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P.200 Comprehensive analysis of IFN-γ and IL-2 production by T cells in acute and chronic HCV infection
Posters: 02c. Hepatitis C - pathogenesis and natural history
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Comprehensive analysis of IFN-y and IL-2 production by T cells in acute and chronic HCV infection R.A. Ffrench 1 *, J. Flynn 1, K. Goy 1, IR Hosseiny 2, Y. Pan 3, W. Rawlinson 3, IR White 4, O. Nguyen 5, M. Hellard 5, J. Kaldor 6, G. Dore 6, A. Lloyd 2 . 1Viral Immunology, Burnet Institute, Melbourne;
2Medical Sciences, UNSW, 3SEALS, POWH, 4BABS, UNSW, Sydney; 5CEPHR, Bumet Institute, Melbourne; 6NCHECR, UNSW, Sydney, Australia Background and Objectives: T-cell responses have been demonstrated to be critical in resolution of acute HCV infection. Memory T-cell responses persist in those that go on to clearance, while effector responses in chronic infection are usually characterized as weak and narrow in specificity. The objective of this study was to comprehensively assess T-cell responses in individuals enrolled in the Australian Trial in Acute HCV (ATAHC) cohort, compared to those with chronic infection, using overlapping peptide pools covering the entire genome. Methods: We have assessed the frequency, specificity and avidity of T-cell responses in individuals acutely infected with HCV but prior to treatment (n = 12), compared to those with chronic HCV infection (n = 32), using stimulation with pools of peptides, corresponding to each of the proteins of HCV and assayed for the production of IFN-y or IL-2 producing cells by ELISPOT. Results: Acutely infected individuals had a lower prevalence of both IFN-y and IL-2 responses than chronically infected individuals, with 7/12 (58%) responding in either assay, compared to 29/32 (90%) chronics. However the magnitude of the responses in the acute infection was greater, with a mean summed response to HCV peptides of 364 SFC/106 PBMC in the IFN-y assay and 387 SFC in IL-2. In chronic infection the mean response was 294 SFC in IFN-y and 215 SFC in IL-2. The breadth of the IFN response was similar, with 3-4 pools positive in both acute and chronic infection. In contrast IL-2 responses were broader in the acute cohort with a median of seven pools positive, compared to three in the chronics. There was significantly higher prevalence of responses to NS4b in acute infection (p=0.02). Discordance in the specificity of the responses in the two assays was evident, with 67% difference in the acute cohort and 86% in the chronic cohort, indicating the likelihood of different effector populations. An inverse correlation between the presence of HCV-specific cell mediated immunity and viral load in the acute cohort prior to treatment was demonstrated. There was a greater range in the functional avidity of the T-cell responses in acute infection compared to chronic infection, where the avidity was higher. Conclusion: Overall the responses in the acute cohort were present in fewer individuals, but of a greater magnitude than in chronic infection. The breadth of the IL-2 response in acute infection was greater, but lower in avidity, than the IFN-y responses. This study sheds light on the early immunopathogenesis of HCV infection that will be useful in vaccine development.
