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394 A promiscuous CD4+ T-cell epitope within HBx protein during chronic HBV infection
05A. Viral Hepatitis
(a) Hepatitis' B
attributable to down-modulation of the CD3? chain. This reversible defect could contribute to both the T cell hyporesponsiveness and the inflammatory milieu characteristic of the HBV-infected liver.
I•
HBeAg DEFECTIVE MUTANTS IN ACTIVE/INACTIVE ANTI-HBe POSITIVE HBV CARRIERS: POSSIBLE ROLE OF PRE-CORE INITIATION MUTANTS
D. Flichman 1, F. Moriconi 1, F. Oliveri 1, R Ciccorossi 1, B. Coco 1, E Colombatto 1, R. Sacco 1, F. Bonino 2, M.R. Brunetto 1. 1UO.
Gastroenterologia e Epatologia, Azienda Ospedaliero Universitaria Pisana, Pisa, Italy," 2Direzione Seientifica Fondazione IRCCS Policlinico Milano, Milano, Italy Background and Aim: Mutations that modulate HBeAg expression at translational or transcriptional levels are present in anti-HBe-positive HBsAg carriers, but their relationship with active and inactive infection is unclear. Materials and Methods: We characterized by direct sequencing Basic Core Promoter (BCP) and pre-Core (pC) HBV mutants in 104 chronic anti-HBe positive carriers, followed prospectively for 25.3 months (range: 12 32) and classified as inactive (26) or active (78) according to 12 month persistence of normal ALT and HBV-DNA levels <5 log10 cp/ml. Results: BCP and pC mutants were found in all cases [1762T: 60.8%; 1764A: 68.6%; 1896A: 72.5%; 1899A: 52.0%; 2bp insertion 1.0%; 1817T: 2.9% and pre-Core initiation codon (PIC): 10.8%]. The 1896A single mutation pattern prevailed in younger than older (>50 years) carriers (29.6% vs. 0.0%, p <0.001). Mutations at nt 1762, 1764 and 1896 had lower prevalence in inactive than active carriers [37.5% vs 67.9% (p 0.009), 45.8% vs 75.6% (p 0.011), 58.3% vs 76.9% (n.s.)] respectively; whereas 1899 mutation was evenly distributed in the 2 groups (50% vs 53%). PIC variants prevailed in inactive HBV genotype D carriers (29.2% vs 5.3%, p <0.003) and were associated with ALT flares in active carriers. Conclusions: BCP/pC mutations are the hallmark of chronic anti-HBe positive HBV infection. Translational pC mutants prevail in younger whereas transcriptional BCP mutants might provide selective viral fitness in older and active HBV carriers. PIC mutations in inactive HBV genotype D carriers might play a role in shifting from active to inactive infection.
I-~
ASSESSMENT OF A REAL-TIME PCR HEPATITIS B VIRUS DNA QUANTITATIVE ASSAY AND COMPARISON WITH TWO COMMERCIAL ASSAYS
K. Hormigos, N. Belguerma, S. Brichler, R D~ny, E. Gordien. Laboratoire de bacteriologic, virologic, hygiene, Assistance Publique des H@itaux de Paris', EA 3406, Universitd Paris' 13, Bobigny, France Background and Aims: Diagnostic assays for the accurate quantitation of hepatitis B virus (HBV) DNA levels from patients undergoing antiviral therapy are useful for monitoring and tailoring therapy. Such assays should give a wide dynamic range and accurate results with all HBV genotypes. However, both the values and the units of measure can vary greatly among different tests. The objective of the present study was to evaluate the new quantitative real time PCR assay, the Affigene | HBV TRENDER (Sangtec Molecular Diagnostic), against two commercial assays, the COBAS Amplicor HBV Monitor assay (Roche Diagnostics) and the version 3.0 branched-DNA (bDNA) assay (Bayer Health Care). We compared these techniques using the international units (IU) established by the World Health Organization. Methods: Eighty-nine consecutive serum samples of patients from the Seine Saint-Denis district of Pards were prospectively collected during two months. HBV DNA extraction and quantification were performed with each test according to manufacturers' recommendations. HBV genotyping
Experimental
S149
were determined by sequencing and phylogenetic analyses of a 410 nucleotides region of the polymerase gene. Statistical analyses using SPSS version 10.0 software package were performed. Results: As expected in this Paris suburb, the five HBV genotypes: A (27%), B/C (10%), D (13.5%) and E (49.5%) were found. The range of quantitation of the HBV Trender was between 1.711ogIU/ml and 8.241ogIU/ml, however the test was able to give values ranging from 1 log IU/ml to 9.5 log IU/ml (• log IU/ml). The assay was fast, easy to use, and able to detect and quantify DNA from genotypes A E equivalently. Considering global results, COBAS technique shows significant lower viral load values compared either to bDNA 3.0 (P 0.002) or to HBV Trender (P 0.015), whereas no difference was found between HBV Trender and bDNA assay. According to genotypes, differences seem to be mainly linked to genotype E (P 0.002). Conclusions: This study raises the question of HBV variability in viral load measurements, and the difference between tests whatever the units of measure used.
