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Characterization of an immunologically conserved epitope from hepatitis C virus E2 glycoprotein recognized by HLA-A2 restrictedcytotoxic T lymphocytes
WORKING
PARTY 5: Immunology
m CHARACTERIZATION OF AN IMMUNOLOGICALLY CONSERVED EPITOPE FROM HEPATITIS C VIRUS E2 GLYCOPRCD’EIN RECOGNIZED BY HLA-A2 RESTRICTED CYTOMXIC T LYMPHOCYTES P. Sarobe. E. Huarte. J.J. Lasarte. E Borras-Cuesta. J. Prieto Eepartment of Internal Medicine, Medical School and University Clinic, University of Navarra, Pamplona, Spain. Background and aims: Identification of epitopes recognized by cytotoxic T lymphocytes (CTL) in hepatitis C virus (HCV) proteins is of great importance, because of their potential use to make HCV vaccines, treat chronic HCV infection or monitor anti-HCV CTL immune responses.The purpose of this work was to characterize new CTL epitopes in HCV structural proteins. Metho& One hundred and seventeen overlapping 14- 15-mer peptides encompassing almost all HCV structural proteins as well as thirty two 9-mer peptides from the same region, predicted as potential binders to HLA-A2 molecules, were synthesized and tested in an HLA-A2 binding assay. Binder peptides were used to stimulate peripheral blood mononuclear cells from an HLA-AZ’ patient chronically infected with HCV. Results: Twenty binder peptides were found among the 9-mer and 14-15-mer groups. Stimulation of peripheral blood mononuclear cells with nine peptides showing high binding ability led to the growth of CD8+ CTL recognizing peptide E2(614-622) in association with HLA-AZ. Five peptides derived from the region E2(614-622) from 26 different viral isolates were all able to bind to HLA-A2 molecules, and all of them but one, containing Phe at position 622, were recognized by E2(614622) specific CTL lines. ConclusionsThese results show that peptide E2(614-622) belongs to a highly conserved region of HCV E2, and might be a good candidate to induce anti-HCV CTL responses in HLA-A2+ subjects.
HCV-ASSOCIATED AUTOIMMUNE HAEMOLYTIC ANAEMIA DO TO WARM-REACTING ANTIBODIES: EVIDENCE OF B-CELL MONOCLONALITY M. Pogaianella. E Benvenuti. S. Baracetti’. E Nascimben’. G. Pozzato’,
E Zorat’. 0. Burrone ICGEB Area Science Park, Trieste, Italy. Inst. Med. Clinica, Trieste, Italy. Nine HCV-posittve patients with Coombs Test- positive haemotytic anaamia were induded in this study. Twenty HCV-pceitive chronic liver diseases (CLD) without mixed cryoglobulinemia (MC), 10 HBV-posittve CLD and 10 alcoholic CLD were used as controls. Total cellular RNA was isolated frcm PBMC using the guanklinium thiianate-phenol-chloroform procedure. The presence of an expanded B-cell clone in the PBMC was investigated by isotypa-specific lmmuncglobulin (lg) fingerprinting procedure. One ug of RNA was reverse transcribed using random hexamers, and then PCR amplified with a daganerata VH FWFtl primer and a Cu speckic primer located 8 nudeo8das downstream from the beginning of the CHf exon. Each Vii PCR product was lab&d by primer extension with two internal 32plabeled prtmers: one corresponding to a conserved sequence in FW3, the other placed in the CDR2 of 51~1 &ted alleles of the VHl-88 locus, located 113 nt upstream from primer in FW3. Thus, the fingerprint of Clpl-related afleles transcripts can be related with the fingerprint of atl Vii transcripts. Two pl of each reaction were analysed on denaturating 8M sequencing gel. All HBV and alcoholic CLD were negatfve. Among 20 patients with HCV-CLD 5 (25%) showed a &cell monoclcnality in PBMC. Among the HCV-patients wtth haemolytic anaemia, 5 (58%) showed a dear monodonality and an additional patient a oligodonality. In all these patients a monocbnal 51~1 gene was found, while in HCV cases without haemolytic anaemia Clpl was polyclonal or not expressed. This study confirms that HCV is able to determine a B-cell monodonality in a large (25%) fraction of infected pakents. How HCV can determine haamolyttc anaamia is still undear, but, since we found a preferenltal usage of 51~1 gene also in chronic lymphocytic leukaamia-associated haemolytic anaemia, it is likely that HCV (like leukaemia) could bs the trigger for n-cnodonal expansion of B-cdl dorms producing autoantibodies. The analysis of light chain and the full-length sequences of variable regions genes could provide more information on pathogenesis on this disorder
ABERRANT MHC CLASS II EXPRESSION AUTOIMMUNE DISEASE?