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Identification of a duplication of the V3 domain in the NS5A region in HCV genotype l b infected patients: prevalence and relation with clinical outcome in two cohorts
H. Le Guillou-Guillemette 1 *, O. Petsaris 2, C. Payan 2, P. Veillon 1, C. Gaudy 3, S. Alain 4, J.C. Trinchet 5, E Lunel 1. 1Laboratory of
Bacteriology-Virology, University Hospital of Angers, Angers cedex 9; 2Department of Microbiology EA3882, University Hospital of Brest, Brest; 3Department of Medical and Molecular Microbiology EA 3250, University of Tours, Tours; 4Department of Bacteriology-Virology, University Hospital Dupuytren, Limoges; 5Department of Hepato-Gastroenterology, Jean Verdier Hospital, Bondy, France Background and Objectives: The role of HCV NS5A in viral replication and resistance to interferon has been demonstrated. Early studies have reported insertions-deletions of small sequences, from 1 to 12 nucleotides long, in different regions of the viral genome. Here, we report a novel insertion in the NS5A V3 domain. To investigate the association between this genetic heterogeneity and a particular stage of the natural history of HCV infection, we
$123 determined the prevalence of this insertion in two groups of patients differing in their clinical status. Methods: NS5A V3 domain was amplified with a real-time or a conventional PCR. Direct sequencing was performed on PCR products. Group 1 included 318 consecutive HCV-lb infected patients [Chronic HCV infection = 287, cirrhosis = 25, hepatocellular carcinoma (HCC) = 6]. Group 2 included 72 patients from the French prospective cohort study on the incidence of HCC (ANRS CO 12 CIRVIR); 19 with a F1-F2 fibrosis stage according to the METAVIR classification, 25 with cirrhosis and 28 with HCC. Results: In the first group, we found an insertion in the V3 domain in 9 patients (2.8%). One of them presented a membranous nephropathy and a non-Hodgkin's lymphoma. Three lengths of inserts were observed in the 9 isolates: 7 presented a 31-amino acid insertion, one had a 27-amino acid insertion and one had a 12-amino acid insertion. The 31-amino acid insertions had a high homology (55-77%) with the V3 domain and might be a duplication of the V3 domain. In the second group studied, we found 7 insertions: 4 insertions in the V3 domain in the 28 patients with HCC (14.3%), 2 insertions in the 25 patients with cirrhosis (8%) and 1 insertion in the 19 F1-F2 fibrosis patients (5.3%). In all isolates, these insertions did not induce disruption of the open reading frame in the NS5A region. Conclusion: We identified an insertion in the V3 domain of the HCV NS5A region, which has not been previously reported. This insertion was observed with a higher frequency in patients with severe liver damage, either cirrhosis or HCC. Further studies are needed to assess whether a correlation exists between this genetic polymorphism in the V3 domain and the progression of the liver disease.
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hepatitis C virus NS3 protease quasispecies heterogeneity predictive of progression from cirrhosis to hepatocellular carcinoma?
S. Vallet 1 *, S. Gouriou 2, G. Nkontchou 3, H. Hotta 4, M. LegrandQuillien 1, M. Beaugrand 3, J. Trinchet 3, J. Nousbaum 5, P. Deny 6, A. Goudeau 7, B. Picard 6, C. Payan 1. 1Microbiologie, CHU
Morvan; 2Bacteriologie-Virologie, Facult6 de M6decine, Brest; 3Hepato-gastroenterologie, CH J. Verdier, Bondy, France, 4Microbiology, University Graduate School of Medicine, Kobe, Japan, 5Hepato-gastroenterologie, CHU La Cavale Blanche, Brest; 6Bacteriologie-Virologie-Hygiene, CH Avicenne, Bobigny; 7Virologie, CHU Bretonneau, Tours, France Background and Objectives: The NS3 protease is essential for hepatitis C virus (HCV) replication, and is therefore one of the most promising targets for specific anti-HCV therapy. Various lines of evidence suggest that the HCV NS3 protease is involved in carcinogenesis. In a previous work, a particular secondary conformation of the amino-terminal 120 residues of the protease was found to be significantly associated with HCC. We aimed to determine whether a particular nucleotide and amino-acid signature pattern of the HCV-lb NS3 protease was predictive of progression from viral cirrhosis to HCC. Methods: The NS3 protease quasispecies structure of 10 HCV-1 b cirrhotic patients (controls) was compared with that of 10 paired HCV-lb cirrhotic patients who went on to display progression to HCC (cases). The secondary structure of the amino-terminal 120 residues of NS3 protease was predicted by computer-assisted Robson analysis with GENETIX-MAC software. Results: NS3 protease genetic complexity and diversity did not differ significantly between cases and controls. Amino-acid substitutions were detected at 20 (11%) and 25 (14%) sites in at least two variants of the NS3 protease in cases and controls, respectively. Significant differences in the percentage of substituted clones were observed for 10 NS3 sites. Mutations Y56F, 171V, T721, Q86P, P89S, S101G/D, R117H, S122G/T/N, V1321 and V1701 were more frequently observed in the NS3 protease sequences of controls than in those of cases. However, these differences did not allow the definition of a specific NS3 profile related to HCC occurrence. The NS3 secondary structure B1-1 previously identified as potentially predictive of HCC was identified with a higher frequency in cases quasispecies (84.2%) than in controls ones (55.9%; p <0.05).
