to be principally hepatotropic, invades the host's lymphatic system and persists in lymphoid cells independently of whether the infection is symptomatic and serologically evident or concealed. In this study, we show, with the woodchuck model of hepatitis B, that hepadnavirus can establish an infection that engages the lymphatic system, but not the liver, and persists in the absence of virus serological markers, including antiviral antibodies. This primary occult infection is caused by wild-type virus invading the host at a quantity usually not greater than 10(3) virions. It is characterized by trace virus replication progressing in lymphatic organs and peripheral lymphoid cells that, with time, may also spread to the liver. The infection is transmissible to virus-naive hosts as an asymptomatic, indefinitely long, occult carriage of small amounts of biologically competent virus. In contrast to residual silent WHV persistence, which normally endures after the resolution of viral hepatitis and involves the liver, primary occult infection restricted to the lymphatic system does not protect against reinfection with a large, liver-pathogenic WHV dose; however, the occult infection is associated with a swift recovery from hepatitis caused by the superinfection. Our study documents that the lymphatic system is the primary target of WHV infection when small quantities of virions invade a susceptible host.
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Affiliation(s)
- Tomasz I Michalak
- Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3V6, Canada.
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42
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Diao J, Slaney DM, Michalak TI. Modulation of the outcome and severity of hepadnaviral hepatitis in woodchucks by antibodies to hepatic asialoglycoprotein receptor. Hepatology 2003; 38:629-38. [PMID: 12939589 DOI: 10.1053/jhep.2003.50370] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/07/2022]
Abstract
Viral hepatitis is frequently accompanied by humoral autoimmune responses toward both organ-nonspecific and liver-specific antigens, but contribution of these reactivities to liver injury remains unrecognized. Infection with woodchuck hepatitis virus (WHV) has been identified as a potent inducer of autoantibodies against asialoglycoprotein receptor (anti-ASGPR), a molecule essentially unique to hepatocytes that mediates clearance of desialylated serum proteins. In this study, we applied the WHV-woodchuck model of hepatitis B to examine the effect of experimentally elicited anti-ASGPR on the progression and the severity of WHV hepatitis in initially healthy animals immunized with the receptor and then infected with WHV and in woodchucks with ongoing chronic WHV hepatitis. The results implied that the induction of anti-ASGPR prior to WHV infection tends to modulate acute viral hepatitis toward chronic outcome and, in animals with established chronic WHV infection, exacerbates histologic severity of liver lesions. The findings also suggest that the liver compromised by chronic hepadnavirus infection might be prone to anti-ASGPR-directed complement-mediated hepatocellular injury and that this is associated with formation of the ASGPR-anti-ASGPR immune complexes on hepatocyte surface. In conclusion, the host's immune response mounted against a hepatocyte-specific autoantigen may modulate both the outcome and the severity of liver injury in viral hepatitis.
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Affiliation(s)
- Jingyu Diao
- Division of Basic Medical Sciences, Molecular Virology and Hepatology Research, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland, Canada
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43
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Menne S, Roneker CA, Tennant BC, Korba BE, Gerin JL, Cote PJ. 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 2003; 45:237-50. [PMID: 12566706 DOI: 10.1159/000067914] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
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-beta-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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Affiliation(s)
- Stephan Menne
- Gastrointestinal Unit, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
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Wang Y, Menne S, Jacob JR, Tennant BC, Gerin JL, Cote PJ. Role of type 1 versus type 2 immune responses in liver during the onset of chronic woodchuck hepatitis virus infection. Hepatology 2003; 37:771-80. [PMID: 12668969 DOI: 10.1053/jhep.2003.50154] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Immune response messenger RNAs (mRNA) were compared in liver during self-limited (resolved) and chronic neonatal woodchuck hepatitis virus (WHV) infection. At week 14 postinfection (mid-acute phase), mRNAs for leukocyte markers (CD3, CD4, CD8), type 1 cytokines and related transcription factors (IFN-gamma, TNF-alpha, STAT4, T-bet), and IL-10 were increased in livers from resolving infections, but mRNAs of other type 1 (IL-2) and type 2 (IL-4, STAT6, and GATA3 markers remained at baseline levels. Increased coexpression of IFN-gamma and TNF-alpha mRNAs correlated in most cases with lower levels of intrahepatic WHV covalently closed circular DNA (cccDNA). At the same time point postinfection, livers from woodchucks that eventually progressed to chronic infection had baseline or slightly elevated levels of CD and type 1 mRNAs, which were significantly lower (or elevated less frequently) compared with resolving woodchucks. Earlier, at week 8, there were no differences between the two outcome settings. During these early time points and at a later stage in chronic infection (15 months), type 2 mRNAs in carrier liver remained at baseline levels or, when elevated, were never in excess of those in resolving woodchucks. In conclusion, the onset and maintenance of neonatal chronic WHV infection are not associated with antagonistic type 2 immunoregulation of type 1 responses in liver. Accordingly, chronicity develops in association with a primary deficiency in the intrahepatic CD responses, especially involving CD8(+) T lymphocytes, and in both extracellular (cytokine) and intracellular (transcriptional) type 1 response mediators. This has relevant implications for future treatment of chronic hepatitis B virus (HBV) infection in humans.
