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Neutralizing antibodies against HCV: liver transplant as an experimental model
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)] HEPATITIS B VIRUS HAS A RECENT NEW WORLD EVOLUTIONARY ORlGtN PL Bollvkv. A Rambaut, N Grasslev, WF Carman and EC Holmes. Wettcome Trust Centre for the Epidemiology of Infectious Disease, University of, Oxford. Institute of Virology, University of Glasgow, UK. The rate of evolution of HBV was determined using phytogenetic methods and care gene sequences from HBeAg positive chronically infected patients and then used to estimate times of divergence of the different mammalian hepadnaviruses and the six known HBV genotypes. Novel statistical methods, reported here, were used to establish 95% confidence intervals. The lowest possible rate of substitution corresponded to an origin of HBV in humans no more than 1000 years ago and a split between the New World and Old World HBV genotypes of no more than 400 years ago. This is in conflict with the widely held view. These results, together with a phylogenetic analysis of 92 core gene sequences from all known hepadnaviruses, are most consistent with cross-species transfer of HBV from rodents to humans in the Americas and a subsequent spread of HBV eastward into the Old World around the time of the European conquest. This is further supported by analysis of hepatitis 0 virus, whose origins cannot predate that of HBV. Gibbon and chimp HBV sequences cluster within the human genotype, suggesting these are cross species transfer events from humans rather than co-speciation. Although the European conquest of the Americas ted to transfer of infectious diseases (influenza, yellow fever, measles and smallpox), with the possible exception of syphilis, HBV is the first case of a New World infection having been transported to the Old World. The relatively recent introduction of HBV into Africa and Asia suggests that the virus enjoyed massive rates of population growth and transmission primarily through horizontal routes.
HUMAN CYTOTOXIC T CELL RECOGNITION OF HEPATITIS C VIRUS (HCV) CORE PROTEIN. M. Jackson, M.F. Bassendine. G.L. Toms, A.G. Diamond. The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. Several groups have identified a number of cytotoxic T lymphocyte (CTL) epitopes derived from HCV core protein by in vitro stimulation with synthetic peptides. Peptide stimulated CTL have been shown to lyse target cells pulsed with peptide and such cells have been generated from both HCV seronegative and seropositive donors. In our studies, we have been interested in studying CTL generated against peptides produced naturally, following intracellular processing of viral protein. Accordingly, we have generated CTL lines from HCV seronegative donors by culturing CD8+ T cells with autologous dendritic cells loaded with recombinant HCV core protein. Analysis of the epitopes recognised by core protein specific CTL has been carried out using synthetic peptides which were selected based on their predicted binding to HLA-A*020 1. Lysis by core protein specific CTL has been demonstrated against five of the predicted epitopes, two of which are previously undefined. The pattern of responses of CTL from HCV seronegative donors is currently being compared with that of HCV infected donors.
1 P/CO5/012 TWO MOTIFS INVOLVED IN CONTROLLING TRANSCRIPTION OF THE HBV S PROMOTER C. Trautwein. CT. Bock. S. Kubicka and M.P. Manns Gastroenterology and Hepatology, Medical School Hannover. FRG. The hepatitis B virus (HBV) S promoter controls S gene transcription. Several mutations were found in this region in a patient who showed cellular retention of viral proteins. Here the transcriptional control of the S promoter of this patient was studied. The mutation consisted of a point mutation located in the CCAAT box (l), a 6 bp deletion (2) located immediately 5’ of the CCAAT box and a second 153 bp deletion (3) located in the preS2 genome. All mutations were cloned either alone or in combination in front of a luciferase ((UC) reporter gene. Transfection experiments in Huh7 cells showed that mutation 1 and 2 alone lead to a reduction in Iuc activity to 25 to 30 % compared to the wl promoter. Therefore both mutations were further investigated. In EMSA experiments, binding of a nuclear protein was impaired due to mutation 1. UV crosslinking and South-Western experiments suggested an approximately 38-40 kd protein for this factor. EMSA experiments wilh overlapping 3 bp deletions showed that the intact CCAAT motif is mandatory for the DNA-binding of this factor. Comouter assisted analvsis. comoetition and suoershift exoeriments with Antibodies directed against pdtential candidates showed ‘that CBFB binds to the CCAAT box. Cotransfection experiments showed that dominant negative CBF suppressed the wt. but not the mut (1) promoter activity and thus also demonstrated on a functional level that CBF-B is essential to control S promoter activity. To investigate the impact of mut (2) on S promoter activity gel shift exoeriments were oerformed. These exoeriments showed that no specilic factor binds’ to this motif and DNA-binding of CBF-B was not altered. Therefore missense mutations, partial deletions and doubling of the deleted motif were introduced in different promoter constructs. These constructs showed that the wi sequence is necessary for full wi promoter activity despite lack of binding of a specific factor. Next cotransfection experiments were performed with mut (2) and dom-neg CBF. Interestingly dom-neg CBF did not suppress Iuc activity indicating that transcription through CBF-B is already impaired in mut (2). CBF-B binds to the CCAAT motif in the HBV S oromoter. Additionallv a second motif shows wi DNA-binding of CBF-&‘but seems involved’ in controlling the interaction of CBF-B with the basal machinery.
