EASL Abstract Highlights – Dr T. Baumert

Posted on April 21, 2015

EASL Abstract O027

The Expression of Tumor Suppressor PTPRD is Down-Regulated in the Liver of Patients With HCV Infection and in Tumor Lesions of Patients With Hepatocellular Carcinoma

Van Renne, F.H.T. Duong, C. Gondeau, D. Calabrese, N. Fontaine, A. Ababsa, S.C. Durand, P. Pessaux, M.H. Heim, T.F. Baumert, J. Lupberger. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

Background and Aims: Chronic hepatitis C virus (HCV) infection is a leading cause of chronic liver disease including hepatocellular carcinoma (HCC). It is generally assumed that HCV contributes to HCC development directly by viral proteins and indirectly by signal transduction. Cellular signaling cascades are involved in cancerogenesis and the regulation of HCV. Signaling is tightly regulated by protein phosphatases and their aberrant expression is involved in various diseases and syndromes. However, the global impact of HCV infection on the expression of phosphatases and their impact on liver disease are unknown. Therefore, we aim to identify the impact of chronic HCV on protein phosphatase expression pattern
Methods: We studied the expression of 84 pathway or disease-focused phosphatases in liver biopsies of chronic HCV infected patients using using real-time PCR, fluorescence in situ hybridization (FISH) and Western blotting.
Results: We identified 29 phosphatases that are significantly (P < 0.01, Mann–Whitney U-test) deregulated. Among the identified hits were several phosphatases associated with cancerogenesis including the tumor suppressor gene phosphotyrosine phosphatase, receptor type D (PTPRD). PTPRD expression did not correlate with the METAVIR score of the studied biopsies suggesting that PTPRD expression is independent from tissue inflammation and fibrosis. PTPRD expression was impaired in HCV-infected liver tissue but not by HBV. Moreover, PTPRD was downregulated in primary human hepatocytes (PHH) infected with HCVcc (JFH1), while IFN-alpha treatment had no impact on PTPRD. Taken together, the data suggest that HCV specifically and directly impairs PTPRD expression in hepatocytes independent from the innate immune response. Furthermore, we demonstrated that PTPRD expression is impaired in transformed liver cell lines and in tumor lesions of HCC patients, which we confirmed by in
silico analysis of a microarray database (Hoshida et al. N. Engl. J. Med. 2008). These data demonstrate an impaired PTPRD expression associated with HCC.
Conclusions: Interestingly, PTPRD dephosphorylates STAT3, which is activated in the majority of HCCs with poor prognosis. We previously demonstrated that STAT3 has an important proviral effect on HCV infection (Lupberger et al., Hepatology 2013). It is thus conceivable that HCV downregulates PTPRD for its replication and immunoevasion with potential consequences for HCC development.

EASL Abstract O014

A Targeted RNAi Screen Using a High-Throughput Infectious Model System Uncovers Glypican GPC5 as a Host Factor For Hepatitis B And D Virus Entry

E.R. Verrier, C. Bach, L. Heydmann, A. Weiss, M. Renaud, P. Fritsch, F. Habersetzer, G. Abou-Jaoude, D. Durantel, C. Schuster, L. Brino, C. Sureau, M.B. Zeisel, T.F. Baumert. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