AS A REASON
FOR
B. Peleikis. J. Henninaer. M. Blissine. P Schirrmacheri. PR. Galle. A.W. I&&&e 1. Dept of Med, Johannes-Gutenberg University Maim, Germany. ‘Institut of Pathology, University of Koln, Germany.
Like many other parenchymal ceils hepatocytes do not express MHC class II contitutively. However, in autoimmune hepatitis, there is MHC II expression detectable on these cells. Botazzo et al. proposed autoimmunity as a result of aberrant MHC class II expression on parenchymal cells (Lancet, 1982) arguing that there is no immunological tolerance towards antigens in parenchymal cells because
these are normally not presented and detectable by the immune system. In order to analyse aberrant MHC class II expression as a cause for autoimmune hepatitis we generated transgenic mice with constitutive and inducible MHC II expression on hepatocytes. The transgene used was the class II transactivator (CIITA) regulating all MHC II genes and also
other important factors in antigen presentation (EMBO 1997). The murine CIITA transgene in our model is controlled by the human hepatocyte specific CRP promotor inducible by LPS. Nothernblot analysis demonstrate CIITA and MHC II RNA expression in hepatocytes with and without LPS induction. FACS analysis and immunohistology showed marked basal and inducible protein expression of MHC class II. Histology showed no evidence of autoimmune inflammation in CIITA transgenic animals. We can show that mice transgenic for CIITA expression can express MHC II on hepatocytes. These results disprove the hypothesis of aberant MHC II expression as the cause of autoimmune disease.
VIRUS/SELF DOUBLE REACTIVITY CHARACTERISES THE HUMORAL IMMUNE RESPONSE IN AUTOIMMUNE HEPATITIS-2 D.I? Bondanosi. M. Okamotoi. Y. Ma’. R. Williams’. G. Mieli-Vernanis, D. Vereani’ ‘Institute of Hepatology, Royal Free & University College London Medical School, UK. *Department of Child Health, King’s College Hospital, London, UK.
Cytochmme P458 2D6 (CYP206)~sr-~r1 is the immunodominant epitops of LKM-1 autoantibodii, the serokqiil marker of AIH-2, and homology between its core sequence CYP2D&m DPAQPPRand IE 175 Herpes Simplex Virus-l (HSV-1) protein has implicated the virus as possibte trigger for LKM-I generation [J Clin Invest. 1991 Cct;88(4):1379-8)]. Interrogating protein databases, we bund 11 additional viral proteins (Epstein-Barr, Herpes Simplex, Cytomegabvirus, Rubella, Papillomavtms, and Human Adenovitus), sharing 5-6 aa rdentity with the CYP2D6 core sequence. Antibody binding to ISmer biotinylated peptides spanning the motif-containing CYP2D6 and viral sequences was tested by ELISA in 15 AIH-2 children, 95 pathological and 15 demographiilly matched healthy controls. Reactivity to the HSV-1 peptii was not significantly diit in AIH-2 (13115, 87%), pathologiil (81195, 85%) and normal controts (12115, 89%) and reactivity to at least one of the other viral proteins was similarly present in 9115 (69%) AIH-2, in 505 (53%) of the pathological and in 7115 (47%) healthy controls. In contrast, double reactivity to cYi%&57-271 and at least one of the viral peptides was found in 198% of the AIH-2 chitdmn (13115 reacti with the IE 175 HSV-I/ CYP2D6 pair), in 31’95 (3%, riI 1)) of the pathological and in none of the normal controls @kI91). These data show that while reactivity to viral mimics of an immuncdominant autoepitope is common, ~double reactivity to viral/self epitope is highly dii specific and its potential pathogenic role will be explored by inhibitionstudies.