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Comprehensive analysis of IFN-y and IL-2 production by T cells in acute and chronic HCV infection R.A. Ffrench 1 *, J. Flynn 1, K. Goy 1, IR Hosseiny 2, Y. Pan 3, W. Rawlinson 3, IR White 4, O. Nguyen 5, M. Hellard 5, J. Kaldor 6, G. Dore 6, A. Lloyd 2 . 1Viral Immunology, Burnet Institute, Melbourne;
2Medical Sciences, UNSW, 3SEALS, POWH, 4BABS, UNSW, Sydney; 5CEPHR, Bumet Institute, Melbourne; 6NCHECR, UNSW, Sydney, Australia Background and Objectives: T-cell responses have been demonstrated to be critical in resolution of acute HCV infection. Memory T-cell responses persist in those that go on to clearance, while effector responses in chronic infection are usually characterized as weak and narrow in specificity. The objective of this study was to comprehensively assess T-cell responses in individuals enrolled in the Australian Trial in Acute HCV (ATAHC) cohort, compared to those with chronic infection, using overlapping peptide pools covering the entire genome. Methods: We have assessed the frequency, specificity and avidity of T-cell responses in individuals acutely infected with HCV but prior to treatment (n = 12), compared to those with chronic HCV infection (n = 32), using stimulation with pools of peptides, corresponding to each of the proteins of HCV and assayed for the production of IFN-y or IL-2 producing cells by ELISPOT. Results: Acutely infected individuals had a lower prevalence of both IFN-y and IL-2 responses than chronically infected individuals, with 7/12 (58%) responding in either assay, compared to 29/32 (90%) chronics. However the magnitude of the responses in the acute infection was greater, with a mean summed response to HCV peptides of 364 SFC/106 PBMC in the IFN-y assay and 387 SFC in IL-2. In chronic infection the mean response was 294 SFC in IFN-y and 215 SFC in IL-2. The breadth of the IFN response was similar, with 3-4 pools positive in both acute and chronic infection. In contrast IL-2 responses were broader in the acute cohort with a median of seven pools positive, compared to three in the chronics. There was significantly higher prevalence of responses to NS4b in acute infection (p=0.02). Discordance in the specificity of the responses in the two assays was evident, with 67% difference in the acute cohort and 86% in the chronic cohort, indicating the likelihood of different effector populations. An inverse correlation between the presence of HCV-specific cell mediated immunity and viral load in the acute cohort prior to treatment was demonstrated. There was a greater range in the functional avidity of the T-cell responses in acute infection compared to chronic infection, where the avidity was higher. Conclusion: Overall the responses in the acute cohort were present in fewer individuals, but of a greater magnitude than in chronic infection. The breadth of the IL-2 response in acute infection was greater, but lower in avidity, than the IFN-y responses. This study sheds light on the early immunopathogenesis of HCV infection that will be useful in vaccine development.
