- Email: [email protected]
1162 TURMERIC CURCUMIN INHIBITS ENTRY OF ALL HEPATITIS C VIRUS GENOTYPES INTO HUMAN LIVER CELLS
POSTERS suppression of viral replication. The synergistic antiviral effects determined by treating humanized mice with CoPP and peg-IFNa hinted at a potential role for HO-1 induction in antiviral therapy, while providing therapeutic protection of the hepatocytes from HCV-mediated hepatocellular injury. 1162 TURMERIC CURCUMIN INHIBITS ENTRY OF ALL HEPATITIS C VIRUS GENOTYPES INTO HUMAN LIVER CELLS A. Kusuma1 , C.C. Colpitts2 , L.M. Schang2 , H. Rachmawati3 , A. Frentzen1 , S. Pfaender1 , P. Behrendt1 , R.J.P. Brown1 , D. Bankwitz1 , J. Steinmann4 , M. Ott5,6 , P. Meuleman7 , T. Pietschmann1 , E. Steinmann8 . 1 Institute of Experimental Virology, Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany; 2 Department of Biochemistry and Medical Microbiology and Immunology and Li Ka Shing Insitute of Virology, University of Alberta, Edmonton, AB, Canada; 3 School of Pharmacy, Institut Teknologi Bandung, Bandung, Indonesia; 4 Institute of Medical Microbiology, University Hospital Essen, Essen, 5 Translational Research Group, Twincore Centre for Experimental and Clinical Infection Research, 6 5Department of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Hannover, Germany; 7 Center for Vaccinology, Ghent University and Hospital, Ghent, Belgium; 8 Experimental Virology, Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) infection causes severe liver disease and affects more than 160 million individuals worldwide. Individuals undergoing liver organ transplantation face universal reinfection of the graft. Therefore, affordable antiviral strategies targeting the early stages of infection are urgently needed to prevent the recurrence of HCV after liver transplantation. The aim of the study was to determine the antiviral activity of turmeric curcumin, a natural compound with antiviral and anti-cancer activity, against HCV. Methods: The antiviral activity of curcumin and its derivates was evaluated using HCV pseudoparticles (HCVpp) and cell-culture derived HCV (HCVcc) in hepatoma cell lines and primary human hepatocytes. Its mechanism of action was dissected by R18-labelled virions and a membrane fluidity assay. Results: Curcumin treatment had no effect on HCV RNA replication or viral assembly and release. However, co-treatment with curcumin potently inhibited entry of all major HCV genotypes. Similar antiviral activity was also exerted by other curcumin derivates but not by tetrahydrocurcumin suggesting the importance of a,b-unsaturated ketone groups for the antiviral activity. Expression levels of known HCV receptors were unaltered while pre-treating the virus with the compound reduced viral infectivity without viral lysis. Membrane fluidity experiments suggested that curcumin affected the fluidity of the HCV envelope resulting in an impairment of viral binding and fusion. Moreover, Curcumin inhibited cell-to-cell transmission and was effective in combination with other antiviral agents. Conclusion: Turmeric curcumin inhibits HCV entry independent of the genotype and in primary human hepatocytes by affecting the membrane fluidity, and hence impairing the virus binding and fusion ability of HCV.
1163 EDA-STREPTAVIDIN FUSION PROTEIN CONJUGATED TO BIOTINYLATED HCV-NS3 PROTEIN INDUCES STRONG T CELL IMMUNE RESPONSES AGAINST NS3 L. Arribillaga1,2 , M. Martinez1 , L. Villanueva1 , M. Gorraiz1 , M. Durantez1 , F. Rudilla1 , N. Casares1 , T. Lozano1 , B.-C. Francisco1 , P. Sarobe1 , J. Prieto1,3 , J. Lasarte1 . 1 Hepatology and Gene Therapy, CIMA, 2 Formune, 3 CIBERehd, Cl´ınica Universidad de Navarra, Pamplona, Spain E-mail: [email protected] Background and Aims: Recombinant proteins are generally poor immunogens and do not induce strong T cell immune responses if they are not combined with appropriate adjuvants, or modified to improve their capture by dendritic cells (DCs). In previous works we have shown that fusion of an antigen to the extra domain A from fibronectin (EDA) targets the antigen to dendritic cells via TLR4 and improves its immunogenicity. In this work we have prepared a fusion protein between EDA and streptavidin (EDAvidin) to allow its interaction with biotinylated antigens. We have tested the immunogenicity of biotinylated NS3 combined with EDAvidin to induce strong anti-NS3 cellular immune responses as a potential vaccination strategy against HCV infection. Methods: Recombinant EDAvidin, EDA-NS3 fusion protein and NS3 were produced in E. coli and purified by affinity chromatography. NS3 was biotinylated using Sulfo-NHS-biotin reagent. The capacity of EDAvidin to bind to biotinilated proteins was tested by surface Plasmon resonance, by western blot and by ELISA. The efficacy of antigen capture by DC was tested by flow cytometry using biotinylated GFP. Immunogenicity of the recombinant proteins was tested in HHD transgenic mice (expressing human HLA-A2) by ELISPOT and by in vivo killing assays. Results: Recombinant EDAvidin tetramerizes and binds very efficiently to biotynilated proteins. EDAvidin retains the proinflammatory properties of EDA, activating TLR4 signaling pathway. When combined with biotinylated GFP, EDAvidin favours GFP capture by dendritic cells. Immunization of mice with EDAvidin combined with biotinylated NS3 induce a strong anti-NS3 cellular immune responses similar to that induced by the fusion protein EDA-NS3. Conclusions: EDAvidin can bind biotinylated proteins and improve their immunogenicity in vivo. EDAvidin combined with biotinylated NS3 can be considered as a vaccination strategy against hepatitis C virus infection. 1164 INDUCTION OF A PREDIABETIC STATE IN TRANSGENIC MICE EXPRESSING THE HCV PROTEINS H. Lerat1 , A. Gaudin1 , B. Blondeau2 , F. Foufelle2 , C. Magnan3 , J.-M. Pawlotsky1,4 . 1 INSERM U955Eq18, Paris-Est University, Cr´eteil, 2 Centre de Recherche des Cordeliers, UMRS 872 – Paris 6 University, 3 CNRS EAC 4413 Eq2 – University Paris Diderot, Paris, 4 National Reference Center for Viral Hepatitis B, C and Delta, Hˆ opital Henri Mondor, Department of Virology, Cr´eteil, France E-mail: [email protected] HCV infection is an independent risk factor of type-2 diabetes (T2D) in humans. We tried to understand the mechanisms of HCV-related T2D using the FL-N/35 transgenic mouse model expressing the full HCV-ORF. FL-N/35 mice displayed similar baseline glycemia, but significantly higher insulinemia and HOMA-IR than control littermates (WT). Glucose tolerance tests (IPGTT) resulted in significantly higher glycemia levels in transgenic mice, demonstrating that HCV protein expression is associated with glucose intolerance, like in HCVinfected patients. We then assessed whether this pre-diabetic state was due to a defect in b-cell function and/or to insulin resistance (IR).