1
NEUTRALIZING ANTIBODIES AGAINST HCV: LIVER TRANSPLANT AS AN FXPERIMENTAL MODEL. E.Villa . P.But.tafoco.A.Grottola, A.Scarcelli.F.Giannii.F.Mane&i.
Division of Gastroenterology, University of Modena, Italy Hepatitis C virus infection (HCV) is a common event in patients undergoing Ortbotopic Liver Transplantation (OLT). We have previously reported that some pre-OLT antiHCV-negative patients became HCV RNA positive after OLT, they did not develop antibodies against HCV but were able to clear HCV infection, becoming persistently HCV RNA negative (1). In the attempt to understand how it is possible to clear the virus despite heavy immunosuppression, we investigated using the Phage Display Technology (PDT) (2) whether it was possible to evidence antibodies (which could therefore be considered as neutralizing) other than those evidenced with routine tests. Methods: In the attempt to discover unknown antibody reactivities, pre- and post-transplant sera of our patients have been assayed with characterized and mapped HCV-specific phagotopes. On the whole we have tested 45 pre- and postOLT sera of 6 patients, 5 of whom cleared HCV infection (4 pre-OLT antiHCV-negative and 1 pre-OLT antiHCV-positive) while 1 pm-OLT antiHCV-negative patient became persistently HCV infected. Results: We have preliminarly shown that before OLT there are relevant HCV-specific serological reactivities, not detected by routine antibody assays. After OLT it has been possible to evidence multiple heterogeneous reactivities against core, envelope and NS4b regions. Particularly, we have identified a common reactivity against NS4b region in 5 pts who cleared HCV. Conclusion: 1. PDT shows higher sensitivity for antibody detection than routine methods; 2. Antibodies against NS4b region epitopes are associated with the occurrence of HCV clearance. l.Clin.Transpl. 1995;9:160-164;2. J.Immunol. 1996;156: 4504-4513.
)] HEPATITIS B VIRUS HAS A RECENT NEW WORLD EVOLUTIONARY ORlGtN PL Bollvkv. A Rambaut, N Grasslev, WF Carman and EC Holmes. Wettcome Trust Centre for the Epidemiology of Infectious Disease, University of, Oxford. Institute of Virology, University of Glasgow, UK. The rate of evolution of HBV was determined using phytogenetic methods and care gene sequences from HBeAg positive chronically infected patients and then used to estimate times of divergence of the different mammalian hepadnaviruses and the six known HBV genotypes. Novel statistical methods, reported here, were used to establish 95% confidence intervals. The lowest possible rate of substitution corresponded to an origin of HBV in humans no more than 1000 years ago and a split between the New World and Old World HBV genotypes of no more than 400 years ago. This is in conflict with the widely held view. These results, together with a phylogenetic analysis of 92 core gene sequences from all known hepadnaviruses, are most consistent with cross-species transfer of HBV from rodents to humans in the Americas and a subsequent spread of HBV eastward into the Old World around the time of the European conquest. This is further supported by analysis of hepatitis 0 virus, whose origins cannot predate that of HBV. Gibbon and chimp HBV sequences cluster within the human genotype, suggesting these are cross species transfer events from humans rather than co-speciation. Although the European conquest of the Americas ted to transfer of infectious diseases (influenza, yellow fever, measles and smallpox), with the possible exception of syphilis, HBV is the first case of a New World infection having been transported to the Old World. The relatively recent introduction of HBV into Africa and Asia suggests that the virus enjoyed massive rates of population growth and transmission primarily through horizontal routes.
HUMAN CYTOTOXIC T CELL RECOGNITION OF HEPATITIS C VIRUS (HCV) CORE PROTEIN. M. Jackson, M.F. Bassendine. G.L. Toms, A.G. Diamond. The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. Several groups have identified a number of cytotoxic T lymphocyte (CTL) epitopes derived from HCV core protein by in vitro stimulation with synthetic peptides. Peptide stimulated CTL have been shown to lyse target cells pulsed with peptide and such cells have been generated from both HCV seronegative and seropositive donors. In our studies, we have been interested in studying CTL generated against peptides produced naturally, following intracellular processing of viral protein. Accordingly, we have generated CTL lines from HCV seronegative donors by culturing CD8+ T cells with autologous dendritic cells loaded with recombinant HCV core protein. Analysis of the epitopes recognised by core protein specific CTL has been carried out using synthetic peptides which were selected based on their predicted binding to HLA-A*020 1. Lysis by core protein specific CTL has been demonstrated against five of the predicted epitopes, two of which are previously undefined. The pattern of responses of CTL from HCV seronegative donors is currently being compared with that of HCV infected donors.
