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Stephan Menne - One of the best experts on this subject based on the ideXlab platform.
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Infection patterns induced in naive adult woodchucks by virions of woodchuck hepatitis virus collected during either the acute or chronic phase of Infection
Journal of Virology, 2015Co-Authors: Natalia Freitas, Louise Rodrigues, Bhaskar Kallakury, Stephan Menne, Tetyana Lukash, Sam Litwin, Severin O GudimaAbstract:UNLABELLED: The infectivity of Hepadnavirus virions produced during either acute or chronic stages of Infection was compared by testing the ability of the virions of woodchuck hepatitis virus (WHV) to induce productive acute Infection in naive adult woodchucks. Serum WHV collected during acute Infection was compared to virions harvested from WHV-infected woodchucks during either (i) early chronic Infection, when WHV-induced hepatocellular carcinoma (HCC) was not yet developed, or (ii) late chronic Infection, when established HCC was terminal. All tested types of WHV inoculum were related, because they were collected from woodchucks that originally were infected with standardized WHV7 inoculum. Despite the individual differences between animals, the kinetics of accumulation of serum relaxed circular DNA of WHV demonstrated that the virions produced during early or late chronic Infection are fully capable of inducing productive acute Infection with long-lasting high viremia. These findings were further supported by the analysis of such intrahepatic markers of WHV Infection as replicative intermediate DNA, covalently closed circular DNA, pregenomic RNA, and the percentage of WHV core antigen-positive hepatocytes measured at several time points over the course of 17.5 weeks after the inoculation. In addition, the observed relationship between the production of antibodies against WHV surface antigens and parameters of WHV Infection appears to be complex. Taken together, the generated data suggest that in vivo Hepadnavirus virions produced during different phases of chronic Infection did not demonstrate any considerable deficiencies in infectivity compared to that of virions generated during the acute phase of Infection. IMPORTANCE: The generated data suggest that infectivity of virions produced during the early or late stages of chronic Hepadnavirus Infection is not compromised. Our novel results provided several lines of further evidence supporting the idea that during the state of chronic Infection in vivo, the limitations of Hepadnavirus cell-to-cell spread/superInfection (observed recently in the woodchuck model) are not due to the diminished infectivity of the virions circulating in the blood and likely are (i) related to the properties of hepatocytes (i.e., their capacity to support Hepadnavirus Infection/replication) and (ii) influenced by the immune system. The obtained results further extend the understanding of the mechanisms regulating the persistence of Hepadnavirus Infection. Follow-up studies that will further investigate Hepadnavirus cell-to-cell spread as a potential regulator of the chronic state of the Infection are warranted.
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superInfection with woodchuck hepatitis virus strain whvny of livers chronically infected with strain whv7
Journal of Virology, 2015Co-Authors: Louise Rodrigues, Bhaskar Kallakury, Natalia Freitas, Stephan Menne, Severin O GudimaAbstract:The determinants of the maintenance of chronic hepadnaviral Infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral Infection, cell-to-cell spread of Hepadnavirus is at least very inefficient (if it occurs at all), virus superInfection is an unlikely event, and chronic Hepadnavirus Infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. SuperInfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superInfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superInfection was demonstrated by using WHV strain-specific PCR assays and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all superinfected animals during weeks one through six after the superInfection, (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three superinfected woodchucks, and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the superInfection. The results are consistent with the occurrence of continuous but inefficient Hepadnavirus cell-to-cell spread and superInfection during chronic Infection and suggest that the replication space occupied by the superinfecting Hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic Hepadnavirus Infection. IMPORTANCE This study aimed to better understand the determinants of the maintenance of chronic Hepadnavirus Infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) Hepadnavirus superInfection and cell-to-cell spread likely continue to occur and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of Hepadnavirus. The limitations of the cell-to-cell virus spread most likely are mediated at the level of the cells and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic Hepadnavirus Infection. The significance of the continuous but limited Hepadnavirus spread and superInfection for the maintenance of the chronic state of Infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic Hepadnavirus Infection.
