1Median time (years) follow-up between the visit representative of treatment failure and last available visit when the UDPS sample was collected; Q1, 1st quartile; Q3, 3rd quartile; 2Percent of the total (n/N) determined to be WT by each method; 3Includes one patient that was WT by UDPS yet had resistance detectable by PS
Article first published online: 15 OCT 2013
Copyright © 2013 American Association for the Study of Liver Diseases
Special Issue: The 64th Annual Meeting of the American Association for the Study of Liver Diseases: The Liver Meeting 2013
Volume 58, Issue S1, pages 926A–943A, October 2013
How to Cite
(2013), HCV: Virology. Hepatology, 58: 926A–943A. doi: 10.1002/hep.26868
- Issue published online: 1 OCT 2013
- Article first published online: 15 OCT 2013
Hepatitis C virus core protein suppresses mitophagy by interacting with Parkin
Yuichi Hara1, Sohji Nishina1, Izumi Yanatori4, Masanori Ikeda2, Emi Kiyokage5, Yasuyuki Tomiyama1, Kazunori Toida5, Fumio Kishi4, Nobuyuki Kato2, Michio Imamura3, Kazuaki Chayama3, Keisuke Hino1
1Departments of Hepatology and Pancreatology, kawasakimedicalschool, Kurashiki, Japan; 2Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima Univercity, Hiroshima, Japan; 3Department of Tumor Virology, Okayama Univercity, Okayama, Japan; 4Molecular Genetics, Kawasaki Medical School, Okayama, Japan; 5Anatomy, Kawasaki Medical School, Okayama, Japan
BACKGROUND and AIM: Hepatitis C virus (HCV) causes mitochondrial injury and oxidative stress, and impaired mitochondria are selectively eliminated through autophagy-dependent degradation (mitophagy). However, whether HCV infection affects mitophagy in terms of mitochondrial quality control remains unknown. METHODS: The effect of HCV on mitophagy was examined using HCV-JFH1-infected cells, genome-length HCV RNA-replicating cells (OR6 cells), HCV core-expressing cells and the uncoupling reagent carbonyl cyanide mchlorophenylhydrazone as a mitophagy inducer in addition to liver cells from HCV-infected human hepatocyte chimeric mice and. transgenic mice expressing the HCV polyprotein. RESULTS : The results indicated that translocation of the E3 ubiquitin ligase Parkin to the mitochondria was impaired without reduction of PTEN-induced putative kinase 1activity in the presence of HCV infection both in vitro and in vivo. Co-immunoprecipitation revealed that Parkin was associated with the HCV core protein but not other HCV proteins, such as NS3, NS4A and NS5A. Furthermore, a yeast two-hybrid assay identified a specific interaction between the HCV core protein and an N-terminal Parkin fragment that contains one of the amino acids that is essential for its mitochondrial localization. Silencing Parkin suppressed HCV replication suggesting the functional role of interaction between HCV core protein and Parkin in HCV propagation. The suppressed translocation of Parkin to the mitochondria inhibited mitochondrial ubiquitination'decreased the number of mitochondria sequestered in isolation membranes (mitophagosomes), and reduced autophagic degradation activity, which clearly indicated the suppression of mitophagy. However, OR6 cells promoted autophagy under non-selective autophagyinducible conditions (amino acid starvation), as indicated by the increased expression of the microtubule-associated protein light chain 3 (LC3)-II. CONCLUSIONS: Through a direct interaction with Parkin, the HCV core protein suppressed mitophagy by inhibiting the translocation of Parkin to the mitochondria. These results have implications for the amplification and sustainability of mitochondria-induced oxidative stress observed in patients with HCV-related chronic liver disease and an increased risk of hepatocarcinogenesis.
