875 IDENTIFICATION AND PROFILE OF POTENT AND SELECTIVE INHIBITORS OF HCV NS5A PROTEIN

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Poster Session − Saturday, April 25

872 HCV-CORE PROTEIN MODULATES CYTOTOXIC T CELL RESPONSE THROUGH PD1/PD-L1 PATHWAY S. Benito1 , T. Parra-Cid1 , J. Miquel2 , E. Sanz-de-Villalobos2 , M. Calvino1 , M.L. Garc´ıa-Buey3 , F. Gonz´alez-Mateos2 , A. Gonz´alezPraetorius4 , S. Albertos5 , J.R. Larrubia1,2 . 1 Liver Research Unit, 2 Department of Gastroenterology, Guadalajara University Hospital. University of Alcal´a, Guadalajara, 3 Department of Gastroenterology, La Princesa University Hospital. Autonomous University, Madrid, 4 Department of Microbiology, Guadalajara University Hospital. University of Alcal´a, Guadalajara, 5 Department of Gastroenterology, Fundaci´on Jim´enez-D´ıaz. Autonomous University, Madrid, Spain E-mail: [email protected] Background and Aims: Certain viral proteins could exert an immunomodulatory effect on cytotoxic T cell response through negativecostimulatory molecule up-regulation. The role of HCV-core protein to modulate PD1/PD-L1 pathway on CD8+ cells was analysed. Methods: Peripheral blood lymphocytes (PBL) from 37 hepatitis C virus (HCV) infected subjects and 12 healthy controls were studied. Peripheral HCV-core protein concentration was estimated from HCV viral load (One HCV-core pg/mL is equivalent to 8.000 HCV-RNA IU/mL). Directly exvivo, PBL were stained with anti-CD8 and anti-PD1 mAbs. PD-1 mean fluorescence intensity (MFI) was correlated with the estimated peripheral HCV-core levels. PD-1 expression on CD8+ cells from healthy controls after stimulation with CD3/CD28 mAbs, in the presence of either HCVcore or B-gal proteins, was analysed. The effect of HCV-core induced PD-1 up-regulation on CD8+ cell proliferation ability, according to PD-1/PD-L1 blocking, was also tested. In 3 HLA-A2+ HCV infected subjects, the role of HCV core protein in modulating PD-1 expression on HCV-specific CD8+ cells, and its effect on proliferation after specific in-vitro challenge, according to PD-1/PD-L1 blocking, were also analysed. HCV-specific CD8+ cells were visualised by staining with HLA-A2/peptide multimeric complexes. Proliferation ability was assessed by Ki-67 expression on total and HCV-specific CD8+ cells. Stained T cells were analysed by flowcytometry. Non-parametric tests were used for statistical analysis. Results: A significant positive correlation between HCV-core protein level and PD1 MFI was found (r = 0.501; p < 0.001). Specific and nonspecific TCR stimulation in the presence of HCV-core protein noticeably enhanced PD-1 expression on CD8+ cells with respect to incubation with B-gal (p < 0.05). PD-1 up-regulation on total and HCV-specific CD8+ cells, induced by incubation with HCV-core, translated into a significant proliferation impairment after TCR stimulation (p < 0.01), which was restored by PD-1/PD-L1 blocking with anti-PD-L1 mAb (p < 0.05). Conclusions: HCV-core protein level correlates with PD-1 expression on CD8+ cells in chronic hepatitis C (CHC). HCV-core protein enhances PD-1 expression, primarily induced by TCR engagement. This mechanism correlates with proliferation impairment, which is restored by PD1/PD-L1 pathway blocking. All these data suggest that HCV-core protein could impair CD8+ cell function in CHC infection through PD1/PD-L1 pathway. 873 LOW PRE-TREATMENT NUMBERS OF CD4+/PD-1+ LYMPHOCYTES AND T REGULATORY LYMPHOCYTES PREDICT SUCCESSFUL RESPONSE TO PEGYLATEDINTERFERON/RIBAVIRIN THERAPY IN CHILDREN WITH CHRONIC HEPATITIS C (CH-C) I. Carey, D. Cebecauerova, S. Bansal, M.S. Longhi, P. Subramaniam, G. Mieli-Vergani, D. Vergani. Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, London, UK E-mail: [email protected] Background: Chronic hepatitis C virus (HCV) infection in children is a slowly progressive mild disease with potentially severe acceleration in adulthood. Effective therapy with pegylated-interferon (Peg-IFN) and ribavirin prevents disease progression. Successful control of viral replication depends on efficient immune reactivity involving several types of cells: expression of the programmed death (PD-1) receptor indicates that