PARTY 5: Immunology
m CHARACTERIZATION OF AN IMMUNOLOGICALLY CONSERVED EPITOPE FROM HEPATITIS C VIRUS E2 GLYCOPRCD’EIN RECOGNIZED BY HLA-A2 RESTRICTED CYTOMXIC T LYMPHOCYTES P. Sarobe. E. Huarte. J.J. Lasarte. E Borras-Cuesta. J. Prieto Eepartment of Internal Medicine, Medical School and University Clinic, University of Navarra, Pamplona, Spain. Background and aims: Identification of epitopes recognized by cytotoxic T lymphocytes (CTL) in hepatitis C virus (HCV) proteins is of great importance, because of their potential use to make HCV vaccines, treat chronic HCV infection or monitor anti-HCV CTL immune responses.The purpose of this work was to characterize new CTL epitopes in HCV structural proteins. Metho& One hundred and seventeen overlapping 14- 15-mer peptides encompassing almost all HCV structural proteins as well as thirty two 9-mer peptides from the same region, predicted as potential binders to HLA-A2 molecules, were synthesized and tested in an HLA-A2 binding assay. Binder peptides were used to stimulate peripheral blood mononuclear cells from an HLA-AZ’ patient chronically infected with HCV. Results: Twenty binder peptides were found among the 9-mer and 14-15-mer groups. Stimulation of peripheral blood mononuclear cells with nine peptides showing high binding ability led to the growth of CD8+ CTL recognizing peptide E2(614-622) in association with HLA-AZ. Five peptides derived from the region E2(614-622) from 26 different viral isolates were all able to bind to HLA-A2 molecules, and all of them but one, containing Phe at position 622, were recognized by E2(614622) specific CTL lines. ConclusionsThese results show that peptide E2(614-622) belongs to a highly conserved region of HCV E2, and might be a good candidate to induce anti-HCV CTL responses in HLA-A2+ subjects.
HCV-ASSOCIATED AUTOIMMUNE HAEMOLYTIC ANAEMIA DO TO WARM-REACTING ANTIBODIES: EVIDENCE OF B-CELL MONOCLONALITY M. Pogaianella. E Benvenuti. S. Baracetti’. E Nascimben’. G. Pozzato’,
E Zorat’. 0. Burrone ICGEB Area Science Park, Trieste, Italy. Inst. Med. Clinica, Trieste, Italy. Nine HCV-posittve patients with Coombs Test- positive haemotytic anaamia were induded in this study. Twenty HCV-pceitive chronic liver diseases (CLD) without mixed cryoglobulinemia (MC), 10 HBV-posittve CLD and 10 alcoholic CLD were used as controls. Total cellular RNA was isolated frcm PBMC using the guanklinium thiianate-phenol-chloroform procedure. The presence of an expanded B-cell clone in the PBMC was investigated by isotypa-specific lmmuncglobulin (lg) fingerprinting procedure. One ug of RNA was reverse transcribed using random hexamers, and then PCR amplified with a daganerata VH FWFtl primer and a Cu speckic primer located 8 nudeo8das downstream from the beginning of the CHf exon. Each Vii PCR product was lab&d by primer extension with two internal 32plabeled prtmers: one corresponding to a conserved sequence in FW3, the other placed in the CDR2 of 51~1 &ted alleles of the VHl-88 locus, located 113 nt upstream from primer in FW3. Thus, the fingerprint of Clpl-related afleles transcripts can be related with the fingerprint of atl Vii transcripts. Two pl of each reaction were analysed on denaturating 8M sequencing gel. All HBV and alcoholic CLD were negatfve. Among 20 patients with HCV-CLD 5 (25%) showed a &cell monoclcnality in PBMC. Among the HCV-patients wtth haemolytic anaemia, 5 (58%) showed a dear monodonality and an additional patient a oligodonality. In all these patients a monocbnal 51~1 gene was found, while in HCV cases without haemolytic anaemia Clpl was polyclonal or not expressed. This study confirms that HCV is able to determine a B-cell monodonality in a large (25%) fraction of infected pakents. How HCV can determine haamolyttc anaamia is still undear, but, since we found a preferenltal usage of 51~1 gene also in chronic lymphocytic leukaamia-associated haemolytic anaemia, it is likely that HCV (like leukaemia) could bs the trigger for n-cnodonal expansion of B-cdl dorms producing autoantibodies. The analysis of light chain and the full-length sequences of variable regions genes could provide more information on pathogenesis on this disorder
ABERRANT MHC CLASS II EXPRESSION AUTOIMMUNE DISEASE?
