05c: VIRAL HEPATITIS − c) HEPATITIS C − EXPERIMENTAL (IMMUNOLOGY) S321 882 SELECTIVE INDUCTION OF ADAPTIVE REGULATROY T-CELLS IN CHRONIC HEPATITIS C B. Langhans 1 , I. Braunschweiger 1 , S. Arndt 1 , W. Schulte 1 , J. Satoguina 2 , N. Vidovic 3 , A. Hoerauf 2 , J. Oldenburg 3 , T. Sauerbruch 1 , U. Spengler 1 . 1 Department of Internal Medicine I, 2 Institute of Medical Microbiology, Immunology and Parasitology, 3 Institute for Experimental Hematology and Transfusion Medicine, University of Bonn, Bonn, Germany E-mail: bettina.langhans@ukb.uni-bonn.de Background and Aims: Weak T-cell responses to hepatitis C virus (HCV) antigens are a hallmark of chronic hepatitis C. Regulatory CD4 + T-cell (Tr-cell) subpopulations have been proposed to contribute to this T-cell dysfunction and may determine the outcome of HCV infection. Methods: We studied ex vivo phenotypic and functional characteristics of CD4 + Tr-cell populations in PBMC from patients with chronic (n = 31) and self-limited HCV infection (n = 12). Tr-cells in bulk cultures were compared with individual Tr-cell clones (Tr-TCC) obtained from both patient groups. Results: Direct phenotypic analysis revealed that CD4 + T-cells expressing Foxp3, GITR and CTLA-4 were markedly less frequent in both CD25 high and CD25 int CD4 + Tr-cell subsets from patients with self-limited HCV infection than in the corresponding subsets of patients with chronic HCV infection and healthy controls. In contrast to self-limited HCV infection, in vitro stimulation of PBMC from patients with chronic hepatitis C using HCV core protein resulted in a significant expansion of Foxp3- and CTLA- 4-expressing CD25 + Tr-cells and revealed a relationship between IL-10 production and numbers of CD25 int CD4 + Tr-cells. Characterisation of CD4 + T-cells from patients with chronic hepatitis C revealed putative Tr-TCC which were hypoproliferative and produced significant amount of IL-10. Among TCC which did not suppress reporter TCC, 10 Tr-TCC significantly inhibited proliferation of both TH1 and TH2 reporter T-cells; 2 Tr-TCC also produced TGF-b1 and suppressed IFN-g secretion. All Tr-TCC were activated in a HCV core-specific, HLA-DR restricted fashion. All Tr-TCC were Foxp3 + and − with the exception of one CD25 high clone − were CD25 int . In contrast, cloning of CD4 + T-cells from patients with self-limited hepatitis C resulted in 9 hypoproliferative IL-10-producing TCC, which resembled the phenotype of Tr-TCC obtained in chronic hepatitis C. However, none of the TCC from self-limited hepatitis C inhibited proliferation of autologous reporter cells. Conclusions: Our data demonstrate that functionally active HCV core- specific CD4 + Tr-cells exist exclusively in patients with chronic hepatitis C but not in self-limited HCV infection. Such HCV-specific Tr-cells may prevent the immune system from ultimately eliminating HCV. 883 VACCINATION AGAINST HEPATITIS C VIRUS WITH A RECOMBINANT FUSION PROTEIN CONTAINING THE EXTRA DOMAIN A FROM FIBRONECTIN AND THE HEPATITIS C VIRUS NS3 PROTEIN C. Mansilla 1 , M. Martinez 1 , M. Gorraiz 1 , N. Casares 1 , L. Arribillaga 1 , I. Echeverria 1 , P. Sarobe 1,2 , F. Borr´ as-Cuesta 1,2 , J. Prieto 1,2,3 , J. Lasarte 1,2 . 1 Hepatology and Gene Therapy, CIMA, 2 Universidad de Navarra, 3 Clinica Universitaria, Pamplona, Spain E-mail: jjlasarte@unav.es Background and Aims: Infection by hepatitis C virus (HCV) is character- ized by its high tendency to chronicity, which is usually associated with a low or absent T-cell response against viral antigens. It has been described that immune response specific for non-structural protein NS3 from HCV was associated with viral clearance. We have demonstrated that fusion of an antigen to the extra domain A from fibronectin (EDA) targets the antigen to dendritic cells and improves its immunogenicity. Thus, we tested if covalent linkage between EDA and NS3 might constitute an alternative for vaccination against HCV infection. Methods: Recombinant plasmids expressing NS3 or EDA-NS3 under the control of CMV promoter were prepared. Recombinant NS3 and the fusion protein EDA-NS3 were produced