immune cells are exhausted and have limited anti-viral function; low frequency of CD127+cells is linked to viral persistence; T regulatory cells (T-regs) (CD4+CD25+FOXP3+) are thought to dampen HCV-specific immune responses. Aims: To investigate whether pre-treatment numbers of CD4+/PD-1+, CD4+/CD127+ and CD4+CD25+FOXP3+ cells and baseline HCV-specific immune responses can predict outcome of therapy with Peg-IFN/ribavirin in children with CH-C. Patients: Eighteen children (9 males, median age 13.5 years) with perinatally acquired CH-C were treated with Peg-IFN/ribavirin. Patients were divided into two groups according to treatment response: 13 responders (R) and 5 non-responders (NR). Five HBV-infected children (4 males, median age 14.2 years) served as controls. Methods: The pre-treatment numbers of CD4+/PD-1+, CD4+/CD127+ and CD4+CD25+FOXP3+ cells were determined by flow-cytometry on peripheral blood mononuclear cells (PBMC). Baseline production of IFN-g and IL-10 in PBMC after exposure to genotype specific HCV antigens (core, NS3, NS4 and NS5) and non-specific stimuli (phytohemagglutinin, bacterial lipopolysacharide and tetanus toxoid antigen) was evaluated by Elispot and intracellular cytokine staining. Results: Baseline numbers of CD4+/PD-1+ cells and of T-regs were lower in R than NR (6.5±0.8 vs. 9.8±1.0%, p = 0.04; 1.2±0.5 vs.2.3±0.4%, p = 0.01 respectively), while the number of CD4+CD127+ and IFN-g producing cells after stimulation with HCV antigens was similar in R and NR. In contrast, number of IL-10 producing cells after stimulation with HCV core was higher in NR than R (CD4+/IL-10+: 2.1±0.3 vs. 3.7±0.9%, p = 0.04). The number of CD4+/PD-1+ cells correlated with the numbers of T-regs (r = 0.44, p = 0.04) and HCV-core specific IL-10 producing cells (r = 0.51, p = 0.03). Conclusions: Low pre-treatment numbers of CD4+/PD-1+, T-regs and low HCV-specific IL-10 producing cells are associated with successful antiviral treatment and may predict therapy response. 874 Withdrawn

875 IDENTIFICATION AND PROFILE OF POTENT AND SELECTIVE INHIBITORS OF HCV NS5A PROTEIN R. Colonno1 , M. Bencsik1 , E. Peng1 , N. Huang1 , J. Williams1 , M. Zhong1 , A. Huq1 , R. Wobbe2 , R. Fathi2 , L. Li1 . 1 Presidio Pharmaceuticals, San Francisco, CA, 2 XTL Biopharmaceuticals, Valley Cottage, NY, USA E-mail: [email protected] Background: With response rates of ~50% and tolerance issues associated with standard of care treatments involving the combination of pegylated IFN and ribavirin, there is a clear need for potent, small molecule inhibitors that target multiple viral targets and can be administered orally in future combination therapies. Here we report the discovery and preclinical profile of a series of novel NS5A inhibitors. Methods: HCV antiviral potency assessments, combination studies and resistance emergence studies utilized standard 3-day HCV replicon systems (genotype 1a and 1b). Initial preclinical pharmacokinetic screening was determined following both IV and PO single dosing in rats at 2 and 10 mg/kg, respectively, along with an assessment of liver concentrations. For spectrum testing, a series of HCV 1b replicon constructs were made in which the NS5A gene of HCV 1b was replaced with the NS5A genes of other HCV genotypes. Results: Through medicinal chemistry efforts, an advanced series of lead inhibitors of NS5A were identified that exhibited potent and selective activity (EC50 < 1 nM) in the both the HCV 1a and 1b replicon assays. Antiviral activity was maintained against other HCV genotypes beyond 1a and 1b in transient replicon assays. No significant activity was observed against control viruses such as BVDV. Cellular cytotoxicity levels (CC50) were in excess of 10 uM in several cell lines, yielding a selectivity index of >10,000. NS5A inhibitors were additive in combination studies involving IFN and inhibitors of other viral proteins. Lead compounds showed oral

