- Email: [email protected]
P0675 : An ex vivo model of human liver slices culture for evaluating liver fibrogenesis
POSTERS flunarizine. Shuffling structural proteins between GT1a, GT2b and GT2a chimeric viruses revealed that GT2a-derived E1-E2 genes confer susceptibility to flunarizine. Resistance mutations map to the viral envelope proteins, thus indicating that flunarizine targets glycoprotein function(s). Pretreatment of target cells, but not of virus particles inhibited HCV entry and time of addition experiments indicated that flunarizine acts at a late stage of entry. Since flunarizine interfered with HCV infection through low-pHtriggered fusion at the plasma membrane, we conclude it inhibits HCV independent of endocytosis. Finally, trans-complemented HCV particles (HCV-TCP) carrying glycoproteins from GT2a patients were inhibited by flunarizine and flunarizine-resistant viruses were more prone to neutralization by patient-derived antibodies. Conclusions: Flunarizine may be an alternative treatment option for GT2a- and possibly other GT2-subtype infected individuals. P0675 AN EX VIVO MODEL OF HUMAN LIVER SLICES CULTURE FOR EVALUATING LIVER FIBROGENESIS 2 S. Lagaye1 , J. Gaston1 , J. Guechot ´ , P.-P. Massault3 , J.-C. Vaillant4 , S. Pol1,5 . 1 INSERM UMS20, Institut Pasteur, 2 Pˆ ole de Biologie M´edicale et Pathologie, APHP, Hˆ opital Saint-Antoine, 3 Service de Chirurgie digestive, H´epato-biliaire et Endocrinienne, AP-HP, Groupe Hospitalier Cochin, 4 Service de Chirurgie digestive et H´epato bilio pancr´eatique, APHP, Groupe Hospitalier La Piti´e Salp´etri`ere, 5 Unit´e d’H´epatologie, APHP, Groupe Hospitalier Cochin, Paris, France E-mail: [email protected] Background and Aims: We extended our ex-vivo model of human liver slices culture infected with HCV (Hepatology 2012) from 10 to 21 days and evaluated the markers of fibrosis in HCV-infected liver slices as compared to uninfected liver slices (CRTL) in presence of Ethanol or not. Methods: Non-infected liver slices, obtained from human liver resection and cut in 350 mm-thick slices (2.7×106 cells per slice), were cultivated for up to 21 days: either in presence or not of ethanol (EtOH) (1mM, 5mM, 25mM), or infected with HCVcc supernatant [Con1/C3 (genotype1b)] (MOI = 0.1) in presence or not of ethanol (1mM, 5mM, 25mM). Expression of liver phenotypic markers and fibrosis markers [Tumor growth factor beta (TGFb), Heat shock protein 47 (Hsp47), Alpha smooth muscle actin (aSma) and Procollagen1 A1 (Procl1A1)] were checked by RT-qPCR. The amount of Hyaluronic acid and TGFb were determined by ELISA assays. Results: Hepatocyte-specific gene expression is maintained in human non infected liver slices culture for 21 days. In uninfected liver slices treated with EtOH, the gene expression of TGFb, Hsp47, aSma and Procl1A1 increased overtime, in a dose-dependent manner up to 3, 3, 4, 4 times, respectively, at day 21, at 25mM EtOH, compared to uninfected non treated control liver slices. Human infected liver slices replicated, expressed efficiently HCVcc and produced high titers of progeny virions (HCVpc) by day 21. In HCV infected liver slices, the gene expression of TGFb, Hsp47, aSma and Procl1A1 were increasing overtime and exhibited at day 21, a significant 3, 3.5, 3.6, 2.6 fold increase, respectively as compared to uninfected liver slices. At day 10, the amount of hyaluronic acid (HA) reached 799 mg/L, more than a 7-fold increase compared to uninfected slices. At day 21, the expression of the TGFb protein, intracellular and in the culture supernatant, were significantly 4 and 2 times increased (up to 152 pg/mg tissue and 1100 pg/mg tissue, respectively) as compared to uninfected liver slices. In HCV infected liver slices treated with EtOH, the gene expression of TGFb, Hsp47, aSma and Procl1A1 increased significantly in a dose-dependent manner, up to day 21, around 5, 6, 7, 5 times, respectively, at 25mM EtOH, compared to controls. Conclusions: This ex vivo model, supporting hepatocyte-specific gene expression for 21 days, provides a powerful tool for studying
