- Email: [email protected]
P212 STEM CELL-DERIVED HEPATOCYTES AS A NOVEL IN VITRO MODEL TO STUDY HEPATOTROPIC VIRUSES
POSTERS Results: Our methylation assay identified an additional two CpG sites immediately downstream from rs12979860. SVR patients CC at rs12979860 with low hepatocellular MxA levels (“favourable” group N = 16) had higher degrees of methylation at all 3 CpG sites compared to NR patients (“unfavourable” group N = 6, homozygous T at rs12979860 with high hepatocellular MxA levels (graph 1). However overall methylation levels were not different between SVR and NR patients across the whole group (2.5% higher in SVR patients). Conclusions: Our data demonstrate higher degrees of methylation in three CpG sites for SVR patients homozygous C for rs12979860 when hepatocellular MxA protein (ISG) levels are low. These findings suggest that rs12979860 genotype influences methylation which ultimately has an effect on ISG expression and treatment response to IFN- a. P210 ALTERATIONS IN CXCR3/CXCL10 BUT NOT CCR5 IN HCV ARE ASSOCIATED WITH VIRAL INHIBITION WITH INTERFERON-FREE DIRECT ACTING ANTIVIRAL THERAPY M. Ow1,2 , D. Hegazy1 , M.E. Cramp1,2 . 1 Hepatology Research Group, Peninsula Medical School, University of Plymouth, 2 South West Liver Unit, Derriford Hospital, Plymouth, United Kingdom E-mail: [email protected] Background and Aims: CXCR3 and CCR5 are important in recruiting cytotoxic T cells to the liver. In HCV, interferon treatment increases peripheral expression of CXCR3+ /CCR5+ on T cells. This could be a direct effect of controlling HCV or a response to interferon. This study examined the effect of viral suppression on CXCR3/CCR5 expression in HCV patients on interferon-free direct acting antiviral (DAA) therapy. Methods: Ten HCV patients on 12 or 24 weeks of DAA therapy were sampled at baseline, weeks 1, 2, 4 and 8, and end of treatment (EOT). CXCR3+ /CCR5+ -expressing CD8 T cells were quantified on flow cytometry and serum chemokine levels determined by ELISA. Results: Viral load (VL) decline was greatest at week 1 (mean decline 4 log10 IU/mL) and all patients had no detectable virus at EOT. Serum CXCL10 levels correlated with VL, falling sharply at week 1 and remained low during treatment (baseline 355±250pg/mL vs. week 1 183±159, p = 0.001). There was a corresponding increase in the percentage of peripheral CXCR3+ CD8+ T cells, peaking at week 4 (baseline 41±12% of CD8 T cells vs. week 4 55±12%, p = 0.04). There was no significant change in the CCR5+ CD8+ T cell population. Conclusions: Interferon-free DAA therapy led to a rapid decline in CXCL10 within 7 days, with a subsequent increase in the expression of CXCR3+ CD8+ T cells. These changes correlated with a decline in VL. This suggests that HCV may impair components of the chemokine/chemokine receptor pathway, which are restored with viral suppression. However, there was no effect on CCR5; previously observed changes may be interferon-driven. P211 HOST mTORC1 SIGNALING IS REQUIRED FOR HEPATITIS C VIRUS (HCV) RNA REPLICATION 2 S. Stohr ¨ 1 , L. Sandmann1 , S. Pfander ¨ , E. Dazert3 , M.P. Manns1 , E. Steinmann2 , T. von Hahn1 , S. Ciesek1 . 1 Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 2 Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany; 3 Cell Growth and Development Biozentrum, University Basel, Basel, Switzerland E-mail: steffi[email protected]
Background and Aims: HCV has a narrow host range and primarily replicates in human hepatocytes. One reason for this is that all S136
stages of HCV’s replication cycle depend on various cellular host factors. Several unknown host factors are likely involved and may represent novel targets for antiviral therapy. Here we investigated the relevance of the mammalian Target of Rapamycin (mTOR) pathway on the HCV lifecycle. Methods: HCV infectivity, RNA replication and virus release was analyzed using the JFH1-based culture model and employing reporter assays and a core ELISA. Experiments were performed using Huh-7.5 cells and DMSO-differentiated Huh-7.5 cells. Lentiviral pseudotypes with HCV glycoproteins (HCVpp) were employed to assess modulation of HCV entry. Short hairpin RNA were used to selectively downregulate components of the mTOR signaling pathway. Results: The mTOR inhibitor Rapamycin inhibited HCV RNA replication and release of novel HCV particles in a dose dependent manner and by up to a 5,000-fold while infectivity of HCVpp was not affected. Treatment with Rapamycin led to reduced production of HCV core protein. Importantly, HCV RNA replication in DMSOdifferentiated Huh-7.5 cells was also decreased confirming that inhibition is not due to anti-proliferative effects. Knockdown of mTORC1 component Raptor but not mTORC2 component Rictor reduces HCV RNA replication and particle release. Conversely, overexpression of Raptor enhances HCV replication up to 10 fold suggesting that host mTORC1 signaling is important for efficient HCV replication. Conclusions: These data demonstrate that mTORC1 signaling regulates HCV RNA replication and that the mTOR antagonists might be useful in HCV positive patients after liver transplantation. P212 STEM CELL-DERIVED HEPATOCYTES AS A NOVEL IN VITRO MODEL TO STUDY HEPATOTROPIC VIRUSES N. Helsen1 , J. Paeshuyse2 , Y. Debing2 , K. Dallmeier2 , J. Neyts2 , C.M. Verfaillie1 . 