1 P/CO5/012 TWO MOTIFS INVOLVED IN CONTROLLING TRANSCRIPTION OF THE HBV S PROMOTER C. Trautwein. CT. Bock. S. Kubicka and M.P. Manns Gastroenterology and Hepatology, Medical School Hannover. FRG. The hepatitis B virus (HBV) S promoter controls S gene transcription. Several mutations were found in this region in a patient who showed cellular retention of viral proteins. Here the transcriptional control of the S promoter of this patient was studied. The mutation consisted of a point mutation located in the CCAAT box (l), a 6 bp deletion (2) located immediately 5’ of the CCAAT box and a second 153 bp deletion (3) located in the preS2 genome. All mutations were cloned either alone or in combination in front of a luciferase ((UC) reporter gene. Transfection experiments in Huh7 cells showed that mutation 1 and 2 alone lead to a reduction in Iuc activity to 25 to 30 % compared to the wl promoter. Therefore both mutations were further investigated. In EMSA experiments, binding of a nuclear protein was impaired due to mutation 1. UV crosslinking and South-Western experiments suggested an approximately 38-40 kd protein for this factor. EMSA experiments wilh overlapping 3 bp deletions showed that the intact CCAAT motif is mandatory for the DNA-binding of this factor. Comouter assisted analvsis. comoetition and suoershift exoeriments with Antibodies directed against pdtential candidates showed ‘that CBFB binds to the CCAAT box. Cotransfection experiments showed that dominant negative CBF suppressed the wt. but not the mut (1) promoter activity and thus also demonstrated on a functional level that CBF-B is essential to control S promoter activity. To investigate the impact of mut (2) on S promoter activity gel shift exoeriments were oerformed. These exoeriments showed that no specilic factor binds’ to this motif and DNA-binding of CBF-B was not altered. Therefore missense mutations, partial deletions and doubling of the deleted motif were introduced in different promoter constructs. These constructs showed that the wi sequence is necessary for full wi promoter activity despite lack of binding of a specific factor. Next cotransfection experiments were performed with mut (2) and dom-neg CBF. Interestingly dom-neg CBF did not suppress Iuc activity indicating that transcription through CBF-B is already impaired in mut (2). CBF-B binds to the CCAAT motif in the HBV S oromoter. Additionallv a second motif shows wi DNA-binding of CBF-&‘but seems involved’ in controlling the interaction of CBF-B with the basal machinery.
1
NEUTRALIZING ANTIBODIES AGAINST HCV: LIVER TRANSPLANT AS AN FXPERIMENTAL MODEL. E.Villa . P.But.tafoco.A.Grottola, A.Scarcelli.F.Giannii.F.Mane&i.
Division of Gastroenterology, University of Modena, Italy Hepatitis C virus infection (HCV) is a common event in patients undergoing Ortbotopic Liver Transplantation (OLT). We have previously reported that some pre-OLT antiHCV-negative patients became HCV RNA positive after OLT, they did not develop antibodies against HCV but were able to clear HCV infection, becoming persistently HCV RNA negative (1). In the attempt to understand how it is possible to clear the virus despite heavy immunosuppression, we investigated using the Phage Display Technology (PDT) (2) whether it was possible to evidence antibodies (which could therefore be considered as neutralizing) other than those evidenced with routine tests. Methods: In the attempt to discover unknown antibody reactivities, pre- and post-transplant sera of our patients have been assayed with characterized and mapped HCV-specific phagotopes. On the whole we have tested 45 pre- and postOLT sera of 6 patients, 5 of whom cleared HCV infection (4 pre-OLT antiHCV-negative and 1 pre-OLT antiHCV-positive) while 1 pm-OLT antiHCV-negative patient became persistently HCV infected. Results: We have preliminarly shown that before OLT there are relevant HCV-specific serological reactivities, not detected by routine antibody assays. After OLT it has been possible to evidence multiple heterogeneous reactivities against core, envelope and NS4b regions. Particularly, we have identified a common reactivity against NS4b region in 5 pts who cleared HCV. Conclusion: 1. PDT shows higher sensitivity for antibody detection than routine methods; 2. Antibodies against NS4b region epitopes are associated with the occurrence of HCV clearance. l.Clin.Transpl. 1995;9:160-164;2. J.Immunol. 1996;156: 4504-4513.