637 Rapid Selection of Escape Mutations Within NS3 1406 CTL Epitope During Acute Hepatitis C with Sequence Variability Due to Immune Pressure

05C. VIRAL HEPATITIS – C) HEPATITIS C – EXPERIMENTAL (IMMUNOLOGY) cytokine staining (ICS) and culture supernatants were also examined by ELISA for IL-12p70. TLR2 expression on monocytes and lipopeptide stimulated MoDC was determined by flow cytometry. Results: Lipopeptides were able to induce specific CD8+ T cell responses in HLA-A2 transgenic mice (1000 spots/106 cells vs 50–100 spots/106 cells non-lipidated peptide, p < 0.05). Lipopeptides consistently activated human MoDC from both healthy individuals and HCV infected patients (77% vs 44% non-lipidated peptide, p < 0.05). The lipopeptide-pulsed human DC were found to secrete high levels of the pro-inflammatory cytokine IL-12p70 (75% vs 25% non-lipidated peptide, p < 0.05) and were able to activate antigen-specific IFN-g production by autologous CD8+ T cells obtained from a hepatitis C patient (12.0%±8.2% for influenza and 11.4%±1.4% HCV). Conclusion: These results show that DC from HCV patients can be matured and antigen loaded with TLR2-targeting lipopeptides for effective presentation of CD8+ T cell epitopes and that DC function is not impaired by HCV. 636 COMBINATORIAL DIAGNOSTICS – A MEANS TOWARDS HCV ANALYSIS D. Siman-Tov1 , E. Spectorman1 , J.M. Gershoni1 , R. Zemel2 , R. Tur-Kaspa2,3 . 1 Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, 2 Molecular Hepatology Laboratory, Felsenstein Medical Research Center, Rabin Medical Center, Petah Tiqva, 3 Department of Medicine D and Liver Institute, Rabin Medical Center, Petah Tiqva, Israel E-mail: rturkaspa@clalit.org.il Sero-diagnosis of infectious disease can be accomplished by solid phase immunoassay (ELISA) using a mixture of viral antigens. Confirmation can be performed by western blots, which are in a sense “antigen arrays”. Combinatorial Diagnostics takes this logic one step forward, to realize the potential value of breaking down viral antigens into collections of isolated epitopes and screening them individually. Our goal was to produce a novel platform for solid-phase immunodiagnostics in which the immobilized “capture-probes” are panels of pathogendefining phage-displayed peptides and analysis of the binding human antibodies. A variety of phage-displayed peptides representing the core, envelope and non-structural antigens of HCV has been produced. Over 70 different peptides were screened for specificity and sensitivity leading to a panel of peptides of preferred diagnostic potential. These were tested against serum samples derived from HCV infected and healthy non infected individuals. HCV RNA and genotyping were performed by commercial kits. The panel of HCV peptides was screened against 36 confirmed positive sera, and 15 negative sera (for anti-HCV antibodies). All 36 positives sera scored positive, while all the healthy individuals sera scored negative using the epitope array. Moreover, the array was found effective in discrimination between HCV genotypes. 26 out of the 36 positive samples were analyzed for HCV RNA and genotyping. In 15 cases the HCV genotype predictions based on the array performance was identical to the PCR based genotype, there was only one mismatch. In 3 cases the array assay could not predict the genotype. It is of interest that in 3 patients the PCR analysis reveled very low level of viremia while according to the array assay they had remarkably high serological scores in the epitope array. In other 4 patients HCV RNA level was negative and the serological score was low (resolved infection). Combinatorial diagnostics using phage displayed peptides has proven to be an effective platform for sero-diagnosis of HCV infection. Epitope arrays can be used to genotype HCV. Epitope arrays in conjunction with PCR allowed detection of selected group of patients that appear to be able to maintain minimal viremia with concomitant high serological scores. S237 637 RAPID SELECTION OF ESCAPE MUTATIONS WITHIN NS3 1406 CTL EPITOPE DURING ACUTE HEPATITIS C WITH SEQUENCE VARIABILITY DUE TO IMMUNE PRESSURE A. Ulsenheimer1 , G. Paranhos-Baccala2 , F. Komurian-Pradel2 , H.M. Diepolder1 , M. Heeg1 , B. Raziorrouh1 , R. Zachoval1 , T. Berg3 , M.C. Jung1 , N.H. Gruener1 . 1 Department of Internal Medicine II, Klinikum Grosshadern and Institue of Immunolgy, University of Munich, Munich, Germany; 2 Emerging Pathogens Department of BioMerieux, IFR128 BioSciences Lyon Gerland, Lyon, France; 3 Department of Hepatology and Gastroenterology, CVK, Charite Universitatsmedizin Berlin, Berlin, Germany E-mail: axel@ulsenheimer.de Background and Aims: Up to now the sequence evolution of well known CD8+ epitopes and their influence on the immune response and course of disease during the very early phase of acute hepatitis C virus infection is not well characterised. Methods: In this study four patients (HLAA2+) with acute hepatitis C virus infection were longitudinally analysed with respect to the sequence evolution of HCV within the NS3 region, the viral load and the HCV specific CD8+ T cell response (tetramer assay, Elispot assay, proliferation). Two to six samples of each patient were analysed within the first six months after onset of symptomatic disease. Results: In three out of four patients there was a change of the viral sequence within the NS3 1406 epitope during the first weeks after disease onset. In one case there was a 100x fold increase of the viral load at the time point when a mutation occured. In this patient there were several mutations of the 1406 epitope detectable within the first six weeks after onset of disease. In contrast the sequence analysis of the NS3 region apart of the 1406 epitope showed only a few mutations. In all patients there were tetramer positive CD8+ HCV specific T cells (1406) detectable (up to 10% of the CD8+ cells). After the appearance of mutations there were no new HCV specific CD8+ T cells detectable which were directed against the altered aminoacid sequence. In addition we were able to show a diminished CD8+ T cell response e.g. loss of gamma interferon production after stimulation with mutated peptide sequences. Importantly there was no loss of function after stimulation with the wild type sequence. Conclusions: The adaption of the virus to a new host is characterised by a high and rapid variability within 1406 epitope. The dynamic of mutations and fluctuations in viral load during acute disease and the strong CD8+ immune response are an important hint for the relevance of the 1406 epitope. The influence of the HLA background of the infected and infecting person on the disease outcome and the frequency of mutations should be addressed in future studies. 638 PARVOVIRUS B19 INFECTION: EVIDENCE FOR INTRAHEPATIC LONG-TERM PERSISTENCE BUT NO ASSOCIATION WITH DISEASE PROGRESSION IN CHRONIC HEPATITIS C C. Wang1 , A. Heim2 , V. Schlaphoff1 , P.V. Suneetha1 , H. Jiang1 , M. Krueger1 , P. Fytili1 , K. Stegmann1 , T. Bock3 , M.P. Manns1 , H. Wedemeyer1 . 1 Department of Gastroenterology, Hepatology und Endocrinology, 2 Department of Virology, Medical School Hannover, Hannover, 3 Department of Molecular Pathology, Institute of Pathology, University Hospital of Tuebingen, Tuebingen, Germany E-mail: chirs66@hotmail.com Background and Aims: Parvovirus B19 is the causative agent for fifth disease. Recently, it was reported that PV-B19 may play an important role in the pathogenesis of HBV in Vietnamese patients. We here aimed to investigate whether PV-B19 infection may be a co-factor for disease progression in European patients with chronic hepatitis C. Methods: 91 serum samples from well characterized and histologically staged chronic hepatitis C patients and 50 serum samples from chronic