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423 Multigenotype HCV NS3 recombinant vaccinia viruses as a model for evaluation of cross-genotype immunity induced by HCV vaccines in the mouse
05C. Viral Hepatitis'
(c9 Hepatitis' C Experimental (immunology)
comparable to apparently recovered anti-HCV-positive individuals; and (5) just discrete frequencies of HCV NS3-specific CD8+ T-cells were detected in the eleven HLA-A2-positive apparently recovered individuals (8 SVR and 3 asymptomatic anti-HCV carriers). Conclusion: HCV-specific T-cells are detectable in apparently recovered individuals in whom HCV RNA can persist detectable in the liver indicating that HCV replication is prolonged in face of an insufficient or inadequate virus-specific CD4+ and CD8+ T-cell responses.
14-~-1 MULTIGENOTYPE HCV-NS3 R E C O M B I N A N T VACCINIA VIRUSES AS A MODEL FOR EVALUATION OF CROSSGENOTYPE IMMUNITY INDUCED BY HCV VACCINES IN THE MOUSE C. Eisenbach 1, A. Freyse 1, C.M. Lupu 1, K. Weigand 1, E. Ernst 1, B. Hoyler1, W. Stremmel 1, J.J. Bugert 2'3, J. Encke 1. 1Dept. of
Gastroenterology, 2Dept. of Virology, University of Heidelberg, Heidelberg, Germany," 3Dept. of Medical Microbiology, Cardiff'University School of Medicine, Cardiff; UK Background: The development of an effective prophylactic vaccine against the hepatitis C virus (HCV) remains a major problem despite considerable efforts. Progress has been hampered by the lack of a convenient animal model and until recently by the limitations in growing the virus in cell culture. Therefore, experimental vaccines are frequently tested in mice, often employing HLA-A2.1 transgenic strains. Surrogate infections with HCV-recombinant vaccinia viruses (rVV) that replicate in the mouse have been used in a number of studies to evaluate vaccine potency in vivo. Although a lot has been learned about various vaccination strategies the genetic variability of HCV has been largely neglected. Methods: We constructed a set of 16 vaccinia viruses recombinant for the HCV non-structural protein 3 (NS3) derived from patient isolates of HCV genotypes la, lb, 2, 3 and 4, respectively, with an amino acid homology ranging from 0.800 to 0.998 as verified by direct sequencing. BALB/c mice immunized with recombinant NS3 protein derived from a genotype lb isolate were challenged with the NS3-rVV and compared to mock immunized animals. The amount of viruses recovered from mouse ovaries after surrogate infection were taken as a measure of the specific immunity induced. Results: Mice challenged with genotype 1 recombinant viruses showed a decrease in rVV titers ranging from factor 3.8 8.5 for subtype la and 32 54 for subtype lb. In animals challenged with genotype 2, 3, 4 or wild type VV no change in titers could be detected. Conclusions: Our proposed infection model is a convenient and reliable tool to analyse the induction of cross-genotype immunity by experimental vaccination of mice. The protection seen in our study has been limited to individual genotypes with lack of cross-genotype immunity. We conclude that for the development of a potential vaccine great care must be taken to cover the genetic broadness of HCV.
14~-1 A CROSSTALK BETWEEN Toil-LIKE A N D Fas RECEPTORS LIGANDS - MODULATORY EFFECT OF IGF-I G. Kocic 1, T. Jevtovic 1, D. Pavlovic 1, R. Kocic 2, G. Bjelakovic 1.