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Affiliation(s)
- Yun Wang
- Division of Molecular Virology and Immunology, Department of Microbiology and Immunology, Georgetown University Medical Center, Rockville, MD 20850, USA.
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Mulrooney PM, Michalak TI. Quantitative detection of hepadnavirus-infected lymphoid cells by in situ PCR combined with flow cytometry: implications for the study of occult virus persistence. J Virol 2003; 77:970-9. [PMID: 12502813 PMCID: PMC140851 DOI: 10.1128/jvi.77.2.970-979.2003] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
The detection of small amounts of viral pathogens in infected cells by classical PCR is hampered by a partial loss of virus nucleic acid due to extraction and by difficulties in discrimination between truly intracellular virus genome material and that possibly adhered to the cell surface. These impediments limit reliable identification of virus traces within infected cells, which are typically encountered in latent and persistent occult infections. In this study, hepadnavirus-specific in situ PCR combined with the enzymatic elimination of extracellular virus and flow cytometry permitted detection of viral genomes in lymphoid cells without nucleic acid isolation and allowed quantification of infected cells during the course of persistent infection with woodchuck hepatitis virus (WHV). The validity of the procedure was confirmed by hybridization analysis of the in situ-amplified viral sequences. The results showed that hepadnavirus can be directly detected within lymphoid cells not only in serologically accountable infection, but also years after recovery from viral hepatitis and in the course of primary occult virus carriage. Percentages of infected peripheral lymphoid cells in symptomatic WHV hepatitis fluctuate between 3.4 and 20.4% (mean +/- standard error of the mean, 9.6% +/- 1.7%), whereas those in persistent, serologically mute WHV infection range from 1.1 to 14.6% (mean +/- standard error of the mean, 4.8% +/- 0.8%) (P = 0.005). The data obtained provide further evidence that WHV infection continues indefinitely in the lymphatic system independently of whether it is symptomatic or concealed. They document that hepadnavirus can be detected in a significant proportion of circulating lymphoid cells in both immunovirologically apparent as well as occult persistent infection.
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Affiliation(s)
- Patricia M Mulrooney
- Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Canada A1B 3V6
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Menne S, Wang Y, Butler SD, Gerin JL, Cote PJ, Tennant BC. Real-time polymerase chain reaction assays for leukocyte CD and cytokine mRNAs of the Eastern woodchuck (Marmota monax). Vet Immunol Immunopathol 2002; 87:97-105. [PMID: 12052347 DOI: 10.1016/s0165-2427(02)00121-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Real-time polymerase chain reaction (PCR) assays were developed for woodchuck leukocyte cluster of differentiation (CD) and cytokine mRNA expression. Plasmid DNA standards of each marker (CD3, CD4, CD8, IL-2, IFN-gamma, TNF-alpha, IL-4, IL-10), and RNA standards from mitogen-stimulated woodchuck peripheral blood mononuclear cells (PBMCs) were used to validate and optimize the assays for TaqMan 7700 and iCycler PCR instruments. The complementary DNAs (cDNAs) produced by reverse transcription (RT) of RNA were quantified by real-time PCR against the plasmid DNA standards (6-8 log range) with detection of as few as 10-50 copies of amplicon cDNA per reaction. Analysis of unstimulated and concanavalin A-stimulated woodchuck PBMC demonstrated increased CD and cytokine mRNA expression following mitogenic activation. A liver sample from a woodchuck hepatitis virus (WHV) infected woodchuck with histologically confirmed acute hepatitis had increased intrahepatic CD and cytokine mRNAs compared to liver from an uninfected control woodchuck. The real-time PCR assays were highly specific for the woodchuck markers in PBMC and liver samples and were equally applicable for use in alternate real-time PCR instrumentation. These assays will enable the high-throughput analyses of mRNA markers during WHV infection, and thereby facilitate continued modelling of the immunopathogenesis and immunotherapy of human hepatitis B virus (HBV) infection.