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superInfection with woodchuck hepatitis virus strain whvny of livers chronically infected with strain whv7
Journal of Virology, 2015Co-Authors: Louise Rodrigues, Bhaskar Kallakury, Natalia Freitas, Stephan Menne, Severin O GudimaAbstract:The determinants of the maintenance of chronic hepadnaviral Infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral Infection, cell-to-cell spread of Hepadnavirus is at least very inefficient (if it occurs at all), virus superInfection is an unlikely event, and chronic Hepadnavirus Infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. SuperInfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superInfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superInfection was demonstrated by using WHV strain-specific PCR assays and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all superinfected animals during weeks one through six after the superInfection, (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three superinfected woodchucks, and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the superInfection. The results are consistent with the occurrence of continuous but inefficient Hepadnavirus cell-to-cell spread and superInfection during chronic Infection and suggest that the replication space occupied by the superinfecting Hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic Hepadnavirus Infection. IMPORTANCE This study aimed to better understand the determinants of the maintenance of chronic Hepadnavirus Infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) Hepadnavirus superInfection and cell-to-cell spread likely continue to occur and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of Hepadnavirus. The limitations of the cell-to-cell virus spread most likely are mediated at the level of the cells and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic Hepadnavirus Infection. The significance of the continuous but limited Hepadnavirus spread and superInfection for the maintenance of the chronic state of Infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic Hepadnavirus Infection.
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identification of an intrahepatic transcriptional signature associated with self limiting Infection in the woodchuck model of hepatitis b
Hepatology, 2013Co-Authors: Simon P Fletcher, Paul J. Cote, Daniel J Chin, Donavan T Cheng, Palanikumar Ravindran, Hans Bitter, Lore Gruenbaum, Klaus Klumpp, Stephan MenneAbstract:The woodchuck model of hepatitis B virus (HBV) Infection displays many characteristics of human Infection and has particular value for characterizing the host immune responses during the development of chronic Infection. Using the newly developed custom woodchuck microarray platform, we compared the intrahepatic transcriptional profiles of neonatal woodchucks with self-limiting woodchuck hepatitis virus (WHV) Infection to those woodchucks progressing to persistent WHV Infection. This revealed that WHV does not induce significant intrahepatic gene expression changes during the early-acute stage of Infection (8 weeks), suggesting it is a stealth virus. At the mid-acute phase of Infection (14 weeks), resolution was associated with induction of a prominent cytotoxic T-cell signature. Strikingly, this was accompanied by high-level expression of PD-1 and various other inhibitory T-cell receptors, which likely act to minimize liver damage by cytotoxic T cells during viral clearance. In contrast to the expression of perforin and other cytotoxic effector genes, the interferon-γ (IFN-γ) signaling response in the mid-acute phase was comparable to that in chronically infected adult animals. The absence of a strong IFN-α/β transcriptional response indicated that type I IFN is not a critical mediator of self-limiting Infection. Nevertheless, a number of antiviral genes, including viperin, were differentially expressed during resolving Infection, suggesting that a subset of IFN-stimulated genes (ISG) may play a role in the control of WHV replication. Conclusion: We identified new immune pathways associated with the clearance of Hepadnavirus Infection revealing novel molecular targets with potential for the therapeutic treatment of chronic hepatitis B. (HEPATOLOGY 2013)
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identification of cd4 and cd8 t cell epitopes of woodchuck hepatitis virus core and surface antigens in balb c mice
Vaccine, 2010Co-Authors: Laura Ochoacallejero, Pablo Sarobe, Jesus Prieto, Juan José Lasarte, Stephan Menne, Africa Vales, Itziar Otano, Cristina Olague, Gloria GonzalezaseguinolazaAbstract:Abstract A therapeutic vaccine against chronic hepatitis B virus (HBV) Infection requires the development of a strong and multispecific Th1 cell immune response. Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) closely resemble HBV Infection and represent the best animal model for this Hepadnavirus-induced disease. Using the BIMAS “HLA Peptide Binding Predictions” program, we have identified and further characterized novel H-2d-restricted CD8+ epitopes within the WHV core (peptides C#12–21, C#18–32, C#19–27, C#61–69) and surface antigens (peptides preS2#10–18, preS2#27–35, S#76–84, S#133–140 and S#257–265), respectively. These peptides bind to H-2d with high efficiency and upon immunization of mice with peptide and Freund's adjuvant they induce the development of IFN-γ producing T cells. More importantly, WHV core peptides C#19–27 and C#61–69 and WHV surface peptides S#133–140 and S#257–265 were also recognized by CD8+ T cells after immunization of mice with DNA/PEI nanoparticles. Direct stimulation of splenocytes obtained from such DNA-immunized mice with peptides C#18–32, S#76–84, and S#257–265 resulted in significant production of IFN-γ. Thus, we have identified T cell determinants in mice from WHV core and surface antigens that have important value for designing and evaluating an effective vaccine against Hepadnavirus Infection.