in E. coli. The recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. HHD transgenic mice (expressing the human HLA-A2 molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(I:C) and anti-CD40 agonistic antibodies. ELISPOT and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. Intrahepatic expression of HCV- NS3 RNA was measured after a hydrodynamic injection with a plasmid encoding HCV NS3. Results: Immunization of mice with the plasmids expressing EDA-NS3, but not NS3 alone, induced strong T cell responses against the main HLA- A2 restricted cytotoxic T cell determinants from NS3. The recombinant EDA-NS3 fusion protein, but not NS3, was able to activate in vitro matura- tion of bone marrow derived dendritic cells (overexpression of maturation markers and production of IL-12) as well as the production of TNF-a by the THP-1 monocyte cell line. Immunization of HHD mice with EDA-NS3 fusion protein induced both CD4+ and CD8+ T cell responses against NS3 (measured by ELISPOT and by conventional chromium releases assay) and, when immunized with poly(I:C) and anti-CD40 antibodies, was able to down-regulate the intrahepatic expression of HCV-NS3 RNA. Conclusions: The recombinant EDA-NS3 fusion protein may be consid- ered for the development of prophylactic or therapeutic vaccines against HCV infection. 884 INTERFERON RELATED THYROID DISEASE IN 3414 NAIVE PATIENTS WITH CHRONIC HEPATITIS C F. Morisco 1 , D. Amoruso 2 , M. Tartaglione 3 , A. De Luna 4 , P. Andreone 5 , C. Coppola 6 , N. Passariello 7 , C. Carella 8 , A. Scuteri 5 , C. Iannaccone 9 , N. Caporaso 2 . 1 Department of Food Science, University of Naples ‘Federico II’, Portici, 2 Department of Clinical and Experimental Medicine, University of Naples ‘Federico II’, 3 Department of Gastroenterology, Hepatology and Pancreas Unit, Cardarelli Hospital, Naples, 4 Ambulatory of Hepatologic Disease, U.O.S.C. n.8 SERT-ASL SA 2, Battipaglia, 5 Department of Internal Medicine, Cardioangiology and Hepatology, University of Bologna, Bologna, 6 Department of Hepatology and Interventional Ultrasound, San Leonardo Hospital, Castellammare di Stabia, 7 Department of Internal Medicine, I Policlinic of Naples, 8 Department of Endocrinology, Second University of Naples, Naples, 9 Biostatistics Department Quintiles, Milan, Italy E-mail: filomena.morisco@unina.it Background: Autoimmune thyroid disorders (ATD) have been widely reported as adverse effects of interferon therapy in patients with chronic hepatitis C (CHC). The incidence, risk factors and the clinical impact of ATD on therapeutic outcomes are however still controversial. Methods: 3414 naive patients were enrolled in the PROBE: an obser- vational study, designed to assess the efficacy and safety of pegylated interferons (PEG-IFN) plus ribavirin (RBV) in a non-selected population of patients with CHC. Clinical, biochemical, and virological parameters were determined at entry, 6 and 12 months after the beginning of treatment and 6 months after the end of therapy. ATD was assessed by the presence of the thyroid antibodies (TAbs)-TgAb and TPOAb. Response to therapy was evaluated by sustained virological response (SVR) (HCV-RNA unde- tectable 6 months after the end of therapy). Results: Before antiviral treatment, 6.0% of patients were positive for TAbs. Over the course of treatment, 5.8% of the patients acquired positivity for the TAbs. Overall, ATD was present in 11.4% of the patients (390/3414). ATD was more frequent in female (55.6% female vs 36.8% male; p < 0.01), in patients with a BMI  30 (15.1% vs 8.6%; p < 0.01), and in patients with antinuclear antibodies (14.9% vs 7.6%; p < 0.01). Multivariate analysis, identified female sex, BMI  30, and ANA positivity as independent predictors of autoimmune thyroiditis.Overall, patients with and without ATD achieved a similar SVR rates (59.2% vs 56.6%). However, the rate of SVR of patients with basal ATD and patients who acquired ATD revealed a significant difference (53.4% and 65.9%, respectively; p < 0.01). According to logistic models, ATD developed during the treatment was an independent predictor of SVR, together with