Resistance selection using glecaprevir and pibrentasvir in replicons of major hepatitis C virus genotypes

POSTER PRESENTATIONS not to miR-21 inhibitors, or in mouse primary hepatocytes isolated from either Flox/flox control or miR21 KO mice, following HCV 3a core protein expression. Viral particle production and HCV replication were investigated in the setting of cell cultured-derived Jc1 HCV infection and with the subgenomic replicon pFK_i389LucNS3 in vitro transcript, respectively. Results: Infection by cultured-derived Jc1 HCV, but not expression of the HCV 3a core protein, slightly increased miR-21 expression in Huh-7 cells. However, in both conditions, miR-21 activity and/or bioavailability were significantly increased. Consistent with these observations, PTEN downregulation and lipid accumulation in cytoplasmic droplets induced by HCV 3a core protein were partially prevented in Huh-7 cells treated with miR-21 inhibitors and in mouse primary hepatocytes isolated from miR-21 knockout mice. Finally, both production of infectious particles and replication step of the HCV life cycle were impaired by miR-21 inhibitor in Huh-7 cells. Conclusions: Altogether, our data indicate that miR-21 activation by viral proteins is a key molecular step in HCV 3a-induced steatosis and in HCV life cycle. THU-305 Resistance selection using glecaprevir and pibrentasvir in replicons of major hepatitis C virus genotypes T. Ng1, L. Lu1, T. Reisch1, P. Krishnan1, G. Schnell1, R. Tripathi1, J. Beyer1, T. Dekhtyar1, M. Irvin1, T. Pilot-Matias1, C. Collins1. 1Abbvie Inc., North Chicago, United States E-mail: [email protected] Background and Aims: Glecaprevir (GLE, NS3/4A protease inhibitor identified by AbbVie and Enanta) and Pibrentasvir (PIB, NS5A inhibitor) are next generation HCV direct-acting antiviral agents. Both inhibitors demonstrate pan-genotypic anti-HCV activity and maintain activity against common amino acid substitutions associated with resistance to earlier generation inhibitors of the respective classes. In this report we present the selection and characterization of amino acid substitutions associated with resistance to GLE or PIB in HCV subgenomic replicons with NS3 and/or NS5A from major HCV genotypes (GTs). Methods: The resistance profile of GLE was determined by colony selection using HCV replicons with NS3 from GT1a, 1b, 2a, 2b, 3a, 4a, or 6a. Similar experiments were conducted with PIB using replicons with NS5A from GT1a, 1b, 2a, 2b, 3a, 4a, 5a or 6a. The level of resistance conferred by the selected substitutions was determined by testing drug susceptibility in transient replicon cells engineered with these substitutions. Results: GLE selected a small number of colonies in most of the GTs tested. The common substitutions selected by GLE in NS3 in GT1a, 1b, 2a and 4a were A156T/V. GLE selected A156G as well as Y56H + Q168R in GT3a replicon cells, and D168G/H/V/Y in GT6a. Substitutions A156T/V in GT1a, 1b, 2a and 4a confer >100-fold resistance to GLE, whereas Q168R in GT3a and D168G/H/V/Y in GT6a confer >35-fold resistance. PIB selected no viable colonies in GT1b, 2b, 4a, 5a and 6a. PIB selected Y93H/N in NS5A in GT1a or 3a; these substitutions confer <7-fold resistance to PIB. In addition, PIB selected 2–3 colonies in GT1a or GT2a replicon cells, each harboring NS5A substitutions resulting from 2 nucleotide changes. Conclusions: GLE and PIB each demonstrated a high genetic barrier to selection of resistance in major HCV GTs. The common resistant substitutions selected by GLE in different GTs were those at position A156, but these substitutions are rarely detected clinically, likely due to their low viral fitness. PIB selected no viable colonies in most of the GTs tested. Of the few substitutions selected by PIB, most of them either confer <7-fold resistance to PIB (e.g. Y93H/N), or were present in very low prevalence because they require overcoming a higher genetic barrier to generate, i.e., 2 nucleotide changes.

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THU-306 Transcriptional response to hepatitis C virus infection in the human liver T. Boldanova1,2, A. Suslov2, M.H. Heim1,2, A. Necsulea3. 1Clinic of Gastroenterology and Hepatology, University Hospital Basel; 2 Department of Biomedicine, University of Basel, Basel; 3School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) remains an important model system to investigate mechanisms of host-virus interactions. Experimental work in vitro generated a wealth of data on cell-intrinsic adaptive responses to HCV that could explain consequences of HCV infection such as inflammation, fibrosis, steatosis, insulin resistance and hepatocellular carcinoma. However, due to limited access to liver tissue, few in vitro data have been rigorously validated in vivo. Such a validation is further complicated by a highly variable interferon (IFN) stimulated gene (ISG) expression that can mask cell-intrinsic changes in infected cells. The first aim of our study was to circumvent these difficulties by carefully selecting patients without an activated endogenous IFN system and comparing their transcriptome with normal liver biopsies. The second part of our study addressed a longstanding conundrum in the field: Despite a strong induction of hundreds of ISGs the endogenous IFN system is ineffective against HCV, whereas therapies with pegIFNa were curative in many patients. Two alternative explanations were discussed: Either some critical ISGs are exclusively induced by pegIFNa, or pegIFNa induces the same ISGs but at a higher level than the endogenous IFNs. Methods: We included 25 patients with CHC and 6 controls. CHC samples were separated into a group with high ISG expression and a group without ISG expression. Patients underwent a second biopsy at one of several time points during the first dosing interval of pegIFNa. RNA-seq data were analyzed with R/ Bioconductor software. Results: We could not detect significant changes in cell-intrinsic pathways involved in stress responses, cell cycle regulation, lipid metabolism, DNA repair in liver biopsies from patients with CHC. The same set of ISGs were induced by pegIFNa and the endogenous IFN system, but with lower induction levels in the latter. Finally, we found that several microRNA precursors, including miR122, are significantly down-regulated in response to IFN treatment, suggesting a new mechanism for fine-tuning IFN induced gene expression. Conclusions: This comprehensive transcriptome analysis of liver biopsy from patients with CHC revealed that HCV has no strong effect on the homeostasis of infected cells, that the endogenous IFN response is qualitatively similar to pegIFNa treatment but too weak to clear the infection and that IFN down-regulates miRNA precursor transcripts, thereby fine-tuning ISG expression. THU-307 Exosomal miRNAs derived from umbilical mesenchymal stem cells inhibit hepatitis C virus infection Z. Qi1, X. Qian1, on behalf of The first author, C. Xu2, S. Fang3, P. Zhao1, Y. Wang4, H. Liu4, W. Yuan2. 1Department of Microbiology, Second Military Medical University; 2Department of Orthopedics, Changzheng Hospital Affiliated to Second Military Medical University; 3 Department of Plastic and Reconstruction, Shanghai Changhai Hospital Affiliated to Second Military Medical University; 4Research Center of Developmental Biology, Second Military Medical University, Shanghai, China E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) infects approximately 3% of the world population. Though the development of direct acting antivirals (DAA) have improved the sustainable virological

Journal of Hepatology 2017 vol. 66 | S95–S332