Tomasz I Michalak - One of the best experts on this subject based on the ideXlab platform.
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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
Journal of Virology, 2009Co-Authors: Shashi A Gujar, Tomasz I MichalakAbstract: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 (>103 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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in vitro and in vivo infectivity and pathogenicity of the lymphoid cell derived woodchuck hepatitis virus
Journal of Virology, 2001Co-Authors: Tomasz I MichalakAbstract:Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of Hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (∼10 3 virions) caused immunovirologically undetectable (occult) WHV Infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious Hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of Hepadnavirus Infection, particularly when Infection is induced by low virus doses.
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in vitro and in vivo infectivity and pathogenicity of the lymphoid cell derived woodchuck hepatitis virus
Journal of Virology, 2001Co-Authors: Yuanyee Lew, Tomasz I MichalakAbstract:Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of Hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV Infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious Hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of Hepadnavirus Infection, particularly when Infection is induced by low virus doses.
Severin O Gudima - One of the best experts on this subject based on the ideXlab platform.
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Infection patterns induced in naive adult woodchucks by virions of woodchuck hepatitis virus collected during either the acute or chronic phase of Infection
Journal of Virology, 2015Co-Authors: Natalia Freitas, Louise Rodrigues, Bhaskar Kallakury, Stephan Menne, Tetyana Lukash, Sam Litwin, Severin O GudimaAbstract:UNLABELLED: The infectivity of Hepadnavirus virions produced during either acute or chronic stages of Infection was compared by testing the ability of the virions of woodchuck hepatitis virus (WHV) to induce productive acute Infection in naive adult woodchucks. Serum WHV collected during acute Infection was compared to virions harvested from WHV-infected woodchucks during either (i) early chronic Infection, when WHV-induced hepatocellular carcinoma (HCC) was not yet developed, or (ii) late chronic Infection, when established HCC was terminal. All tested types of WHV inoculum were related, because they were collected from woodchucks that originally were infected with standardized WHV7 inoculum. Despite the individual differences between animals, the kinetics of accumulation of serum relaxed circular DNA of WHV demonstrated that the virions produced during early or late chronic Infection are fully capable of inducing productive acute Infection with long-lasting high viremia. These findings were further supported by the analysis of such intrahepatic markers of WHV Infection as replicative intermediate DNA, covalently closed circular DNA, pregenomic RNA, and the percentage of WHV core antigen-positive hepatocytes measured at several time points over the course of 17.5 weeks after the inoculation. In addition, the observed relationship between the production of antibodies against WHV surface antigens and parameters of WHV Infection appears to be complex. Taken together, the generated data suggest that in vivo Hepadnavirus virions produced during different phases of chronic Infection did not demonstrate any considerable deficiencies in infectivity compared to that of virions generated during the acute phase of Infection. IMPORTANCE: The generated data suggest that infectivity of virions produced during the early or late stages of chronic Hepadnavirus Infection is not compromised. Our novel results provided several lines of further evidence supporting the idea that during the state of chronic Infection in vivo, the limitations of Hepadnavirus cell-to-cell spread/superInfection (observed recently in the woodchuck model) are not due to the diminished infectivity of the virions circulating in the blood and likely are (i) related to the properties of hepatocytes (i.e., their capacity to support Hepadnavirus Infection/replication) and (ii) influenced by the immune system. The obtained results further extend the understanding of the mechanisms regulating the persistence of Hepadnavirus Infection. Follow-up studies that will further investigate Hepadnavirus cell-to-cell spread as a potential regulator of the chronic state of the Infection are warranted.