1Medical Faculty Nis Department of Biochemistry,, 4Medical Faculty Nis Clinic for Endocrinology, Nis, Serbia and Montenegro Background and Aims: The discovery of the Toll-like receptors (TLRs) on hepatocytes and Kupffer cells, reinforced the prominent role of liver as a microbial product-responsive organ, because TLRs represent the transmembrane proteins involved in innate immunity, by detecting a panel of microbial molecules and combating microbial infections. TLR3 recognizes dsRNA, a viral product, TLR9 recognizes unmethylated CpG motifs frequently found in the genome of bacteria and viruses, while TLR7 recognizes small synthetic immune modifiers, self and non-self ssRNA and different sized oligonucleotides. Their ligands are capable of inhibiting viral replication in the liver, applied or promising for clinical use against
$159
viral infections and cancers as vaccine adjuvants. Since in animal models of acute viral hepatitis, apoptosis of hepatocytes is mediated by death receptors, the aim of the present study was to evaluate the effect of the antiapoptotic agent, insulin-like growth factor I (IGF-I) on TLR ligands degradation during Fas-ligand induced apoptosis. Methods: Fas (CD95)-activating antibodies Jo2 (BD Biosciences) were applied i.p. in a dose of 5 g/mice daily for 48h. One group received simultaneously IGF-I (6ng/gBW). Results: The degradation (expressed as U/g prot) of possible TLR ligands increased several times after Fas-antibody application: for synthetic dsRNA (poly I:C) 32.09• vs 11.28• of control (p <0.001); for CpG 15.42• 1.12 vs 9.64• of control (p < 0.05); for polynucleotide (poly U) 17.53• vs 7.67• of control (p<0.001), supporting the finding of silent host immune response in apoptosis. IGF-I given simultaneously with anti-Fas mAb significantly prevented TLR ligands degradation (I:C 12.8• 3.06 p<0.05; CpG 11.22• 3.34 p<0.05; and polyU 15.67• p <0.05). Given alone IGF-I significantly decreased their degradation:(I:C 5.95• p <0.001; CpG 8.85• 1.98 p <0.05; polyU 9.87• p < 0.005). Conclusion: The modulation of TLR activity may play a fundamental role in exploring new therapeutic strategies and vaccines based on the concept that viral hepatitis C prevention may activate the host innate immunity.
14-~-1 E N H A N C E D APOPTOSIS OF CD4+CD25+ T-REGULATORY LYMPHOCYTES IN CHRONIC HCV INFECTION: ASSOCIATION WITH AUTOIMMUNITY Y. Kovalevl., A. Kessel2, R. Perri 2, J.E. Naschitz 3, E. Toubi 2, E. Zuckerman 1. 1Liver Unit Bnai Zion Medical Cente~ Technion
Faculty of Medicine, 2Department of Clinical Immunology, 3Department of Internal Medicine A, Bnai Zion Medical Cente~ Haifa, Israel Background: Chronic HCV infection is associated with a variety of autoimmune phenomena and lymphoproliferation. CD4+ cells constitutively expressing CD25 have a regulatory function. Their ability to expand is a key to the maintenance of immune tolerance, they suppress both TH1 and TH2 responses and their experimental removal leads to spontaneous autoimmune disease in normal rodents. We have previously shown that HCV-related autoimmunity is associated with enhanced apoptosis of both, peripheral T-cells and CD5-negative B-cell whereas CD5+ B-cell expand and are apoptosis-protected. In the present study, we assessed the "apoptotic status" of CD4+CD25+ and its correlation with autoimmunity in chronic HCV infection. Methods: The sensitivity of CD4+CD25+ T-regulatory cells (Tr) to undergo apoptosis in patients with chronic HCV infection (with and without autoimmune markers) and healthy controls was assessed by anexin V binding using triple color flow cytometry (FACS) of fresh peripheral blood mononuclear cells (PBMCs) and by DAPI staining for chromatin condensation of purified Tr cells. HCV-related autoimmunity was defined as the presence of auto-antibodies (ANA, anti-smooth muscle, anti LKM-1, anti mitochondrial, anti cardiolipin and ANCA), rheumatoid factor (RF), cryglobulinemia or other clinical autoimmune manifestations such as vasculitis or Sjogren syndrome. Results: The apoptosis rate of circulating CD4+CD25+ Tr cells was significantly higher (by both methods) in HCV-patients with, than without autoimmune markers (p 0.018). The rate of apoptosis of CD4+CD25+ Tr cells was comparable between control group and HCV-patients without autoimmune markers (p 0.09). In addition, the absolute number of CD4+CD25+ Tr cells in HCV-patients with autoimmunity was lower than in patients without autoimmune markers and controls (p 0.03). The enhanced apoptosis was significantly associated with the occurrence of autoimmune phenomena (i. e. the presence of at least one auto-antibody, cryoglobulin or RF) but not with liver disease severity or viral load. Conclusions: CD4+CD25+ Tr cells undergo enhanced apoptosis in patients with chronic HCV infection compared to healthy controls. The enhanced apoptosis of these cells is correlated with HCV-related autoim-
(c9 Hepatitis' C Experimental (immunology)
comparable to apparently recovered anti-HCV-positive individuals; and (5) just discrete frequencies of HCV NS3-specific CD8+ T-cells were detected in the eleven HLA-A2-positive apparently recovered individuals (8 SVR and 3 asymptomatic anti-HCV carriers). Conclusion: HCV-specific T-cells are detectable in apparently recovered individuals in whom HCV RNA can persist detectable in the liver indicating that HCV replication is prolonged in face of an insufficient or inadequate virus-specific CD4+ and CD8+ T-cell responses.