the onset of fibrosis and for evaluating the potency of new antifibrotic therapies in the native tissue. P0676 CLEARANCE OF PERSISTENT HEPATITIS C VIRUS INFECTION USING A MONOCLONAL ANTIBODY SPECIFIC FOR TIGHT JUNCTION PROTEIN CLAUDIN-1 L. Mailly1,2 , F. Xiao1,2 , J. Lupberger1,2 , G.K. Wilson3 , P. Aubert4,5,6 , F.H. Duong7 , D. Calabrese7 , C. Leboeuf1,2 , I. Fofana1,2 , C. Thumann1,2 , 8 S. Bandiera1,2 , M. Lutgehetmann ¨ , T. Volz8 , C. Davis3 , H.J. Harris3 , C. Mee3 , E. Girardi2,9 , B. Chane-Woon-Ming2,9 , M. Ericsson10 , N. Fletcher3 , R. Bartenschlager11,12 , P. Pessaux1,2,13 , K. Vercauteren14 , P. Meuleman14 , P. Villa2,15 , L. Kaderali16 , S. Pfeffer2,9 , M.H. Heim7 , M. Neunlist4,5,6 , M.B. Zeisel1,2 , M. Dandri8 , J.A. McKeating3 , E. Robinet1,2 , T.F. Baumert1,2,13 . 1 Inserm U1110, 2 Universit´e de Strasbourg, Strasbourg, France; 3 Institute for Biomedical Research, University of Birmingham, Birmingham, United Kingdom; 4 Inserm U913, 5 Universit´e de Nantes, 6 Institut des Maladies de l’Appareil Digestif, CHU Nantes – Hˆ opital Hˆ otel-Dieu, Nantes, France; 7 Department of Biomedicine, University of Basel, Basel, Switzerland; 8 Department of Internal Medicine, University of Hambourg, Hambourg, Germany; 9 Architecture et R´eactivit´e de l’ARN, Institut de Biologie Mol´eculaire et Cellulaire – UPR9002, Strasbourg, France; 10 Electron Microscopy Facility, Harvard Medical School, Boston, United States; 11 Department of Infectious Diseases – Molecular Virology, 12 German Center for Infection Research, Heidelberg University, Heidelberg, Germany; 13 Pˆ ole H´epato-Digestif – Insitut Hospitalo-Universitaire, Hˆ opitaux Universitaires de Strasbourg, Strasbourg, France; 14 Center for Vaccinology, Ghent University and Hospital, Ghent, Belgium; 15 Plateforme de Chimie Biologique Int´egrative de Strasbourg, UMS 3286 CNRS-UdS & FMTS, Illkirch, France; 16 Institute for Medical Informatics and Biometry, Technische Universit¨ at Dresden, Dresden, Germany E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer. Although direct-acting acting antivirals have revolutionized treatment, several challenges remain: these include treatment of certain genotypes, advanced liver disease, resistance and liver graft infection. Tight junction (TJ) proteins claudin-1 and occludin mediate cell entry of HCV. However, the role of TJ proteins as therapeutic target is unknown. Methods: Using a human liver-chimeric mouse model combined with advanced in situ imaging and mechanistic studies we investigated the role of TJ protein claudin-1 as a therapeutic target for HCV infection. Results: Here we report that a monoclonal antibody specific for TJ protein claudin-1 eliminates chronic HCV infection with undetectable resistance and toxicity in a human liver chimeric mouse model. In contrast to DAAs the claudin-1 specific antibody can cure infection in monotherapy. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Importantly, antibody treatment reduces the frequency of HCVinfected hepatocytes in vivo, highlighting the need for de-novo infection via host entry factors to maintain chronic infection. Conclusions: We demonstrate that an antibody targeting a virus receptor can cure chronic HCV infection and uncover TJ proteins as targets for antiviral therapy. This host-targeting strategy provides a simple approach for prevention of liver graft infection and opens a novel perspective for treatment of drug resistance.