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superInfection with woodchuck hepatitis virus strain whvny of livers chronically infected with strain whv7
Journal of Virology, 2015Co-Authors: Louise Rodrigues, Bhaskar Kallakury, Natalia Freitas, Stephan Menne, Severin O GudimaAbstract:The determinants of the maintenance of chronic hepadnaviral Infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral Infection, cell-to-cell spread of Hepadnavirus is at least very inefficient (if it occurs at all), virus superInfection is an unlikely event, and chronic Hepadnavirus Infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. SuperInfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superInfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superInfection was demonstrated by using WHV strain-specific PCR assays and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all superinfected animals during weeks one through six after the superInfection, (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three superinfected woodchucks, and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the superInfection. The results are consistent with the occurrence of continuous but inefficient Hepadnavirus cell-to-cell spread and superInfection during chronic Infection and suggest that the replication space occupied by the superinfecting Hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic Hepadnavirus Infection. IMPORTANCE This study aimed to better understand the determinants of the maintenance of chronic Hepadnavirus Infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) Hepadnavirus superInfection and cell-to-cell spread likely continue to occur and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of Hepadnavirus. The limitations of the cell-to-cell virus spread most likely are mediated at the level of the cells and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic Hepadnavirus Infection. The significance of the continuous but limited Hepadnavirus spread and superInfection for the maintenance of the chronic state of Infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic Hepadnavirus Infection.
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superInfection with woodchuck hepatitis virus strain whvny of livers chronically infected with strain whv7
Journal of Virology, 2015Co-Authors: Louise Rodrigues, Bhaskar Kallakury, Natalia Freitas, Stephan Menne, Severin O GudimaAbstract:The determinants of the maintenance of chronic hepadnaviral Infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral Infection, cell-to-cell spread of Hepadnavirus is at least very inefficient (if it occurs at all), virus superInfection is an unlikely event, and chronic Hepadnavirus Infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. SuperInfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superInfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superInfection was demonstrated by using WHV strain-specific PCR assays and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all superinfected animals during weeks one through six after the superInfection, (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three superinfected woodchucks, and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the superInfection. The results are consistent with the occurrence of continuous but inefficient Hepadnavirus cell-to-cell spread and superInfection during chronic Infection and suggest that the replication space occupied by the superinfecting Hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic Hepadnavirus Infection. IMPORTANCE This study aimed to better understand the determinants of the maintenance of chronic Hepadnavirus Infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) Hepadnavirus superInfection and cell-to-cell spread likely continue to occur and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of Hepadnavirus. The limitations of the cell-to-cell virus spread most likely are mediated at the level of the cells and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic Hepadnavirus Infection. The significance of the continuous but limited Hepadnavirus spread and superInfection for the maintenance of the chronic state of Infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic Hepadnavirus Infection.
Michael Roggendorf - One of the best experts on this subject based on the ideXlab platform.
