POSTERS P0677 CIVACIR HEPATITIS C IMMUNE GLOBULIN (HCIG) POTENTLY NEUTRALIZES INFECTION OF HEPATITIS C VIRUS TRANSPLANT ESCAPE VARIANTS 3 R.G. Tawar1,2 , L. Heydmann1,2 , J. Schuttrumpf ¨ , S. Chavan4 , M.B. Zeisel1,2 , T.F. Baumert1,2,5 . 1 Inserm, U1110, Institut de Recherche sur les Maladies Virales et H´epatiques, Strasbourg, France; 2 University of Strasbourg, Strasbourg, France; 3 Biotest AG, Dreieich, Germany; 4 Clinical Research, Medical Affairs and Drug Safety, Biotest Pharmaceuticals, Boca Raton, Florida, United States; 5 Institut Hospitalo-Universitaire, Pˆ ole H´epato-digestif, Strasbourg, France E-mail:
[email protected] 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 phenoype 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 stateof-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. P0678 IL-8 PRODUCING CD4+ REGULATORY T CELLS STIMULATE ANGIOGENESIS IN CHRONIC HEPATITIS C 1 B. Langhans1 , H.-D. Nischalke1 , B. Kramer ¨ , D.J. Kaczmarek1 , P. Lutz1 , C.P. Strassburg1 , J. Nattermann1 , U. Spengler1 . 1 Department of Internal Medicine I, University of Bonn, Germany, Bonn, Germany E-mail:
[email protected] Background and Aims: In chronic hepatitis C regulatory CD4+ T cells (Tregs) inhibit anti-viral T cell response via soluble IL-10 and IL-35 (Langhans et al., Clin Science 2010). In addition, we demonstrated that CD4+ Tregs enhance fibrosis by modulating the interaction of NK cells and hepatic stellate cells (HSC) and stimulating fibrogenesis in HSC by secreting IL-8 (Langhans et al., J Hepatol 2014 in press). Here, we analyzed in how far intrahepatic CD4+ Tregs can stimulated angiogenesis. Methods: Biopsies and explant livers from patients with chronic hepatitis C (cHCV), chronic hepatitis B (cHBV) and alcoholic liver disease (EtOH) were studied by multiple colour immunofluorescence histology on cryostat sections. S576
Numbers of IL-8+ Foxp3+ CD127low CD25+ CD4+ Tregs were determined quantitatively using flow cytometry in freshly isolated liver infiltrating lymphocytes obtained from unfixed parts of the liver samples. Results: Our quantitative ex vivo analysis revealed that HCVspecific Tregs produce marked amounts of IL-8 (MW±SEM: 650±133 pg/ml). Despite equivalent numbers of CD4+ Tregs, IL-8 producing Tregs were exclusively found in the liver of patients with cHCV, but not cHBV or EtOH. In situ we found a close relationship between Tregs and intrahepatic blood vessels indicating that activated Tregs are focally associated with C31+ endothelial cells in HCV-associated cirrhosis. Of note, Tregs attached to the vessels from outside and not the lumen. In the liver tissue of HCVpatients overall number of CD4+ Tregs were significantly enhanced with advanced fibrosis and correlated with IL-8 producing Tregs (R = 0.7501, p < 0.001). Further in vitro experiments demonstrated that HCV-specific Tregs produce additional potent angiogenic factors such as MMP8, MMP9, TIMP-1 and activin-A and serpinE1. Conclusions: Our data propose that intrahepatic CD4+ Tregs are associated with blood vessels in the liver of patients with chronic hepatitis C and thus have the potential to stimulate angiogenesis by producing IL-8. P0679 DERISKING THE POTENTIAL FOR MITOCHONDRIAL TOXICITY OF NUCLEOSIDE ANALOGS Z. Jin1 , J. Deval1 , J.A. Symons1 , H. Tan1 , K. Shaw1 , S.M. Chanda1 , Q. Zhang1 , Y. Tam1 , L.M. Blatt1 , G. Wang1 , N. Dyatkina1 , L. Beigelman1 , D.B. Smith1 . 1 Alios BioPharma, Inc., South San Francisco, United States E-mail:
[email protected] Background and Aims: Nucleoside analogs play an important role in the treatment of multiple viral diseases. Nucleoside analogs that fail clinically do so for multiple reasons, but primarily for lack of selectivity versus human polymerases resulting in mitochondrial toxicity. In general, nucleoside toxicity appears compound specific and does not appear to be associated with any particular base, sugar or phosphate modification. We have established a screening paradigm designed to assess the potential for mitochondrial toxicity of ribonucleoside analogs. We exemplified the strategy using nucleoside analogs previously advanced for the treatment of chronic hepatitis C (CHC), and applied the strategy to novel nucleoside analogs currently being advanced for the treatment of CHC. Methods: The triphosphate derivative (NTP) of each nucleoside analog was prepared and evaluated for the ability to be incorporated by human mitochondrial RNA polymerase (HMRP). Nucleoside analogs were tested in HepG2 cells for 6–8 days for inhibition of the synthesis of the mitochondrial-DNA encoded protein, cytochrome c oxidase I (COX-I), relative to the nuclear DNA encoded mitochondrial protein, succinate dehydrogenase A subunit (SDH-A). Nucleoside analogs were also assessed for general cytotoxicity in 8-day assays across a panel of cell lines. Results: The NTP’s of 4 -azidocytidine, INX-08189, PSI-7851, AL335 and AL-516 all showed potent inhibition of the HCV NS5B polymerase. 4 -azidocytidine and 2 -Me-guanosine NTP’s were shown to be efficient substrates for incorporation by human mitochondrial RNA polymerase, the NTP’s of 2 -Me-2 -F-uridine, AL-335 and AL-516 were not substrates for HMRP (Figure). 4 -azidocytidine and 2 -Me-guanosine prodrugs were inhibitors of human mitochondrial protein synthesis. 2 -Me-2 -F-uridine prodrug PSI-7851, AL-335 and AL-516 did not inhibit human mitochondrial protein synthesis. For most cell lines, only 2 -Me-guanosine prodrugs showed cytotoxicity.
Journal of Hepatology 2015 vol. 62 | S263–S864