Journal of Hepatology 2013 vol. 58 | S409–S566
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1163 EDA-STREPTAVIDIN FUSION PROTEIN CONJUGATED TO BIOTINYLATED HCV-NS3 PROTEIN INDUCES STRONG T CELL IMMUNE RESPONSES AGAINST NS3 L. Arribillaga1,2 , M. Martinez1 , L. Villanueva1 , M. Gorraiz1 , M. Durantez1 , F. Rudilla1 , N. Casares1 , T. Lozano1 , B.-C. Francisco1 , P. Sarobe1 , J. Prieto1,3 , J. Lasarte1 . 1 Hepatology and Gene Therapy, CIMA, 2 Formune, 3 CIBERehd, Cl´ınica Universidad de Navarra, Pamplona, Spain E-mail: [email protected] Background and Aims: Recombinant proteins are generally poor immunogens and do not induce strong T cell immune responses if they are not combined with appropriate adjuvants, or modified to improve their capture by dendritic cells (DCs). In previous works we have shown that fusion of an antigen to the extra domain A from fibronectin (EDA) targets the antigen to dendritic cells via TLR4 and improves its immunogenicity. In this work we have prepared a fusion protein between EDA and streptavidin (EDAvidin) to allow its interaction with biotinylated antigens. We have tested the immunogenicity of biotinylated NS3 combined with EDAvidin to induce strong anti-NS3 cellular immune responses as a potential vaccination strategy against HCV infection. Methods: Recombinant EDAvidin, EDA-NS3 fusion protein and NS3 were produced in E. coli and purified by affinity chromatography. NS3 was biotinylated using Sulfo-NHS-biotin reagent. The capacity of EDAvidin to bind to biotinilated proteins was tested by surface Plasmon resonance, by western blot and by ELISA. The efficacy of antigen capture by DC was tested by flow cytometry using biotinylated GFP. Immunogenicity of the recombinant proteins was tested in HHD transgenic mice (expressing human HLA-A2) by ELISPOT and by in vivo killing assays. Results: Recombinant EDAvidin tetramerizes and binds very efficiently to biotynilated proteins. EDAvidin retains the proinflammatory properties of EDA, activating TLR4 signaling pathway. When combined with biotinylated GFP, EDAvidin favours GFP capture by dendritic cells. Immunization of mice with EDAvidin combined with biotinylated NS3 induce a strong anti-NS3 cellular immune responses similar to that induced by the fusion protein EDA-NS3. Conclusions: EDAvidin can bind biotinylated proteins and improve their immunogenicity in vivo. EDAvidin combined with biotinylated NS3 can be considered as a vaccination strategy against hepatitis C virus infection. 1164 INDUCTION OF A PREDIABETIC STATE IN TRANSGENIC MICE EXPRESSING THE HCV PROTEINS H. Lerat1 , A. Gaudin1 , B. Blondeau2 , F. Foufelle2 , C. Magnan3 , J.-M. Pawlotsky1,4 . 1 INSERM U955Eq18, Paris-Est University, Cr´eteil, 2 Centre de Recherche des Cordeliers, UMRS 872 – Paris 6 University, 3 CNRS EAC 4413 Eq2 – University Paris Diderot, Paris, 4 National Reference Center for Viral Hepatitis B, C and Delta, Hˆ opital Henri Mondor, Department of Virology, Cr´eteil, France E-mail: [email protected] HCV infection is an independent risk factor of type-2 diabetes (T2D) in humans. We tried to understand the mechanisms of HCV-related T2D using the FL-N/35 transgenic mouse model expressing the full HCV-ORF. FL-N/35 mice displayed similar baseline glycemia, but significantly higher insulinemia and HOMA-IR than control littermates (WT). Glucose tolerance tests (IPGTT) resulted in significantly higher glycemia levels in transgenic mice, demonstrating that HCV protein expression is associated with glucose intolerance, like in HCVinfected patients. We then assessed whether this pre-diabetic state was due to a defect in b-cell function and/or to insulin resistance (IR).
Journal of Hepatology 2013 vol. 58 | S409–S566
S473