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coadministration of gamma interferon with dna vaccine expressing woodchuck hepatitis virus whv core antigen enhances the specific immune response and protects against whv Infection
Journal of Virology, 2001Co-Authors: Felix Siegel, Mengji Lu, Michael RoggendorfAbstract:Plasmid DNA vaccines are novel and powerful tools to induce humoral and cellular immune responses which are protective against bacterial and viral Infections (25, 33; reviewed in reference 8). Altering the route of delivery and coapplication of stimulatory molecules can be used to improve DNA vaccines. DNA vaccines are commonly delivered by either intramuscular injection or intradermal application using a gene gun. The gene gun-mediated propulsion of DNA-coated gold particles into the dermis is an attractive mode of application, since only small amounts of plasmid DNA are needed for vaccination (13, 20, 26, 30, 31). The presence of large numbers of antigen-presenting Langerhans cells makes the skin a major immunological inductive site and may explain the high efficacy of gene gun vaccination (19, 29). The coapplication of plasmids expressing cytokines is an approach to modulate immune response to DNA vaccines (3, 4, 11, 14, 15). It has been demonstrated that gamma interferon (IFN-γ) plasmids support Th1 responses and suppress Th2 responses. Other biological effects of IFN-γ include the induction of major histocompatibility complex class I and II expression on cellular surfaces and hence an enhancement of antigen presentation. IFN-γ also converts various cell types into nonprofessional antigen-presenting cells and triggers the differentiation, maturation, and activation of resting macrophages (reviewed in reference 9). Furthermore, IFN-γ supports tumor necrosis factor alpha effects in a synergistic way. Consequently, it has been shown that the coinjection of IFN-γ and interleukin-12 expression vectors significantly enhances the cellular immune response in mice (15). To evaluate DNA vaccines against hepatitis B, a number of immunogenicity studies and protection studies with different hepatitis B virus proteins have been carried out (1, 2, 7, 17, 18, 34, 35). Immunizations with plasmids expressing hepatitis B virus surface antigen (HBsAg) and hepatitis B virus core antigen (HBcAg) have been shown to induce high antibody titers and substantial T-cell responses in mice (1, 2). Protection from Hepadnavirus Infection by intramuscular DNA immunization has been demonstrated with ducks and woodchucks (22, 32). Antibody titers known to be protective in humans have also been induced by DNA vaccination of chimpanzees using a plasmid expressing HBsAg (6). In previous studies, it has been demonstrated that an immune response against the woodchuck hepatitis virus (WHV) core antigen (WHcAg) primed by DNA vaccination effectively protected woodchucks against subsequent challenge with WHV (22). As the core protein inside the intact viral particle is covered by the surface antigen and therefore is not accessible to neutralizing antibodies, the cellular immune response may have played the major role in this protection. DNA vaccinations appeared to be less effective in large animals than in mice. Immunizations of woodchucks with a plasmid expressing WHcAg (pWHcIm) induced only a low level of WHcAg-specific lymphoproliferative and antibody responses. Therefore, we investigated whether coapplication of the recently characterized woodchuck IFN-γ (21) can improve the efficacy of pWHcIm-based DNA vaccination. We demonstrated that gene gun immunization using WHcAg in combination with woodchuck IFN-γ is sufficient to induce a lymphoproliferative immune response and to suppress viral replication after challenge with WHV.
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immunization of woodchucks with plasmids expressing woodchuck hepatitis virus whv core antigen and surface antigen suppresses whv Infection