Background and Aims: Chronic hepatitis B virus (HBV) infection and its co-infection with hepatitis D virus (HDV) are leading causes of liver disease and cancer world-wide. Viral entry is the first step of infection, plays a key role in spread and control of infection and has been shown to be a viable target for the development of curative therapeutic strategies. HBV and HDV viruses infect exclusively hepatocytes and share the same envelope proteins and entry pathway. Heparan sulfate proteoglycans (HSPGs) have been shown to mediate HBV and HDV attachment at the hepatocyte cell surface before interacting with sodium taurocholate cotransporting polypeptide (NTCP). However, the detailed mechanisms of entry and its host cell-dependency factors are still poorly understood. Using a targeted RNAi screen we aimed to investigate the role of HSPG core proteins in HBV/HDV entry.
Methods: We established a high-throughput HDV infection model using Huh7 cells overexpressing NTCP and susceptible to HDV infection. Unlike previous approaches, this system does not require dimethylsulfoxide or polyethylene glycol to facilitate entry, thus providing a model closely mimicking natural infection. Using a small library of siRNAs targeting the core members of the HSPG family, we performed a targeted screen to evaluate the role of individual HSPG core proteins in HDV entry.
Results: While the silencing of the expression of most HSPG core genes did not result in strong modulation of infection, silencing of Glypican GPC5 expression induced a marked and significant decrease of HDV infection as shown by immunofluorescence of HDV infected Huh7-NTCP+ cells and RT-PCR of HDV RNA. Using HepG2 cells overexpressing NTCP and individual siRNAs, we demonstrate that GPC5 silencing is a host cell-dependency factor for HBV infection. Silencing of GPC5 in HepG2-NTCP cells resulted in a marked decrease of both HBV-positive cells and HBV pregenomic RNA, confirming the functional role of this host cell surface protein for both HDV and HBV infection.
Conclusions: Collectively, this targeted RNAi screen in a high throughput infectious model system uncovers GPC5 as an entry factor for hepatitis B and D viruses. These results advance our understanding of cell entry of HBV and HDV and open new avenues for therapies aiming at HBV cure. Since glypicans have been shown to play a role in the control of cell division and growth regulation, virus-GPC5 interactions may also play a role for pathogenesis of virus-induced liver disease.

EASL Abstract P0676

Clearance of Persistent Hepatitis C Virus Infection Using a Monoclonal Antibody Specific For Tight Junction Protein Claudin-1

Mailly, F. Xiao, J. Lupberger, G.K. Wilson, P. Aubert, F.H. Duong, D. Calabrese, C. Leboeuf, I. Fofana, C. Thumann, S. Bandiera, M. Lutgehetmann, T. Volz, C. Davis, H.J. Harris, C. Mee, E. Girardi, B. Chane-Woon-Ming, M. Ericsson, N. Fletcher, R. Bartenschlager, P. Pessaux, K. Vercauteren, P. Meuleman, P. Villa, L. Kaderali, S. Pfeffer, M.H. Heim, M. Neunlist, M.B. Zeisel, M. Dandri, J.A. McKeating, E. Robinet, T.F. Baumert. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

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 HCV infected 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.

EASL Abstract P0677

Civacir Hepatitis C Immune Globulin (HCIG) Potently Neutralizes Infection of Hepatitis C Virus Transplant Escape Variants

R.G. Tawar, L. Heydmann, J. Schuttrumpf, S. Chavan, M.B. Zeisel, T.F. Baumert. The International Liver Congress™ 2015, 50th annual meeting of the European Association for the Study of the Liver, Vienna, Austria, April 22-26, 2015.

Background and Aims: Hepatitis C virus (HCV) induced endstage liver disease is the major indication for liver transplantation in most countries. However, re-infection of the liver graft is universal. The safety and efficacy of DAAs for prevention of liver graft infection remains to be determined. Biotest Pharmaceutical’s Civacir, a human hepatitis C antibody enriched immune globulin product (HCIG), has been shown to efficiently prevent liver graft infection in a phase III RCT (Terrault et al. AASLD 2014). Using well characterized patient-derived HCV transplant escape variants (Fofana et al. Gastroenterology 2012; Felmlee/Fauvelle et al. EASL 2014) we aimed to study the molecular mechanism of action of HCIG/Civacir.
Methods: Inhibition of Civacir/HCIG-mediated HCV infection was studied using 25 viral variants isolated from patients before and after liver transplantation and state-of-art HCV cell culture models. HCV pseudoparticles (HCVpp) and cell-culture-derived HCV (HCVcc) expressed patient-derived viral envelope glycoproteins from transplant escape variants.
Results: Civacir potently, broadly and dose-dependently neutralized all the patient variants in HCVpp assays including variants displaying a high entry phenotype and resistant to host neutralizing antibodies. The IC50 values (0.0016–1.50 mg/ml) for inhibition were independent of the phenotype of the viral variant indicating that virus neutralization by Civacir/HCIG is not affected by viral escape. Furthermore, at clinically relevant antibody concentrations Civacir potently neutralized HCVcc variants bearing envelopes efficiently escaping patient-derived and monoclonal anti-E2 antibodies.
Conclusions: Patient-derived HCV escape variants resistant to autologous antibodies are potently neutralized by Civacir in state-of-the-art HCV cell culture models. The potent activity of Civacir is likely because of synergy between anti-HCV antibodies derived from different patient donors. Collectively, these results uncover the mechanism of action of HCIG/Civacir and explain its clinical efficacy for prevention of HCV liver graft infection.