I-~A
PROMISCUOUS CD4+ T-CELL EPITOPE WITHIN HBx PROTEIN DURING CHRONIC HBV INFECTION
S.L. Malmassari 1. H. Fontaine 2, S. Pol 2, M.-L. Michel 1. 1Carcinogdn&se H@atique et Virologic Moldculaire, INSERM U370, Institut Pasteug 2Service d'H@atologie, H@ital Necker Enfants Malades, Paris', France HBx protein of hepatitis B virus (HBV) is a critical component of the infection process in vivo and has been implicated in the development of hepatocellular carcinoma. The aim of this study was the detection and characterisation of CD8+ and CD4+ peripheral HBx-specific T cells. For this purpose, chronically infected patients with less than 100,000 HBV copies/ml were enrolled. To analyze the HBx-specific T cells, we used ELISPOT assays or intracellular cytokines staining to measure IFN-?, IL-10 and IL-2 secretion. HLA-A2-restricted peptides, previously characterized in HLA-A2 transgenic mice, were used for in vitro stimulation of PBMC from HLA-A2+ patients. To define new CD4+ and CD8+ T-cell epitopes, other than HLA-A2-restricted, 15-mer peptides corresponding to the HBx consensus sequence were used to stimulate PBMC from all patients. Our results show: (a) a sporadic detection of HLA-A2-restricted HBxspecific CD8+ T cells secreting IFN-? or IL-10; (b) 50% of studied patients (23/46) presented IFN-?-secreting T cells specific for epitopes located in the carboxy-terminal part of HBx; (c) more than 80% (19/23) of the IFN-?-secreting T cells recognizing the carboxy-terminal domain of HBx targeted a single immunodominant epitope; (d) this promiscuous epitope stimulated specific IFN-?-secreting CD4+ T cells; (e) in some patients, the prosmiscuous epitope activated both IFN-?- and IL-10-secreting T cells, (f) IL-2 secretion was not observed under our experimental conditions. Overall, these results show a peripheral low frequency of CD8+ T cells and a predominance of CD4+ T cells specific for HBx. Further characterisation of the immunodominant T-cell response directed against HBx could have important implications for diagnosis purposes and therapeutic vaccine design for chronically infected patients.
I-~
ROLE OF CIRCULATING IL-10 IN ACUTE VIRAL HEPATITIS
G. Nebbial, J. Northfield 2, E. Barnes 2, V. Emery 1, D. Brown 3, C. Willberg 2, G. Dusheiko 3, R Klenerman2, R Fabris 4. 1Department of
Virology, Royal Free and University College Medical School, London, UK," 2peter Medawar Building for Pathogen Research, Nujfield Department of Medicine, University of Oxford, Oxford, UK," SDepartment of Medicine, Royal Free and University College Medical School, London, UK," 4Department of Infectious Diseases, S. Bortolo Hospital, Vicenza, Italy Background: Acute viral hepatitis caused by hepatitis A, B and C viruses may present in a similar fashion clinically, but the long term sequelae are
(a) Hepatitis' B
attributable to down-modulation of the CD3? chain. This reversible defect could contribute to both the T cell hyporesponsiveness and the inflammatory milieu characteristic of the HBV-infected liver.