05c: VIRAL HEPATITIS − c) HEPATITIS C − EXPERIMENTAL (IMMUNOLOGY) bioavailability in animal PK studies, with elevated liver concentrations relative to serum levels, and potential for once daily dosing in humans. Resistance selection studies indicated that high level resistance will likely require multiple substitutions within the NS5A gene. Resistance emergence appeared relatively slowly compared to parallel studies with NS3 inhibitors, suggestive of a higher resistance barrier. Structurally different inhibitors exhibited a range of sensitivities to emerging resistance substitutions. An extensive characterization of these compounds is underway. Conclusion: A series of highly potent and selective NS5A inhibitors have been identified which demonstrated an in vitro and in vivo profile supportive of their advancement and development as clinical candidates. 876 THE C-TERMINUS OF THE HEPATITIS C VIRUS CORE PROTEIN IS ESSENTIAL FOR THE IMMUNOGENICITY OF PRECEDING CD8+ T CELL EPITOPES C. Flechsig, G. Adler, N. Dikopoulos. Department of Internal Medicine I, University Medical Center Ulm, Ulm, Germany E-mail: [email protected] Background and Aims: Infection with the Hepatitis C Virus (HCV) leads to chronic infection in the majority of patients. Still, factors that lead to poor outcome of immune responses against the virus are incompletely understood. The aim of the study was to elucidate differences regarding T cell immunogenicity between different forms of the HCV core protein (HCVc) which occur naturally during infection. Methods: Mice that express the human MHC class I molecule HLAA2 were immunized by intramuscular injection of DNA vaccines coding for different naturally occuring forms of the HCV core protein. After 12 days antigen-specific CD8+ T cell responses were determined by peptide restimulation of spleen cells. A peptide library consisting of overlapping peptides covering the amino acid sequence of the core protein of the HCV genotype 1a was utilized to identify HLA-A2-restricted epitopes. Induction of CD8+ T cell responses against the identified epitopes by the different forms of HCVc was determined. Results: DNA vaccination induced robust HLA-A2 restricted CD8+ T cell responses against the HCVc. The dominant epitope by far was the HCVc132−140 sequence. Besides, T cells recognizing the HCVc35−44 sequence could be identified. Immunization with cationic peptides containing these sequences confirmed the epitopes. C-terminal deletion mutants of HCVc induced less specific CD8+ T cells than the complete protein; the shorter the protein the less specific CD8+ T cells were induced. This effect could not be rescued by stabilisation of antigen expression by fusion of the HCVc to the HSP-binding T-antigen of the SV40 virus. Conclusion: The C-terminus of the HCV core protein is essential for the immunogenicity of CD8+ T cell epitopes located on the preceding part of the protein. The loss of immune induction is not due to impaired antigen expression, as stabilisation of the HCVc through fusion to an HSP-binding domain does not rescue induction of T cell responses. 877 INCREASED REGULATORY POTENTIAL AND IMBALANCE OF THE VDELTA1/VDELTA2 RATIO IN PATIENTS WITH DIFFERENT MANIFESTATIONS OF HEPATITIS C S. Ferri1 , R. Menichella1 , M. Bassi2 , C. De Molo1 , A.C. Dall’Aglio1 , A. Granito1 , G. Pappas1 , C. Quarneti1 , P. Muratori1 , A. Belardinelli3 , F.B. Bianchi1 , M. Lenzi1 , L. Muratori2 . 1 Department of Clinical Medicine, University of Bologna, 2 Department of Clinical Pathology, 3 Department of Immuno-Haematology, S. Orsola-Malpighi Hospital, Bologna, Italy E-mail: [email protected] Introduction: Hepatitis C virus (HCV)-related chronic liver disease (CLD) is characterised by a variety of clinical expressions, ranging from mild hepatitis to extra-hepatic manifestations such as symptomatic mixed cryoglobulinemia. Aim: To determine frequency and function of peripheral regulatory T cells expressing CD4+CD25+hiFOXP3+ (T-reg) and of gd T cell subsets in patients with different manifestations of HCV-related CLD.