14-~-1 MULTIGENOTYPE HCV-NS3 R E C O M B I N A N T VACCINIA VIRUSES AS A MODEL FOR EVALUATION OF CROSSGENOTYPE IMMUNITY INDUCED BY HCV VACCINES IN THE MOUSE C. Eisenbach 1, A. Freyse 1, C.M. Lupu 1, K. Weigand 1, E. Ernst 1, B. Hoyler1, W. Stremmel 1, J.J. Bugert 2'3, J. Encke 1. 1Dept. of
Gastroenterology, 2Dept. of Virology, University of Heidelberg, Heidelberg, Germany," 3Dept. of Medical Microbiology, Cardiff'University School of Medicine, Cardiff; UK Background: The development of an effective prophylactic vaccine against the hepatitis C virus (HCV) remains a major problem despite considerable efforts. Progress has been hampered by the lack of a convenient animal model and until recently by the limitations in growing the virus in cell culture. Therefore, experimental vaccines are frequently tested in mice, often employing HLA-A2.1 transgenic strains. Surrogate infections with HCV-recombinant vaccinia viruses (rVV) that replicate in the mouse have been used in a number of studies to evaluate vaccine potency in vivo. Although a lot has been learned about various vaccination strategies the genetic variability of HCV has been largely neglected. Methods: We constructed a set of 16 vaccinia viruses recombinant for the HCV non-structural protein 3 (NS3) derived from patient isolates of HCV genotypes la, lb, 2, 3 and 4, respectively, with an amino acid homology ranging from 0.800 to 0.998 as verified by direct sequencing. BALB/c mice immunized with recombinant NS3 protein derived from a genotype lb isolate were challenged with the NS3-rVV and compared to mock immunized animals. The amount of viruses recovered from mouse ovaries after surrogate infection were taken as a measure of the specific immunity induced. Results: Mice challenged with genotype 1 recombinant viruses showed a decrease in rVV titers ranging from factor 3.8 8.5 for subtype la and 32 54 for subtype lb. In animals challenged with genotype 2, 3, 4 or wild type VV no change in titers could be detected. Conclusions: Our proposed infection model is a convenient and reliable tool to analyse the induction of cross-genotype immunity by experimental vaccination of mice. The protection seen in our study has been limited to individual genotypes with lack of cross-genotype immunity. We conclude that for the development of a potential vaccine great care must be taken to cover the genetic broadness of HCV.
14~-1 A CROSSTALK BETWEEN Toil-LIKE A N D Fas RECEPTORS LIGANDS - MODULATORY EFFECT OF IGF-I G. Kocic 1, T. Jevtovic 1, D. Pavlovic 1, R. Kocic 2, G. Bjelakovic 1.