I•
HBeAg DEFECTIVE MUTANTS IN ACTIVE/INACTIVE ANTI-HBe POSITIVE HBV CARRIERS: POSSIBLE ROLE OF PRE-CORE INITIATION MUTANTS
D. Flichman 1, F. Moriconi 1, F. Oliveri 1, R Ciccorossi 1, B. Coco 1, E Colombatto 1, R. Sacco 1, F. Bonino 2, M.R. Brunetto 1. 1UO.
Gastroenterologia e Epatologia, Azienda Ospedaliero Universitaria Pisana, Pisa, Italy," 2Direzione Seientifica Fondazione IRCCS Policlinico Milano, Milano, Italy Background and Aim: Mutations that modulate HBeAg expression at translational or transcriptional levels are present in anti-HBe-positive HBsAg carriers, but their relationship with active and inactive infection is unclear. Materials and Methods: We characterized by direct sequencing Basic Core Promoter (BCP) and pre-Core (pC) HBV mutants in 104 chronic anti-HBe positive carriers, followed prospectively for 25.3 months (range: 12 32) and classified as inactive (26) or active (78) according to 12 month persistence of normal ALT and HBV-DNA levels <5 log10 cp/ml. Results: BCP and pC mutants were found in all cases [1762T: 60.8%; 1764A: 68.6%; 1896A: 72.5%; 1899A: 52.0%; 2bp insertion 1.0%; 1817T: 2.9% and pre-Core initiation codon (PIC): 10.8%]. The 1896A single mutation pattern prevailed in younger than older (>50 years) carriers (29.6% vs. 0.0%, p <0.001). Mutations at nt 1762, 1764 and 1896 had lower prevalence in inactive than active carriers [37.5% vs 67.9% (p 0.009), 45.8% vs 75.6% (p 0.011), 58.3% vs 76.9% (n.s.)] respectively; whereas 1899 mutation was evenly distributed in the 2 groups (50% vs 53%). PIC variants prevailed in inactive HBV genotype D carriers (29.2% vs 5.3%, p <0.003) and were associated with ALT flares in active carriers. Conclusions: BCP/pC mutations are the hallmark of chronic anti-HBe positive HBV infection. Translational pC mutants prevail in younger whereas transcriptional BCP mutants might provide selective viral fitness in older and active HBV carriers. PIC mutations in inactive HBV genotype D carriers might play a role in shifting from active to inactive infection.
I-~
ASSESSMENT OF A REAL-TIME PCR HEPATITIS B VIRUS DNA QUANTITATIVE ASSAY AND COMPARISON WITH TWO COMMERCIAL ASSAYS
K. Hormigos, N. Belguerma, S. Brichler, R D~ny, E. Gordien. Laboratoire de bacteriologic, virologic, hygiene, Assistance Publique des H@itaux de Paris', EA 3406, Universitd Paris' 13, Bobigny, France Background and Aims: Diagnostic assays for the accurate quantitation of hepatitis B virus (HBV) DNA levels from patients undergoing antiviral therapy are useful for monitoring and tailoring therapy. Such assays should give a wide dynamic range and accurate results with all HBV genotypes. However, both the values and the units of measure can vary greatly among different tests. The objective of the present study was to evaluate the new quantitative real time PCR assay, the Affigene | HBV TRENDER (Sangtec Molecular Diagnostic), against two commercial assays, the COBAS Amplicor HBV Monitor assay (Roche Diagnostics) and the version 3.0 branched-DNA (bDNA) assay (Bayer Health Care). We compared these techniques using the international units (IU) established by the World Health Organization. Methods: Eighty-nine consecutive serum samples of patients from the Seine Saint-Denis district of Pards were prospectively collected during two months. HBV DNA extraction and quantification were performed with each test according to manufacturers' recommendations. HBV genotyping
Experimental
S149
were determined by sequencing and phylogenetic analyses of a 410 nucleotides region of the polymerase gene. Statistical analyses using SPSS version 10.0 software package were performed. Results: As expected in this Paris suburb, the five HBV genotypes: A (27%), B/C (10%), D (13.5%) and E (49.5%) were found. The range of quantitation of the HBV Trender was between 1.711ogIU/ml and 8.241ogIU/ml, however the test was able to give values ranging from 1 log IU/ml to 9.5 log IU/ml (• log IU/ml). The assay was fast, easy to use, and able to detect and quantify DNA from genotypes A E equivalently. Considering global results, COBAS technique shows significant lower viral load values compared either to bDNA 3.0 (P 0.002) or to HBV Trender (P 0.015), whereas no difference was found between HBV Trender and bDNA assay. According to genotypes, differences seem to be mainly linked to genotype E (P 0.002). Conclusions: This study raises the question of HBV variability in viral load measurements, and the difference between tests whatever the units of measure used.