Journal of Virology, 1999Co-Authors: Mengji Lu, Gero Hilken, Johannes Kruppenbacher, Thekla Kemper, Reinhold Schirmbeck, Joerg Reimann, Michael RoggendorfAbstract:Hepatitis B virus (HBV) causes acute self-limiting and chronic Infection in humans (24). A chronic HBV Infection leads to a high risk for the development of liver cirrhosis and hepatocellular carcinoma (30, 54). The current strategy for preventing HBV Infection is vaccination with hepatitis B surface antigen (HBsAg), which induces virus-neutralizing anti-HBsAg antibodies (28). Though HBsAg is a potent immunogen and induces protective immunity in the majority of vaccines, 5 to 10% of persons who receive the HBsAg vaccine failed to develop anti-HBsAg antibodies. In addition, HBV variants carrying mutations within the HBsAg can escape the neutralization of vaccine-induced anti-HBsAg and establish acute or chronic Infection (3, 4, 6, 25, 29, 43). Therefore, a new vaccine strategy would be desirable to induce a multiple immune response consisting of HBV-specific T helper (Th), cytotoxic T cells (CTLs), and anti-HBsAg antibodies. HBV-specific Th and CTL responses play a pivotal role for the clearance of virus in a primary HBV Infection and may control HBV persisting in unknown reservoirs in patients whose disease is resolved (1, 7, 16, 18, 26, 27, 35, 41, 42, 44, 45). The induction of HBV-specific humoral and cellular immune response by a single vaccine may overcome the nonresponsiveness of individuals to conventional HBsAg vaccines and control immune escape variants of HBV with mutations within HBsAg. DNA vaccination is a powerful method to induce antigen-specific humoral and cellular immune response (14, 56). DNA-induced immune response provides protective immunity to various viruses in animal models (2, 5, 13, 19, 22, 31, 34, 36, 51, 55, 57). Genetic vaccination to HBsAg, HBV core antigen (HBcAg), and HBV e antigen (HBeAg) was evaluated in different animal models. In mice, a single intramuscular injection of plasmids expressing HBsAg is sufficient to induce a long-lasting humoral response to HBsAg and CTL response (10, 12, 40, 50). A plasmid vaccination of chimpanzees led to the production of low anti-HBsAg antibody titers (11, 47). Recently, Triyatni et al. reported that vaccination of ducks with plasmid expressing duck hepatitis B virus (DHBV) surface antigens (DHBsAg) induced antibodies to DHBsAg (55). Anti-DHBsAg antibodies induced by DNA vaccination were able to neutralize virus in vitro. DHBV was removed more rapidly from the bloodstreams of vaccinated ducks after a challenge. Infection of hepatocytes by DHBV was limited or prevented in vaccinated ducks. Therefore, the genetic vaccination was effective to prime an anti-HBsAg antibody response in this model. The vaccination of mice with HBcAg or HBeAg was also effective for inducing specific CTL responses (33). The woodchuck (Marmota monax) model is useful to study immune response to Hepadnavirus and to perform vaccination trials (8, 9, 23, 39, 48, 49, 52). Woodchuck hepatitis virus (WHV) causes acute self-limiting and chronic Infection, like HBV in humans (53). The humoral immune responses to woodchuck hepatitis surface antigen (WHsAg) and core antigen (WHcAg) in acute and chronic WHV Infection have the same features as those of HBV Infection. Anti-WHcAg develops in woodchucks during the early phase of a primary WHV Infection and persists lifelong. Anti-WHsAgs, like anti-HBsAgs, increase at the end of the viremic phase and may provide immunity to a secondary WHV Infection. Recently, T-cell response to WHsAg and WHcAg in woodchucks during acute and chronic WHV Infection was investigated by an in vitro assay to measure the antigen-specific proliferation of peripheral blood mononuclear cells (PBMCs) (8, 32, 38, 39). Multispecific Th response to WHcAg and WHsAg was present during acute WHV Infection but absent in woodchucks with chronic WHV Infection (39). Thus, the Th response to WHV in woodchucks closely resembles the HBV-specific Th response in humans (52). The woodchuck model is informative in the study of immune response induced by vaccines and virus challenge. Immunization of woodchucks with WHsAg-induced anti-WHsAg antibodies provided protection against a subsequent challenge with WHV (9). Interestingly, woodchucks immunized with WHcAg were protected against WHV challenge even though anti-WHcAg antibodies do not possess the ability to neutralize WHV (49, 52). Apparently, WHcAg induced a specific T-cell response which conferred protective immunity (39). We demonstrated that immunization with a peptide containing a T-cell epitope derived from WHcAg leads to the protection of woodchucks against WHV Infection. These results emphasize the significance of T-cell response to the core antigen for control of Hepadnavirus Infection (16, 18). In the present study, we wanted to determine whether vaccination of woodchucks with plasmids expressing WHV proteins can induce a protective immune response to WHV. We vaccinated mice and woodchucks with plasmids expressing WHcAg and WHsAg and investigated the humoral and cellular immune response to WHcAg and WHsAg in woodchucks. The protective efficacy of plasmid vaccination was demonstrated in woodchucks in subsequent challenge experiments.