1 Department of Development and Regeneration, 2 Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium E-mail: [email protected] Background and Aims: The study of hepatotropic viruses is hampered due to the lack of reliable cell culture models. Current cell culture systems mainly rely on the use of hepatoma cell lines and human primary hepatocytes which both have conceivable limitations. Alternative sources of human hepatocytes are human embryonic and induced pluripotent stem cell-derived hepatocytelike cells. Upon differentiation, we already demonstrated that these hepatocyte-like cells can be productively infected with the hepatitis C virus (HCV). However, the efficiency of HCV infection was low and requires further optimization. We aim to improve HCV infection of stem cell-derived hepatocytes and create a robust antiviral drug testing system. Next to HCV, we will investigate if the system is also supporting infection with other hepatotropic viruses. Methods: Different conditions are tested to optimize the hepatic differentiation and subsequently infection with hepatotropic viruses. Upon inoculation, infection is analyzed by conventional techniques. Next to improving the hepatic differentiation, a HCV reporter cell line is created that should allow more high throughput antiviral drug screening. Results: Compared to our published results, the infection with HCV is already improved. We also obtained initial evidence that our stem cell-derived hepatocyte-like cells are permissive for infection with different hepatotropic viruses including the hepatitis E and dengue virus. We are currently exploring whether the hepatitis B and the yellow fever virus replicate in these cultures. Conclusions: Stem cell-derived hepatocyte-like cells support the replication of multiple hepatotropic viruses and provide a more physiologically relevant cell culture model to study the biology of these and inhibition thereof by specific antivirals.
Journal of Hepatology 2014 vol. 60 | S67–S214
Background and Aims: HCV has a narrow host range and primarily replicates in human hepatocytes. One reason for this is that all S136
stages of HCV’s replication cycle depend on various cellular host factors. Several unknown host factors are likely involved and may represent novel targets for antiviral therapy. Here we investigated the relevance of the mammalian Target of Rapamycin (mTOR) pathway on the HCV lifecycle. Methods: HCV infectivity, RNA replication and virus release was analyzed using the JFH1-based culture model and employing reporter assays and a core ELISA. Experiments were performed using Huh-7.5 cells and DMSO-differentiated Huh-7.5 cells. Lentiviral pseudotypes with HCV glycoproteins (HCVpp) were employed to assess modulation of HCV entry. Short hairpin RNA were used to selectively downregulate components of the mTOR signaling pathway. Results: The mTOR inhibitor Rapamycin inhibited HCV RNA replication and release of novel HCV particles in a dose dependent manner and by up to a 5,000-fold while infectivity of HCVpp was not affected. Treatment with Rapamycin led to reduced production of HCV core protein. Importantly, HCV RNA replication in DMSOdifferentiated Huh-7.5 cells was also decreased confirming that inhibition is not due to anti-proliferative effects. Knockdown of mTORC1 component Raptor but not mTORC2 component Rictor reduces HCV RNA replication and particle release. Conversely, overexpression of Raptor enhances HCV replication up to 10 fold suggesting that host mTORC1 signaling is important for efficient HCV replication. Conclusions: These data demonstrate that mTORC1 signaling regulates HCV RNA replication and that the mTOR antagonists might be useful in HCV positive patients after liver transplantation. P212 STEM CELL-DERIVED HEPATOCYTES AS A NOVEL IN VITRO MODEL TO STUDY HEPATOTROPIC VIRUSES N. Helsen1 , J. Paeshuyse2 , Y. Debing2 , K. Dallmeier2 , J. Neyts2 , C.M. Verfaillie1 . 1 Department of Development and Regeneration, 2 Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium E-mail: [email protected] Background and Aims: The study of hepatotropic viruses is hampered due to the lack of reliable cell culture models. Current cell culture systems mainly rely on the use of hepatoma cell lines and human primary hepatocytes which both have conceivable limitations. Alternative sources of human hepatocytes are human embryonic and induced pluripotent stem cell-derived hepatocytelike cells. Upon differentiation, we already demonstrated that these hepatocyte-like cells can be productively infected with the hepatitis C virus (HCV). However, the efficiency of HCV infection was low and requires further optimization. We aim to improve HCV infection of stem cell-derived hepatocytes and create a robust antiviral drug testing system. Next to HCV, we will investigate if the system is also supporting infection with other hepatotropic viruses. Methods: Different conditions are tested to optimize the hepatic differentiation and subsequently infection with hepatotropic viruses. Upon inoculation, infection is analyzed by conventional techniques. Next to improving the hepatic differentiation, a HCV reporter cell line is created that should allow more high throughput antiviral drug screening. Results: Compared to our published results, the infection with HCV is already improved. We also obtained initial evidence that our stem cell-derived hepatocyte-like cells are permissive for infection with different hepatotropic viruses including the hepatitis E and dengue virus. We are currently exploring whether the hepatitis B and the yellow fever virus replicate in these cultures. Conclusions: Stem cell-derived hepatocyte-like cells support the replication of multiple hepatotropic viruses and provide a more physiologically relevant cell culture model to study the biology of these and inhibition thereof by specific antivirals.
Journal of Hepatology 2014 vol. 60 | S67–S214