I-~A
PROMISCUOUS CD4+ T-CELL EPITOPE WITHIN HBx PROTEIN DURING CHRONIC HBV INFECTION
S.L. Malmassari 1. H. Fontaine 2, S. Pol 2, M.-L. Michel 1. 1Carcinogdn&se H@atique et Virologic Moldculaire, INSERM U370, Institut Pasteug 2Service d'H@atologie, H@ital Necker Enfants Malades, Paris', France HBx protein of hepatitis B virus (HBV) is a critical component of the infection process in vivo and has been implicated in the development of hepatocellular carcinoma. The aim of this study was the detection and characterisation of CD8+ and CD4+ peripheral HBx-specific T cells. For this purpose, chronically infected patients with less than 100,000 HBV copies/ml were enrolled. To analyze the HBx-specific T cells, we used ELISPOT assays or intracellular cytokines staining to measure IFN-?, IL-10 and IL-2 secretion. HLA-A2-restricted peptides, previously characterized in HLA-A2 transgenic mice, were used for in vitro stimulation of PBMC from HLA-A2+ patients. To define new CD4+ and CD8+ T-cell epitopes, other than HLA-A2-restricted, 15-mer peptides corresponding to the HBx consensus sequence were used to stimulate PBMC from all patients. Our results show: (a) a sporadic detection of HLA-A2-restricted HBxspecific CD8+ T cells secreting IFN-? or IL-10; (b) 50% of studied patients (23/46) presented IFN-?-secreting T cells specific for epitopes located in the carboxy-terminal part of HBx; (c) more than 80% (19/23) of the IFN-?-secreting T cells recognizing the carboxy-terminal domain of HBx targeted a single immunodominant epitope; (d) this promiscuous epitope stimulated specific IFN-?-secreting CD4+ T cells; (e) in some patients, the prosmiscuous epitope activated both IFN-?- and IL-10-secreting T cells, (f) IL-2 secretion was not observed under our experimental conditions. Overall, these results show a peripheral low frequency of CD8+ T cells and a predominance of CD4+ T cells specific for HBx. Further characterisation of the immunodominant T-cell response directed against HBx could have important implications for diagnosis purposes and therapeutic vaccine design for chronically infected patients.
I-~
ROLE OF CIRCULATING IL-10 IN ACUTE VIRAL HEPATITIS
G. Nebbial, J. Northfield 2, E. Barnes 2, V. Emery 1, D. Brown 3, C. Willberg 2, G. Dusheiko 3, R Klenerman2, R Fabris 4. 1Department of
Virology, Royal Free and University College Medical School, London, UK," 2peter Medawar Building for Pathogen Research, Nujfield Department of Medicine, University of Oxford, Oxford, UK," SDepartment of Medicine, Royal Free and University College Medical School, London, UK," 4Department of Infectious Diseases, S. Bortolo Hospital, Vicenza, Italy Background: Acute viral hepatitis caused by hepatitis A, B and C viruses may present in a similar fashion clinically, but the long term sequelae are