- Email: [email protected]
cellular models
$6
Journal of Clinical Virology 2006, Vol 36 (suppl 2)
SOl, but not POI, was associated with protective immunity against hepadnavirus and that WHV traces carried in both POI and SOl were infectious and could be transmitted to virus-naive hosts. Conclusion: POI and SOl have clearly distinctive molecular and serological characteristics allowing for their confident identification in experimental hepadnaviral infection.
I-0---.~ Therapeutic vaccination in chronic hepatitis B: pre-clinical studies in the woodchuck M. Roggendorf*, M. Lu. Institute of Virology, University of Duisburg-Essen, Germany Interferon ,Y, and nucleoside analogues are available for treatment of chronic hepatitis B virus (HBV) infection but do not lead to a satisfactory result. New findings about the immunological control of HBV during acute infection suggest the pivotal role of T-cell mediated immune responses. Several preclinical and clinical trials were undertaken to explore the possibility to stimulate specific immune responses in chronically infected animals and patients by vaccination. Vaccinations with commercially available H BV vaccines in patients and immunisation of woodchuck with core protein or surface protein of WHV did not result in effective control of HBV WHV infection, suggesting that new formulations of therapeutic vaccines are needed. Some new approaches combining antiviral treatments with nucleosidanalogs, DNA vaccine, and protein vaccines were tested in the woodchuck model. It could be shown that therapeutic vaccinations are able to stimulate specific B- and T-cell responses and to achieve transient suppression of viral replication. These results suggest the great potential of therapeutic vaccination in combination with antivirals to reach an effective and sustained control of HBV infection.
I-0---.~ Expansion of HBV S gene heterogeneity in chimpanzees after experimental inoculation with HBV sT126N mutant
Abstracts, 12th ISHVLD sequences from CH6413 indicated that the heterogeneity of the sT126N population increased from an average of 15% to 41% during this infection. Conclusion: Heterogeneity in the S gene was observed after homogeneous cloned HBV DNA containing sT126N was inoculated into CH256 and the serum of CH256 inoculated to CH6413. The sT126N mutation, however, remained invariant during both infection experiments. This is the first observation of substantial heterogeneity of the HBV genome generated after induction of infection with HBV sT126N mutant in the absence of a wild type virus.
[-0--.-.-.-.-.-.-~HBV/HCV: Animal/cellular models J. Petersen *. Department of Medicine, University Hospital
Hamburg-Eppendorf, Germany The lack of reliable in vitro infection systems and convenient animal models has hindered progress in HBV and HCV research and the development of new treatment options. Due to various restraints encountered using chimpanzees and other models of HBV infection that are based on HBV-related viruses, and due to the necessity to work with small and wellcharacterized animal systems, most recent developments focused on mice. Various strains of transgenic mice were developed and proved to be very useful to elucidate mechanisms of HBV replication and pathogenesis. Novel, exciting alternative mouse models such as the upa model have been recently developed, which allow not only studies on viral replication, but also to investigate mechanisms of viral entry and clearance of HBV and possibly HCV, although for hepatitis C virus, chimpanzees remain the only recognized animal model up to now. Studies performed in chimpanzees played a critical role in the discovery of HCV and are continuing to play an essential role in defining the natural history of this important human pathogen. Recently, a system of efficient culture was established that allows replication of subgenomic molecules of HCV RNA in a cell line of human hepatoma and generation of infectious particles.
A. Araujo, J. Yokosawa, J. Spelbring, V. Tzaneva, S. Kamili, K. Krawczynski, H. Fields, Y. Khudyakov'. Laboratory Branch
[-0--.-~ Determinants of infectivity in the antigenic loop of hepatitis B virus envelope proteins
Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, USA
G. Abou Jaoud~*, C. Sureau. Virology, INTS, Paris, France
Background and Objectives: Mutations within or in proximity to the "a" determinant region may cause antigenic modifications of the HBsAg protein, thereby preventing neutralization of the HBV by vaccine and diminishing detection by commercially available diagnostic assays. Consequently, HBV S gene mutants may pose a potential risk to public health. Despite a large body of research on the biological significance of HBV mutations available in the extant literature, very limited information is known regarding the features of mutant HBV infections in the absence of infection by wild type virus. Our objectives were to study the serological and molecular characteristics of infection caused by S-gene HBV mutant T126N (sT126N) in experimentally infected chimpanzees. Methods: Two nafve chimpanzees (CH256 and CH257) were intrahepatically inoculated with cloned HBVDNA containing the T126N mutation within the S gene (HBV sT126N). A heterogenous population of HBV sT126N obtained from viremic plasma from CH256 was intravenously inoculated into a third nafve chimpanzee (CH6413). Serial serum samples were tested for HBV DNA by PCR, for ALT activity, and for markers of infection. PCR