1Medical Faculty Nis Department of Biochemistry,, 4Medical Faculty Nis Clinic for Endocrinology, Nis, Serbia and Montenegro Background and Aims: The discovery of the Toll-like receptors (TLRs) on hepatocytes and Kupffer cells, reinforced the prominent role of liver as a microbial product-responsive organ, because TLRs represent the transmembrane proteins involved in innate immunity, by detecting a panel of microbial molecules and combating microbial infections. TLR3 recognizes dsRNA, a viral product, TLR9 recognizes unmethylated CpG motifs frequently found in the genome of bacteria and viruses, while TLR7 recognizes small synthetic immune modifiers, self and non-self ssRNA and different sized oligonucleotides. Their ligands are capable of inhibiting viral replication in the liver, applied or promising for clinical use against
$159
viral infections and cancers as vaccine adjuvants. Since in animal models of acute viral hepatitis, apoptosis of hepatocytes is mediated by death receptors, the aim of the present study was to evaluate the effect of the antiapoptotic agent, insulin-like growth factor I (IGF-I) on TLR ligands degradation during Fas-ligand induced apoptosis. Methods: Fas (CD95)-activating antibodies Jo2 (BD Biosciences) were applied i.p. in a dose of 5 g/mice daily for 48h. One group received simultaneously IGF-I (6ng/gBW). Results: The degradation (expressed as U/g prot) of possible TLR ligands increased several times after Fas-antibody application: for synthetic dsRNA (poly I:C) 32.09• vs 11.28• of control (p <0.001); for CpG 15.42• 1.12 vs 9.64• of control (p < 0.05); for polynucleotide (poly U) 17.53• vs 7.67• of control (p<0.001), supporting the finding of silent host immune response in apoptosis. IGF-I given simultaneously with anti-Fas mAb significantly prevented TLR ligands degradation (I:C 12.8• 3.06 p<0.05; CpG 11.22• 3.34 p<0.05; and polyU 15.67• p <0.05). Given alone IGF-I significantly decreased their degradation:(I:C 5.95• p <0.001; CpG 8.85• 1.98 p <0.05; polyU 9.87• p < 0.005). Conclusion: The modulation of TLR activity may play a fundamental role in exploring new therapeutic strategies and vaccines based on the concept that viral hepatitis C prevention may activate the host innate immunity.
14-~-1 E N H A N C E D APOPTOSIS OF CD4+CD25+ T-REGULATORY LYMPHOCYTES IN CHRONIC HCV INFECTION: ASSOCIATION WITH AUTOIMMUNITY Y. Kovalevl., A. Kessel2, R. Perri 2, J.E. Naschitz 3, E. Toubi 2, E. Zuckerman 1. 1Liver Unit Bnai Zion Medical Cente~ Technion
Faculty of Medicine, 2Department of Clinical Immunology, 3Department of Internal Medicine A, Bnai Zion Medical Cente~ Haifa, Israel Background: Chronic HCV infection is associated with a variety of autoimmune phenomena and lymphoproliferation. CD4+ cells constitutively expressing CD25 have a regulatory function. Their ability to expand is a key to the maintenance of immune tolerance, they suppress both TH1 and TH2 responses and their experimental removal leads to spontaneous autoimmune disease in normal rodents. We have previously shown that HCV-related autoimmunity is associated with enhanced apoptosis of both, peripheral T-cells and CD5-negative B-cell whereas CD5+ B-cell expand and are apoptosis-protected. In the present study, we assessed the "apoptotic status" of CD4+CD25+ and its correlation with autoimmunity in chronic HCV infection. Methods: The sensitivity of CD4+CD25+ T-regulatory cells (Tr) to undergo apoptosis in patients with chronic HCV infection (with and without autoimmune markers) and healthy controls was assessed by anexin V binding using triple color flow cytometry (FACS) of fresh peripheral blood mononuclear cells (PBMCs) and by DAPI staining for chromatin condensation of purified Tr cells. HCV-related autoimmunity was defined as the presence of auto-antibodies (ANA, anti-smooth muscle, anti LKM-1, anti mitochondrial, anti cardiolipin and ANCA), rheumatoid factor (RF), cryglobulinemia or other clinical autoimmune manifestations such as vasculitis or Sjogren syndrome. Results: The apoptosis rate of circulating CD4+CD25+ Tr cells was significantly higher (by both methods) in HCV-patients with, than without autoimmune markers (p 0.018). The rate of apoptosis of CD4+CD25+ Tr cells was comparable between control group and HCV-patients without autoimmune markers (p 0.09). In addition, the absolute number of CD4+CD25+ Tr cells in HCV-patients with autoimmunity was lower than in patients without autoimmune markers and controls (p 0.03). The enhanced apoptosis was significantly associated with the occurrence of autoimmune phenomena (i. e. the presence of at least one auto-antibody, cryoglobulin or RF) but not with liver disease severity or viral load. Conclusions: CD4+CD25+ Tr cells undergo enhanced apoptosis in patients with chronic HCV infection compared to healthy controls. The enhanced apoptosis of these cells is correlated with HCV-related autoim-