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Patients and Methods: We studied 43 HCV RNA-positive patients [HCV] not on antiviral treatment (14 with normal ALT [N], 24 with ALT >3× upper normal levels [A] and 5 with symptomatic cryoglobulinemia [C]; median age 60 years, 51% females) and 19 healthy controls [HC] (median age 41 years, 42% females). Surface (CD4, CD25, CD3, gd TCR, Vd1, Vd2) and intracellular (FOXP3) markers were evaluated by flow cytometry on fresh whole blood. T-regs and CD4+CD25- cells from 6 [HCV] and 6 [HC] were separated using immuno-magnetic beads. CD4+CD25- cells were cultured alone and at 4/1 ratio together with T-regs; CD4+CD25cell proliferation and IFN-g production were evaluated by flow cytometry after five days culture. Results: T-regs from [N] showed increased FOXP3 expression compared to [A] (mean fluorescence intensity, MFI, 2603 vs 2122 respectively, p = 0.027); T-regs from [HCV] were more effective than those from [HC] in inhibiting both target cell proliferation (84% vs 45%, p = 0.008) and IFN-g production (55% vs 20%, p = 0.08). Despite a numerically similar global gd population, in [A] the Vd1/Vd2 ratio was inverted compared to [HC] (1.90 vs 0.50, p < 0.001), due to a reduced Vd2 pool (1.8% vs 4.9% of circulating CD3, p = 0.038) together with an enriched Vd1 subset (2.2% vs 1.2% of circulating CD3, p = 0.022); [N] showed a Vd1/Vd2 ratio similar to [HC] (0.57) and lower than [A] (p = 0.01) whereas [C] presented with an even more evident imbalance (11.22, p = 0.002 vs [HC]). Conclusion: The increased regulatory potential displayed by [HCV] cells suggests an impaired immunological response, possibly leading to the persistence of the infection. The inverted Vd1/Vd2 ratio in patients with active disease as [A] and [C] points to the disruption of gd T cell balance in the pathogenesis of inflammation.

878 HCV-RELATED MIXED CRYOGLOBULINEMIA AND BAFF PROMOTER POLYMORPHISM C. Giannini, L. Gragnani, A. Piluso, P. Caini, A. Petrarca, M. Monti, G. Laffi, A.L. Zignego. MASVE Center − Department of Internal Medicine, University of Florence, Florence, Italy E-mail: carlo.giannini@unifi.it Mixed cryoglobulinemia(MC), a benign, but pre-lymphomatous condition, is strongly associated with Hepatitis C virus (HCV) infection. In fact, a majority of MC patients exhibit HCV markers (>90%) and approximately 50% of HCV patients exhibit a wide range of MC markers/symptoms. Several pathogenetic mechanisms for HCV-related MC have been proposed, but the reasons why cryoglobulins appear in only half of all HCV patients are still unclear. Suggested key determinants include host genetic background or viral factors. B-cell activating factor (BAFF), a TNF-a family member, plays an essential role in B-lymphocyte development and survival. Impairment of BAFF regulation has been associated with human autoimmune disorders (Sj¨ogren syndrome, SLE and rheumatoid arthritis). Recent reports found elevated serum BAFF levels in HCV patients with B-cell lymphoproliferative disorders (MC and B cell non-Hodgkin lymphoma). A polymorphism −871C/T was detected in the BAFF promoter, and the presence of the mutated −871T genotype was associated with higher BAFF mRNA levels in monocytes. Molecular studies showed an increased transcriptional activity of the mutated promoter. The aim of the study was to define the role of the −871C/T polymorphism in HCV-related MC patients as genetic contributor to its pathogenesis. We analysed the BAFF serum levels by ELISA and the presence of the −871 mutation using RFLP analysis in 123 HCV patients: 57 with HCVassociated MC and 66 HCV chronic carriers without any evidence of cryoglobulins or other autoimmune/LPDs. We noted a significantly higher prevalence of T allele homozygosity in MC patients (p < 0.0005), as well as the presence of the T allele (homozygous TT plus heterozygous TC) in respect to HCV carriers without MC (p = 0.004). This result was consistent with the higher serum levels found in MC patients compared to patients with the sole HCV infection. In conclusion, these results emphasize the potential contribution of the genetic background in the development of HCV-related LPDs. The transcriptional activation induced by the mutated BAFF promoter can be