813 HEPATITIS C VIRUS-SPECIFIC CELLULAR IMMUNITY DOES NOT PROTECT AGAINST FUTURE HCV INFECTION IN ANTI-HCV NEGATIVE INJECTING DRUG USERS

POSTERS performed using a non-targeted multiple platform methodology combining ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS) and gas chromatography/mass spectrometry (GC/MS). Metabolites were identified by automated comparison of ion features to a reference of chemical standard entries. Welch’s two-sample t-tests were used to identify biochemicals that differed significantly between HCV infected and un-infected cells. Results: A total of 250 metabolites were detected and quantified, of which 73 were differentially regulated. At the 24-hour time point, there was a significant increase in a number of metabolites involved in nucleotide synthesis and RNA replication. NAD levels were also significantly increased along with several amino acids. A number of lipid metabolic pathways were disrupted by HCV infection, resulting in an increase in cholesterol and sphingolipid levels, altered phospholipid metabolism and a possible disruption in mitochondrial fatty acid transport. Fluctuations in methylthioadenosine (MTA) levels were also noted, along with alterations in the glutathione synthesis pathway. Conclusions: These results suggest that elevated metabolism and altered energy status are associated with early HCV infection. These findings also provide new information on the effect HCV infection has on the disruption of lipid metabolism. 813 HEPATITIS C VIRUS-SPECIFIC CELLULAR IMMUNITY DOES NOT PROTECT AGAINST FUTURE HCV INFECTION IN ANTI-HCV NEGATIVE INJECTING DRUG USERS R. Sacks-Davis1,2 , C. Aitken1,2 , P. Higgs1,2,3 , S. Moneer4 , J. Flynn5,6 , V. Suppiah7,8 , L. Tracy9 , R. Ffrench5,6 , D. Bowden9 , H. Drummer4,10,11 , J. George7 , M. Bharadwaj4 , M. Hellard1,2 . 1 Centre for Population Health, Burnet Institute, 2 Department of Epidemiology and Preventative Medicine, Monash University, Melbourne, VIC, 3 National Centre for HIV Epidemiology & Clinical Research, University of New South Wales, Darlinghurst, NSW, 4 Department of Microbiology and Immunology, University of Melbourne, Parkville, 5 Centre for Immunology, Burnet Institute, 6 Department of Immunology, Monash University, Melbourne, VIC, 7 Storr Liver Unit, Westmead Millennium Institute, 8 Institute for Immunology and Allergy Research, Westmead Millennium Institute, University of Sydney, Sydney, NSW, 9 Victorian Infectious Diseases Reference Laboratory, North Melbourne, 10 Centre for Virology, Burnet Institute, Melbourne, 11 Department of Microbiology, Monash University, Clayton, VIC, Australia E-mail: [email protected] Background and Aims: Findings of large proportions of hepatitis C virus (HCV) anti-HCV negative injecting drug users (IDUs) with detectable HCV-specific cellular immune responses raise the question of whether some people have immunity to HCV infection. We conducted a longitudinal study to determine whether antiHCV negative IDUs with detectable HCV-specific cellular immune responses were more or less likely than other anti-HCV negative participants to develop persistent HCV infection. Methods: Cohort study participants who were anti-HCV antibody and HCV RNA negative at baseline and had at least one further HCV test were eligible for participation. HCV-specific cellular immune responses were measured at baseline and one subsequent time point in an ex vivo interferon-g enzymelinked immunospot (ELISpot) assay with pooled HCV peptides. Participants with responses of at least 25 spot forming cells per million were classified as ELISpot positive. Potential behavioral, social, environmental and genetic variation (single nucleotide polymorphism in the IL28 region) that might correlate with ELISpot positivity were examined. Results: Of 53 anti-HCV negative participants, 20 were HCV ELISpot positive at baseline. Age, gender and HCV risk behaviors did not differ by HCV ELISpot status. Similar rates of HCV incidence were S326