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t cell response to woodchuck hepatitis virus whv antigens during acute self limited whv Infection and convalescence and after viral challenge
Journal of Virology, 1998Co-Authors: Stephan Menne, Jan Maschke, H Grossewilde, Michael RoggendorfAbstract:The Infection of woodchucks with woodchuck hepatitis virus (WHV) provides an experimental model to study early immune responses during Hepadnavirus Infection that cannot be tested in patients. The T-cell response of experimentally WHV-infected woodchucks to WHsAg, rWHcAg, and WHcAg peptides was monitored by observing 5-bromo-2′-deoxyuridine and [2-3H]adenine incorporation. The first T-cell responses were directed against WHsAg 3 weeks after Infection; these were followed by responses to rWHcAg including the immunodominant T-cell epitope of WHcAg (amino acids 97 to 110). Maximal proliferative responses were detected when the animals seroconvered to anti-WHs and anti-WHc (week 6). A decrease in the T-cell response to viral antigens coincided with clearance of viral DNA. Polyclonal rWHcAg-specific T-cell lines were established 6, 12, 18, and 24 weeks postInfection, and their responses to WHcAg peptides were assessed. Five to seven peptides including the immunodominant epitope were recognized throughout the observation period (6 months). At 12 months after Infection, T-cell responses to antigens and peptides were not detected. Reactivation of T-cell responses to viral antigens and peptides occurred within 7 days after challenge of animals with WHV. These results demonstrate that a fast and vigorous T-cell response to WHsAg, rWHcAg, and amino acids 97 to 110 of the WHcAg occurs within 3 weeks after WHV Infection. The peak of this response was associated with viral clearance and may be crucial for recovery from Infection. One year after Infection, no proliferation of T cells in response to antigens was observed; however, the WHV-specific T-cell response was reactivated after challenge of woodchucks with WHV and may be responsible for protection against WHV reInfection.
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the early host innate immune response to duck hepatitis b virus Infection
Journal of General Virology, 2010Co-Authors: Rahma Tohidiesfahani, Karen Vickery, Yvonne CossartAbstract:The early phase after hepatitis B virus Infection could play a crucial role in clearance and/or persistence of the virus, particularly in neonates. This work compared the early phase of duck hepatitis B virus Infection in 1-day-old (D1) and 28-day-old (D28) ducks to determine whether differences in viral or host innate immune response can be related to the difference in outcome. In the first phase, almost immediately after inoculation, virus was taken up by components of the reticulo-endothelial systems, particularly liver-specific macrophages, Kupffer cells. Very early after Infection, the induction of alpha interferon by infected hepatocytes occurred and was rapidly reinforced by recruitment of effector lymphocytes, which directly or indirectly caused apoptosis, eliminating infected hepatocytes, as was seen in mature birds. In addition, a lack of lymphocytic infiltration of the liver was found in D1 ducks, which supports the suggestion that the innate immune network is less effective in D1 ducks. Taken together, these results suggest that failure of the co-ordinated innate immune response rather than a defect in induced antiviral cell-mediated immunity may be the key factor which makes baby ducks vulnerable to persistence of Hepadnavirus Infection.
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cellular immune response of ducks to duck hepatitis b virus Infection
Journal of Medical Virology, 1999Co-Authors: Karen Vickery, Yvonne Cossart, R M W DixonAbstract:Duck hepatitis B virus (DHBV) has been a useful model for Hepadnavirus Infection. There have been few studies on immunity to DHBV and none describing the cell-mediated immune response by acute and chronically infected ducks. A duck hepatitis B antigen-specific blastogenesis assay was used to measure DHBV antigen-specific responses of duck peripheral blood (PBMC) and splenic mononuclear cells (SMCs) from uninfected control ducks, ducks acutely or chronically infected with DHBV, and ducks immune to DHBV. A comparison of the group mean responses by PBMC to DHBV surface antigen (DHBsAg) found that the immune group was significantly different to the other three groups (controls or unexposed, P < 0.0001; acutely infected, P< 0.01; chronically infected, P < 0.01). The responses to DHBsAg by PBMC of the acute group (P< 0.01) were significantly different also to that of the unexposed group. For DHBV core antigen (DHBcAg), significant differences in the responses were found between immune ducks and unexposed (P < 0.0005) and acutely infected (P < 0.05) groups. The SMC showed a significant difference between unexposed ducks and immune ducks (P< 0.05) in the group mean responses to DHBsAg. The responses to DHBcAg were significantly different between the immune group and the acute (P < 0.01) and unexposed (P < 0.01) groups. The group mean of unexposed ducks was also significantly different to that of acutely infected ducks (P < 0.01). This study indicates that the cellular immune response in immune animals differs from acutely and chronically infected ducks. Further studies of these differences may provide some explanations for the differing outcomes of DHBV Infection.