AS A REASON
FOR
B. Peleikis. J. Henninaer. M. Blissine. P Schirrmacheri. PR. Galle. A.W. I&&&e 1. Dept of Med, Johannes-Gutenberg University Maim, Germany. ‘Institut of Pathology, University of Koln, Germany.
Like many other parenchymal ceils hepatocytes do not express MHC class II contitutively. However, in autoimmune hepatitis, there is MHC II expression detectable on these cells. Botazzo et al. proposed autoimmunity as a result of aberrant MHC class II expression on parenchymal cells (Lancet, 1982) arguing that there is no immunological tolerance towards antigens in parenchymal cells because
these are normally not presented and detectable by the immune system. In order to analyse aberrant MHC class II expression as a cause for autoimmune hepatitis we generated transgenic mice with constitutive and inducible MHC II expression on hepatocytes. The transgene used was the class II transactivator (CIITA) regulating all MHC II genes and also
other important factors in antigen presentation (EMBO 1997). The murine CIITA transgene in our model is controlled by the human hepatocyte specific CRP promotor inducible by LPS. Nothernblot analysis demonstrate CIITA and MHC II RNA expression in hepatocytes with and without LPS induction. FACS analysis and immunohistology showed marked basal and inducible protein expression of MHC class II. Histology showed no evidence of autoimmune inflammation in CIITA transgenic animals. We can show that mice transgenic for CIITA expression can express MHC II on hepatocytes. These results disprove the hypothesis of aberant MHC II expression as the cause of autoimmune disease.
VIRUS/SELF DOUBLE REACTIVITY CHARACTERISES THE HUMORAL IMMUNE RESPONSE IN AUTOIMMUNE HEPATITIS-2 D.I? Bondanosi. M. Okamotoi. Y. Ma’. R. Williams’. G. Mieli-Vernanis, D. Vereani’ ‘Institute of Hepatology, Royal Free & University College London Medical School, UK. *Department of Child Health, King’s College Hospital, London, UK.
Cytochmme P458 2D6 (CYP206)~sr-~r1 is the immunodominant epitops of LKM-1 autoantibodii, the serokqiil marker of AIH-2, and homology between its core sequence CYP2D&m DPAQPPRand IE 175 Herpes Simplex Virus-l (HSV-1) protein has implicated the virus as possibte trigger for LKM-I generation [J Clin Invest. 1991 Cct;88(4):1379-8)]. Interrogating protein databases, we bund 11 additional viral proteins (Epstein-Barr, Herpes Simplex, Cytomegabvirus, Rubella, Papillomavtms, and Human Adenovitus), sharing 5-6 aa rdentity with the CYP2D6 core sequence. Antibody binding to ISmer biotinylated peptides spanning the motif-containing CYP2D6 and viral sequences was tested by ELISA in 15 AIH-2 children, 95 pathological and 15 demographiilly matched healthy controls. Reactivity to the HSV-1 peptii was not significantly diit in AIH-2 (13115, 87%), pathologiil (81195, 85%) and normal controts (12115, 89%) and reactivity to at least one of the other viral proteins was similarly present in 9115 (69%) AIH-2, in 505 (53%) of the pathological and in 7115 (47%) healthy controls. In contrast, double reactivity to cYi%&57-271 and at least one of the viral peptides was found in 198% of the AIH-2 chitdmn (13115 reacti with the IE 175 HSV-I/ CYP2D6 pair), in 31’95 (3%, riI 1)) of the pathological and in none of the normal controls @kI91). These data show that while reactivity to viral mimics of an immuncdominant autoepitope is common, ~double reactivity to viral/self epitope is highly dii specific and its potential pathogenic role will be explored by inhibitionstudies.