observed amongst HCV ELISpot positive participants (28.7 per 100 py, 95% CI 12.9, 63.8) and those who were HCV ELISpot negative (19.4 per 100 py, 95% CI 8.1, 46.6). Genetic variations in the IL28B region previously identified as associated with viral clearance were no more prevalent amongst participants who were ELISpot positive and anti-HCV antibody negative, HCV RNA negative than those who were anti-HCV antibody positive or HCV RNA positive. Conclusions: HCV-specific cellular immune responses were observed in more than one third of anti-HCV negative IDUs but our data contain no evidence that this subgroup of IDUs is protected from future HCV infection. 814 INDUCTION OF EPIGENETIC CELL MODIFICATIONS MEDIATED BY HEPATITIS C VIRUS J. Sheldon1 , A. Madejon2 , F. Antonucci3 , C. Perales1 , J. Garc´ıa3 1 Samaniego2 , E. Domingo1 , A. Sanchez-Pacheco ´ . Department of Virology and Microbiology, Centro de Biologia Molecular Severo Ochoa, CSIC-Universidad Autonoma de Madrid, 2 Hepatology Unit, Hospital Carlos III. CIBERehd, 3 Department of Biochemistry, Universidad Autonoma de Madrid-Instituto de Investigaciones Biom´edicas A. Sols (UAM-CSIC), Madrid, Spain E-mail: [email protected] Background and Aims: Epigenetic regulation mechanisms are essential for the modulation of eukaryotic gene expression. These processes are regulated by nuclear enzymatic activities such as kinases, acetyltranferases or demethylases. There are not too much knowledges about if HCV, an infectious agent with a cytoplasmic replicative cycle can induce epigenetic modifications in the host cell. Material and Methods: Huh 7.5 cells were infected with Jc1 virus supernatant (HCV cell culture infectious clone) or DMEM media and passaged until >90% cells were infected. In these cells several epigenetic markers were analyzed. Phosphorylation of histone-3 in serine-10 position (H3Ser10ph) and methylation of histone 4 in lysine-20 residue (H4K20me3) was tested by western blot analysis using specific antibodies against the histone modifications. The expression levels of EGFR and IGBP3 genes were tested by reverse-transcription/quantitative-real time PCR. All the assays were performed in triplicate in independent experiments Results: Data obtained showed that the infection of the Huh 7.5 cells with the Jc1 is associated with the induction of several epigenetic changes. The levels of H3Ser10ph were higher in the mock compared to the infected cells. In addition H4K20me3 levels were significantly reduced (70%) in the infected cells when compared to the mock. The level of EGFR mRNA expressed was also 75% lower in the infected cells, while the IGBP3 expression didn’t change by Jc1 infection. Conclusions: Our data shows that HCV infection can induce epigenetic modifications within the host cell. We found both H3Ser10ph and H4K20me3 levels were reduced in the presence of HCV infection. These epigenetic modifications correlated with changes in the expression levels of EGFR gene. Since HCV replicates in the cytoplasm, this induction process should occur by indirect regulation of nuclear enzymatic activities implicated in the epigenetic regulation. 815 IDENIX NS5A HCV REPLICATION INHIBITORS WITH LOW PICOMOLAR, PAN-GENOTYPIC IN VITRO ANTIVIRAL ACTIVITY C.B. Dousson1 , C. Chapron2 , D. Standring2 , J.P. Bilello2 , J. McCarville2 , M. La Colla2 , M. Seifer2 , C. Parsy1 , D. Dukhan1 , C. Pierra1 , D. Surleraux1 . 1 Idenix Pharmaceuticals, Montpellier, France; 2 Idenix Pharmaceuticals, Cambridge, MA, USA E-mail: [email protected] Background: The NS5A protein is essential to the life cycle of the hepatitis C virus (HCV) and provides an important antiviral drug

Journal of Hepatology 2011 vol. 54 | S209–S361