fragments of 354 bp containing part of the S gene from different time points were cloned and at least 32 clones were sequenced to assess HBV genome heterogeneity. Collectively, 640 clones were sequenced from the infected animals. Results: Inoculation of monoclonal HBV sT126N mutant in CH256 resulted in acute infection that quickly resolved. Sequencing of PCR fragments from serum taken at 3 time points showed that the sT126N mutation was invariant; however, mutations at some other positions were observed. The majority of these mutations were nonsynonymous mutations both in the S and P genes. Two sequences were found that contained stop codons in the P gene. No evidence of HBV infection was observed in CH 257. Inoculation to CH6413 of serum from CH 256 obtained during the period of viremia resulted in a prolonged infection with persistent antigenemia. Analysis of
Background and Objectives: The main infectivity determinant on the hepatitis B virus (HBV) envelope proteins has been mapped to the N-terminal pre-S1 domain of the large envelope protein. It is assumed to constitute a primary receptor-binding site responsible for tissue and species specificity. Methods: Using the hepatitis delta virus (HDV) as a surrogate model to investigate the role of the HBV envelope proteins at viral entry, we recently demonstrated that a second determinant of infectivity resides in the antigenic loop (AGL) of HBV envelope proteins (Abou-Jaoud~ G., Sureau C., 2005, J. Virol. 79 10460-6). Results: In the study reported here, we further characterized the AGL-infectivity determinant by introducing single amino acid substitutions between residues 119 and 164, and by testing the mutant-HDV particles for infectivity in human hepatocyte cultures or the HepaRG cell line. We identified residues, such as Gl19, P120, R122, K141, P153 and W156 that are critical to in vitro infectivity. In addition, single amino acid substitutions of serine for cysteine residues at positions -121, -124, -137, -138, -139, -147 and -149 previously described as being engaged in disulfide-bounds, were permissive to HDV assembly but detrimental to infectivity. To investigate whether isomerization of the envelope protein disulphide-bonds was involved at viral entry, we conducted infection assays in the presence of alkylators such as NEM, DTNB or MTSET, or reducing agents such as DTT or TCER We observed a dosedependent effect of the treatment when the inhibitors (alkylators or reducing agent) were provided during exposure of the virus to the hepatocytes, but no inhibition was recorded when inhibitors were added to the cells post-inoculation. Conclusion: Overall, our data demonstrate that in addition to a putative receptor-binding site located in the pre-S1 domain of the large envelope protein, the AGL common to all three viral envelope proteins, is essential for infectivity. Whether its role at viral entry is associated to disassembly of the viral envelope after attachment, or
Journal of Clinical Virology 2006, Vol 36 (suppl 2)
SOl, but not POI, was associated with protective immunity against hepadnavirus and that WHV traces carried in both POI and SOl were infectious and could be transmitted to virus-naive hosts. Conclusion: POI and SOl have clearly distinctive molecular and serological characteristics allowing for their confident identification in experimental hepadnaviral infection.
I-0---.~ Therapeutic vaccination in chronic hepatitis B: pre-clinical studies in the woodchuck M. Roggendorf*, M. Lu. Institute of Virology, University of Duisburg-Essen, Germany Interferon ,Y, and nucleoside analogues are available for treatment of chronic hepatitis B virus (HBV) infection but do not lead to a satisfactory result. New findings about the immunological control of HBV during acute infection suggest the pivotal role of T-cell mediated immune responses. Several preclinical and clinical trials were undertaken to explore the possibility to stimulate specific immune responses in chronically infected animals and patients by vaccination. Vaccinations with commercially available H BV vaccines in patients and immunisation of woodchuck with core protein or surface protein of WHV did not result in effective control of HBV WHV infection, suggesting that new formulations of therapeutic vaccines are needed. Some new approaches combining antiviral treatments with nucleosidanalogs, DNA vaccine, and protein vaccines were tested in the woodchuck model. It could be shown that therapeutic vaccinations are able to stimulate specific B- and T-cell responses and to achieve transient suppression of viral replication. These results suggest the great potential of therapeutic vaccination in combination with antivirals to reach an effective and sustained control of HBV infection.
I-0---.~ Expansion of HBV S gene heterogeneity in chimpanzees after experimental inoculation with HBV sT126N mutant
Abstracts, 12th ISHVLD sequences from CH6413 indicated that the heterogeneity of the sT126N population increased from an average of 15% to 41% during this infection. Conclusion: Heterogeneity in the S gene was observed after homogeneous cloned HBV DNA containing sT126N was inoculated into CH256 and the serum of CH256 inoculated to CH6413. The sT126N mutation, however, remained invariant during both infection experiments. This is the first observation of substantial heterogeneity of the HBV genome generated after induction of infection with HBV sT126N mutant in the absence of a wild type virus.