Journal of Hepatology 2015 vol. 62 | S263–S864
S575
the onset of fibrosis and for evaluating the potency of new antifibrotic therapies in the native tissue. P0676 CLEARANCE OF PERSISTENT HEPATITIS C VIRUS INFECTION USING A MONOCLONAL ANTIBODY SPECIFIC FOR TIGHT JUNCTION PROTEIN CLAUDIN-1 L. Mailly1,2 , F. Xiao1,2 , J. Lupberger1,2 , G.K. Wilson3 , P. Aubert4,5,6 , F.H. Duong7 , D. Calabrese7 , C. Leboeuf1,2 , I. Fofana1,2 , C. Thumann1,2 , 8 S. Bandiera1,2 , M. Lutgehetmann ¨ , T. Volz8 , C. Davis3 , H.J. Harris3 , C. Mee3 , E. Girardi2,9 , B. Chane-Woon-Ming2,9 , M. Ericsson10 , N. Fletcher3 , R. Bartenschlager11,12 , P. Pessaux1,2,13 , K. Vercauteren14 , P. Meuleman14 , P. Villa2,15 , L. Kaderali16 , S. Pfeffer2,9 , M.H. Heim7 , M. Neunlist4,5,6 , M.B. Zeisel1,2 , M. Dandri8 , J.A. McKeating3 , E. Robinet1,2 , T.F. Baumert1,2,13 . 1 Inserm U1110, 2 Universit´e de Strasbourg, Strasbourg, France; 3 Institute for Biomedical Research, University of Birmingham, Birmingham, United Kingdom; 4 Inserm U913, 5 Universit´e de Nantes, 6 Institut des Maladies de l’Appareil Digestif, CHU Nantes – Hˆ opital Hˆ otel-Dieu, Nantes, France; 7 Department of Biomedicine, University of Basel, Basel, Switzerland; 8 Department of Internal Medicine, University of Hambourg, Hambourg, Germany; 9 Architecture et R´eactivit´e de l’ARN, Institut de Biologie Mol´eculaire et Cellulaire – UPR9002, Strasbourg, France; 10 Electron Microscopy Facility, Harvard Medical School, Boston, United States; 11 Department of Infectious Diseases – Molecular Virology, 12 German Center for Infection Research, Heidelberg University, Heidelberg, Germany; 13 Pˆ ole H´epato-Digestif – Insitut Hospitalo-Universitaire, Hˆ opitaux Universitaires de Strasbourg, Strasbourg, France; 14 Center for Vaccinology, Ghent University and Hospital, Ghent, Belgium; 15 Plateforme de Chimie Biologique Int´egrative de Strasbourg, UMS 3286 CNRS-UdS & FMTS, Illkirch, France; 16 Institute for Medical Informatics and Biometry, Technische Universit¨ at Dresden, Dresden, Germany E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer. Although direct-acting acting antivirals have revolutionized treatment, several challenges remain: these include treatment of certain genotypes, advanced liver disease, resistance and liver graft infection. Tight junction (TJ) proteins claudin-1 and occludin mediate cell entry of HCV. However, the role of TJ proteins as therapeutic target is unknown. Methods: Using a human liver-chimeric mouse model combined with advanced in situ imaging and mechanistic studies we investigated the role of TJ protein claudin-1 as a therapeutic target for HCV infection. Results: Here we report that a monoclonal antibody specific for TJ protein claudin-1 eliminates chronic HCV infection with undetectable resistance and toxicity in a human liver chimeric mouse model. In contrast to DAAs the claudin-1 specific antibody can cure infection in monotherapy. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Importantly, antibody treatment reduces the frequency of HCVinfected hepatocytes in vivo, highlighting the need for de-novo infection via host entry factors to maintain chronic infection. Conclusions: We demonstrate that an antibody targeting a virus receptor can cure chronic HCV infection and uncover TJ proteins as targets for antiviral therapy. This host-targeting strategy provides a simple approach for prevention of liver graft infection and opens a novel perspective for treatment of drug resistance.
Journal of Hepatology 2015 vol. 62 | S263–S864
S575