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Affiliation(s)
- Stephan Menne
- Gastrointestinal Unit, Department of Clinical Sciences, College of Veterinary Medicine, Room C2 005 VMC, Cornell University, Ithaca, NY 14853, USA.
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Abstract
Occult hepatitis B virus (HBV) infection is characterized by presence of HBV infection with undetectable hepatitis B surface antigen (HBsAg). Serum HBV level is usually less than 104 copies/mL in these patients. Diagnosis of occult HBV infection requires sensitive HBV-DNA PCR assay. Several possibilities have been hypothesized as the mechanisms of occult HBV infection. These include: (i) mutations of HBV-DNA sequence; (ii) integration of HBV-DNA into host's chromosomes; (iii) infection of peripheral blood mononuclear cells by HBV; (iv) formation of HBV-containing immune complex; (v) altered host immune response; and (vi) interference of HBV by other viruses. The precise prevalence of occult HBV infection remains to be defined. The clinical implications of occult HBV infection involve different clinical aspects. First of all, occult HBV infection harbours potential risk of HBV transmission through blood transfusion, haemodialysis, and organ transplantation. Second, it may serve as the cause of cryptogenic liver disease, contribute to acute exacerbation of chronic hepatitis B, or even fulminant hepatitis. Third, it is associated with development of hepatocellular carcinoma. Fourth, it may affect disease progression and treatment response of chronic hepatitis C. Most of the previous studies utilized retrospective observation without control groups, and lacked direct association of occult HBV infection with specific pathological changes and disease progression. Highly sensitive, quantitative, and functional molecular analyses of HBV, combined with a well-designed prospective clinical assessment will provide the best approach for the future study of occult HBV infection.
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Affiliation(s)
- Ke-Qin Hu
- Transplantation Institute and Division of Gastroenterology, Loma Linda University Medical Center and Jerry L. Pettis Memorial VA Medical Center, Loma Linda, California 92354, USA.
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Abstract
The immune response initiated by the T-cell response to viral antigens is thought to be fundamental for viral clearance and disease pathogenesis in hepatitis B virus (HBV) infection. The T-cell response during acute self-limited hepatitis B in people is characterised by a vigorous, polyclonal, and multispecific cytotoxic and helper-T-cell response. By contrast, the immune response in chronic carriers, not able to eliminate the virus, is weak or undetectable. Thus a dominant cause of viral persistence could be the existence of a weak antiviral immune response. Methodological progress in animal models allows more precise investigation of the mechanisms by which the immune system resolves viral infection or develops chronic infection. Although clearance of most virus infections is widely thought to indicate the killing of infected cells by virus-specific T cells, data suggest that non-cytolytic intracellular viral inactivation by cytokines released by virus-inactivated lymphomononuclear cells could have an important role in the clearance of this virus without killing the infected cell. Additional factors that could contribute to viral persistence, which have been partly proven in animal models, are viral inhibition of antigen processing or presentation, modulation of the response to cytotoxic mediators, immunological tolerance to viral antigens, viral mutations, and infection of immunologically privileged sites. In view of the central role of cellular immunity in disease pathogenesis, strategies have been proposed to manipulate this cellular immune response in favour of protection from disease.
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Affiliation(s)
- Maria-Christina Jung
- Institute for Immunology and Medical Departments, University of Munich, Munich, Germany.
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