[-0--.-.-.-.-.-.-~HBV/HCV: Animal/cellular models J. Petersen *. Department of Medicine, University Hospital
Hamburg-Eppendorf, Germany The lack of reliable in vitro infection systems and convenient animal models has hindered progress in HBV and HCV research and the development of new treatment options. Due to various restraints encountered using chimpanzees and other models of HBV infection that are based on HBV-related viruses, and due to the necessity to work with small and wellcharacterized animal systems, most recent developments focused on mice. Various strains of transgenic mice were developed and proved to be very useful to elucidate mechanisms of HBV replication and pathogenesis. Novel, exciting alternative mouse models such as the upa model have been recently developed, which allow not only studies on viral replication, but also to investigate mechanisms of viral entry and clearance of HBV and possibly HCV, although for hepatitis C virus, chimpanzees remain the only recognized animal model up to now. Studies performed in chimpanzees played a critical role in the discovery of HCV and are continuing to play an essential role in defining the natural history of this important human pathogen. Recently, a system of efficient culture was established that allows replication of subgenomic molecules of HCV RNA in a cell line of human hepatoma and generation of infectious particles.
A. Araujo, J. Yokosawa, J. Spelbring, V. Tzaneva, S. Kamili, K. Krawczynski, H. Fields, Y. Khudyakov'. Laboratory Branch
[-0--.-~ Determinants of infectivity in the antigenic loop of hepatitis B virus envelope proteins
Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, USA
G. Abou Jaoud~*, C. Sureau. Virology, INTS, Paris, France
Background and Objectives: Mutations within or in proximity to the "a" determinant region may cause antigenic modifications of the HBsAg protein, thereby preventing neutralization of the HBV by vaccine and diminishing detection by commercially available diagnostic assays. Consequently, HBV S gene mutants may pose a potential risk to public health. Despite a large body of research on the biological significance of HBV mutations available in the extant literature, very limited information is known regarding the features of mutant HBV infections in the absence of infection by wild type virus. Our objectives were to study the serological and molecular characteristics of infection caused by S-gene HBV mutant T126N (sT126N) in experimentally infected chimpanzees. Methods: Two nafve chimpanzees (CH256 and CH257) were intrahepatically inoculated with cloned HBVDNA containing the T126N mutation within the S gene (HBV sT126N). A heterogenous population of HBV sT126N obtained from viremic plasma from CH256 was intravenously inoculated into a third nafve chimpanzee (CH6413). Serial serum samples were tested for HBV DNA by PCR, for ALT activity, and for markers of infection. PCR fragments of 354 bp containing part of the S gene from different time points were cloned and at least 32 clones were sequenced to assess HBV genome heterogeneity. Collectively, 640 clones were sequenced from the infected animals. Results: Inoculation of monoclonal HBV sT126N mutant in CH256 resulted in acute infection that quickly resolved. Sequencing of PCR fragments from serum taken at 3 time points showed that the sT126N mutation was invariant; however, mutations at some other positions were observed. The majority of these mutations were nonsynonymous mutations both in the S and P genes. Two sequences were found that contained stop codons in the P gene. No evidence of HBV infection was observed in CH 257. Inoculation to CH6413 of serum from CH 256 obtained during the period of viremia resulted in a prolonged infection with persistent antigenemia. Analysis of
Background and Objectives: The main infectivity determinant on the hepatitis B virus (HBV) envelope proteins has been mapped to the N-terminal pre-S1 domain of the large envelope protein. It is assumed to constitute a primary receptor-binding site responsible for tissue and species specificity. Methods: Using the hepatitis delta virus (HDV) as a surrogate model to investigate the role of the HBV envelope proteins at viral entry, we recently demonstrated that a second determinant of infectivity resides in the antigenic loop (AGL) of HBV envelope proteins (Abou-Jaoud~ G., Sureau C., 2005, J. Virol. 79 10460-6). Results: In the study reported here, we further characterized the AGL-infectivity determinant by introducing single amino acid substitutions between residues 119 and 164, and by testing the mutant-HDV particles for infectivity in human hepatocyte cultures or the HepaRG cell line. We identified residues, such as Gl19, P120, R122, K141, P153 and W156 that are critical to in vitro infectivity. In addition, single amino acid substitutions of serine for cysteine residues at positions -121, -124, -137, -138, -139, -147 and -149 previously described as being engaged in disulfide-bounds, were permissive to HDV assembly but detrimental to infectivity. To investigate whether isomerization of the envelope protein disulphide-bonds was involved at viral entry, we conducted infection assays in the presence of alkylators such as NEM, DTNB or MTSET, or reducing agents such as DTT or TCER We observed a dosedependent effect of the treatment when the inhibitors (alkylators or reducing agent) were provided during exposure of the virus to the hepatocytes, but no inhibition was recorded when inhibitors were added to the cells post-inoculation. Conclusion: Overall, our data demonstrate that in addition to a putative receptor-binding site located in the pre-S1 domain of the large envelope protein, the AGL common to all three viral envelope proteins, is essential for infectivity. Whether its role at viral entry is associated to disassembly of the viral envelope after attachment, or