• I-~-]
Identification of a duplication of the V3 domain in the NS5A region in HCV genotype l b infected patients: prevalence and relation with clinical outcome in two cohorts
H. Le Guillou-Guillemette 1 *, O. Petsaris 2, C. Payan 2, P. Veillon 1, C. Gaudy 3, S. Alain 4, J.C. Trinchet 5, E Lunel 1. 1Laboratory of
Bacteriology-Virology, University Hospital of Angers, Angers cedex 9; 2Department of Microbiology EA3882, University Hospital of Brest, Brest; 3Department of Medical and Molecular Microbiology EA 3250, University of Tours, Tours; 4Department of Bacteriology-Virology, University Hospital Dupuytren, Limoges; 5Department of Hepato-Gastroenterology, Jean Verdier Hospital, Bondy, France Background and Objectives: The role of HCV NS5A in viral replication and resistance to interferon has been demonstrated. Early studies have reported insertions-deletions of small sequences, from 1 to 12 nucleotides long, in different regions of the viral genome. Here, we report a novel insertion in the NS5A V3 domain. To investigate the association between this genetic heterogeneity and a particular stage of the natural history of HCV infection, we
$123 determined the prevalence of this insertion in two groups of patients differing in their clinical status. Methods: NS5A V3 domain was amplified with a real-time or a conventional PCR. Direct sequencing was performed on PCR products. Group 1 included 318 consecutive HCV-lb infected patients [Chronic HCV infection = 287, cirrhosis = 25, hepatocellular carcinoma (HCC) = 6]. Group 2 included 72 patients from the French prospective cohort study on the incidence of HCC (ANRS CO 12 CIRVIR); 19 with a F1-F2 fibrosis stage according to the METAVIR classification, 25 with cirrhosis and 28 with HCC. Results: In the first group, we found an insertion in the V3 domain in 9 patients (2.8%). One of them presented a membranous nephropathy and a non-Hodgkin's lymphoma. Three lengths of inserts were observed in the 9 isolates: 7 presented a 31-amino acid insertion, one had a 27-amino acid insertion and one had a 12-amino acid insertion. The 31-amino acid insertions had a high homology (55-77%) with the V3 domain and might be a duplication of the V3 domain. In the second group studied, we found 7 insertions: 4 insertions in the V3 domain in the 28 patients with HCC (14.3%), 2 insertions in the 25 patients with cirrhosis (8%) and 1 insertion in the 19 F1-F2 fibrosis patients (5.3%). In all isolates, these insertions did not induce disruption of the open reading frame in the NS5A region. Conclusion: We identified an insertion in the V3 domain of the HCV NS5A region, which has not been previously reported. This insertion was observed with a higher frequency in patients with severe liver damage, either cirrhosis or HCC. Further studies are needed to assess whether a correlation exists between this genetic polymorphism in the V3 domain and the progression of the liver disease.
~ l s
hepatitis C virus NS3 protease quasispecies heterogeneity predictive of progression from cirrhosis to hepatocellular carcinoma?
S. Vallet 1 *, S. Gouriou 2, G. Nkontchou 3, H. Hotta 4, M. LegrandQuillien 1, M. Beaugrand 3, J. Trinchet 3, J. Nousbaum 5, P. Deny 6, A. Goudeau 7, B. Picard 6, C. Payan 1. 1Microbiologie, CHU
Morvan; 2Bacteriologie-Virologie, Facult6 de M6decine, Brest; 3Hepato-gastroenterologie, CH J. Verdier, Bondy, France, 4Microbiology, University Graduate School of Medicine, Kobe, Japan, 5Hepato-gastroenterologie, CHU La Cavale Blanche, Brest; 6Bacteriologie-Virologie-Hygiene, CH Avicenne, Bobigny; 7Virologie, CHU Bretonneau, Tours, France Background and Objectives: The NS3 protease is essential for hepatitis C virus (HCV) replication, and is therefore one of the most promising targets for specific anti-HCV therapy. Various lines of evidence suggest that the HCV NS3 protease is involved in carcinogenesis. In a previous work, a particular secondary conformation of the amino-terminal 120 residues of the protease was found to be significantly associated with HCC. We aimed to determine whether a particular nucleotide and amino-acid signature pattern of the HCV-lb NS3 protease was predictive of progression from viral cirrhosis to HCC. Methods: The NS3 protease quasispecies structure of 10 HCV-1 b cirrhotic patients (controls) was compared with that of 10 paired HCV-lb cirrhotic patients who went on to display progression to HCC (cases). The secondary structure of the amino-terminal 120 residues of NS3 protease was predicted by computer-assisted Robson analysis with GENETIX-MAC software. Results: NS3 protease genetic complexity and diversity did not differ significantly between cases and controls. Amino-acid substitutions were detected at 20 (11%) and 25 (14%) sites in at least two variants of the NS3 protease in cases and controls, respectively. Significant differences in the percentage of substituted clones were observed for 10 NS3 sites. Mutations Y56F, 171V, T721, Q86P, P89S, S101G/D, R117H, S122G/T/N, V1321 and V1701 were more frequently observed in the NS3 protease sequences of controls than in those of cases. However, these differences did not allow the definition of a specific NS3 profile related to HCC occurrence. The NS3 secondary structure B1-1 previously identified as potentially predictive of HCC was identified with a higher frequency in cases quasispecies (84.2%) than in controls ones (55.9%; p <0.05).