Thomas F. Baumert, MD is Professor of Medicine, head of Inserm Research Unit U1110, Institute of Viral and Liver Diseases, head of the Laboratory of Excellence HEPSYS at the University of Strasbourg and Physician at the Center for Digestive Disease and Hepatology at the Strasbourg University Hospitals.

He received his MD from the University of Heidelberg, Germany. Following his doctoral thesis at the German Cancer Research Center (DKFZ) in Heidelberg (headed at that time by Nobel laureate Prof. H. zur Hausen) and his internship in Internal Medicine at the Ludwig Maximilians University in Munich, he was a postdoctoral fellow in Dr. T. J. Liang’s laboratory at the Dept. of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston and the Liver Diseases Branch at the National Institutes of Health, Bethesda,USA. He subsequently joined the Department of Medicine at the University Hospital in Freiburg, Germany to become a board-certified internist and gastroenterologist, associate professor and to establish his laboratory focussing on the molecular pathogenesis of hepatitis B and C virus infection. In 2006, he relocated to the University of Strasbourg in France as full professor of medicine to create and head a new Inserm research unit on virus-host interactions and liver disease and establish a highly recognized program on translational virology and hepatology. Most recently he spent a sabbatical as a research scholar at Mass General Hospital, the BROAD institute and Harvard Medical School, Boston.His laboratory focuses on the modelling and discovery of the biological circuits underlying viral persistence and virus-induced disease such as chronic inflammation and cancer. As a model he uses chronic hepatitis B and C virus infection – a major cause of chronic liver disease and cancer world-wide. The understanding of the molecular mechanisms of virus-host interactions and disease biology has allowed his laboratory to uncover novel strategies for prevention and treatment of chronic viral hepatitis. He has received several awards including the Hans Popper Young Investigator Award, the Chair of Excellence and Laboratory of Excellence Awards of the French National Research Agency and the Infectious Disease Award of the German Infectious Disease Society. Most recently he has been awarded the Prix Galien (2013) for the discovery of host-targeting entry inhibitors as a novel strategy for prevention and treatment of HCV infection. He has published more than 150 scientific articles including Nature Medicine, N. Engl. J. Med., Cell Host & Microbe, PNAS, J. Clin. Invest. and J. Exp. Med.. He is an inventor on 11 patents and patent applications and scientific founder of Vironexx, a biotech company focussing on discovery and preclinical development of host-targeting antivirals. In his translational work he focuses on novel approaches for prevention and treatment of virus-induced liver disease. Research in his laboratory is supported by the European Commission through the ERC, FP7 and INTERREG IV programs, Inserm, the Agence Nationale de Recherches sur le Sida, hépatites virales B et C (ANRS) and the Agence Nationale de Recherche (ANR), France as well as industry. He is an Associate Editor for the Journal of Hepatology (IF 9.8) and serves as a member of the editorial board of several journals in the field of Hepatology and Virology. He actively participates in structuring national and international research programs in Virology and Hepatology by serving as on various review and policy committees at the European Association for the Study of Liver Disease (EASL) as well as Inserm, CNRS, and ANRS France (ATIP-AVENIR).

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