Kazuaki Chayama - Consulting: Abbvie; Grant/Research Support: Dainippon Sumitomo, Chugai, Mitsubishi Tanabe, DAIICHI SANKYO, Toray, BMS, MSD; Speaking and Teaching: Chugai, Mitsubishi Tanabe, DAIICHI SANKYO, KYORIN, Nihon Medi-Physics, BMS, Dainippon Sumitomo, MSD, ASKA, Astellas, AstraZeneca, Eisai, Olympus, GlaxoSmithKline, ZERIA, Bayer, Minophagen, JANSSEN, JIMTO, TSUMUTA, Otsuka, Taiho, Nippon Kayaku, Nippon Shinyaku, Takeda, AJINOMOTO, Meiji Seika, Toray
The following people have nothing to disclose: Yuichi Hara, Sohji Nishina, Izumi Yanatori, Masanori Ikeda, Emi Kiyokage, Yasuyuki Tomiyama, Kazunori Toida, Fumio Kishi, Nobuyuki Kato, Michio Imamura, Keisuke Hino
Quercetin inhibits HCV replication by modulating lipid droplets morphology and core protein co-localization
Angela Rojas1, Jose A. Del Campo1, Marta Garcίa-Valdecasas1, Sophie Clement2, Francesco Negro3, Manuel Romero-Gómez1
1Hospital de Valme, Sevilla, Spain; 2Division of Clinical Pathology, Geneve, Switzerland; 3Division of Clinical Pathology and Gastroenterology and Hepatology, University Hospital, Geneve, Switzerland
Aims: To investigate the role of the flavonoid quercetin (Q) on modulation of lipid droplets (LDs) size and morphology and HCV core protein localization and (3) HCV life cycle focusing on entry and replication. Methods: The morphology of LDs and core localisation were studied by immunofluorescence using confocal microscopy on Huh-7 cells transduced with lentivectors encoding the core protein of HCV genotype3a and treated with quercetin for 48h at different concentrations (50μM-100μM). LDs analysis was done using MetaMorph Microscopy-Software. To study the effects of quercetin on viral replication (iRNA), Huh7 cells were infected with Jc1 ccHCV particles (1Moi) and subsequently treated with quercetin for 48 and 72h. NS3 and core protein levels were evaluated by immunoblot. HCV entry was assessed using HCV pseudoparticies(HCVpp), which are lentivectors harboring HCV entry proteins and containing luciferase as reporter gene. Results: LDs morphology(area, radium, and volume) and distribution were modified by quercetin in Huh7. Quercetin treatment could impede the core 3a- induced increase of LD size in cells transduced with lentivirus expressing the Core genotype 3a protein [LD area (μm2): 3a:109.8±33.72; 3aQ50μM: 79.90±36(p<0.001); 3aQ100μM: 72, 6±35.4(p<0, 0003); radium(μm): 3a: 5.85±0.88; 3aQ50μM: 4.91 ±1.15(p<0.001) 3aQ100μM: 4.65±1.22 (p<0.0002), voiumen (μm3) 3a: 894.7±418.5; 3aQ50μM: 577.03±379.26(p<0.003) 3aQ100μM: 505.4±355.2(p<0.0006)]. In these cells'the typical localization of the core protein around the LDs was almost fully inhibited by quercetin, Core protein rather displaying a punctated pattern throughout the cytoplasm. While quercetin inhibited ccHCV replication by more than 75% and 85% when cells were treated with 50μM-100μM respectively in comparison with untreated cells, it did not impact the entry of HCVpp. As well, quercetin decreased Core and NS3 protein level expression Conclusion: Quercetin has a major effect of LD morphology and interferes with HCV-induced steatosis. Besides, it decreases viral replication, core and NS3 proteins expression and avoided the co-localization between core and lipid droplets, without impact on viral entry. Therefore, this flavonoid could be considered as a new drug for hepatitis C treatment.
Francesco Negro - Advisory Committees or Review Panels: Roche, MSD, Gilead, Boehringer Ingelheim; Grant/Research Support: Roche, Gilead
Manuel Romero-Gomez - Advisory Committees or Review Panels: Roche Farma, SA, MSD, SA, Janssen, SA., ABBOTT, SA; Grant/Research Support: Ferrer, SA
The following people have nothing to disclose: Angela Rojas, Jose A. Del Campo, Marta Garda-Valdecasas, Sophie Clement
Syndecan 4 is the primary heparan sulfate proteoglycan mediating HCV entry through interaction with apolipoprotein E
Mathieu Lefevre1,2, Daniel Felmlee1,2, Thomas F. Baumert1,2, Catherine Schusfer1,3
1UMR1110 virus-host interactions and liver disease, Inserm, Strasbourg, France; 2UMR1110 virus-host inteactions and liver disease, University of Strasbourg, Strasbourg, Fronce; 3Pôle Hêpato-digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
In hepatitis C virus (HCV) infected patients, virions are associated with very low density lipoprotein (VLDL)-type lipoproteins forming an infectious lipo-viro-particle (LVP). Apolipoprotein E (apoE), a major component of VLDL, interacts with heparan sulfate proteoglycans (HSPG) at the hepatocyte cell surface. As well, apoE is present at the surface of the LVP playing a crucial role in HCV infectivity. We aimed to investigate the role of apoE and its functional regions in HCV infectivity and to identify the syndecan (Sdc) involved in the HCV entry process. First, using adenoviral vectors expressing wild type or mutant apoE, we complemented apoE expression in Huh7.5.1 depleted cells from the endogenous apoE. Increasing amounts of apoE lead to a dose-dependent increase in HCV infectivity, the more apoE was expressed the more HCV particles were infectious, demonstrating the primary role of apoE in HCV infectivity. ApoE mutated in the HSPG binding domain (HSPG-BD) as well as competition experiment using a peptide mimicking the HSPGBD confirmed the HSPG dependency for HCV infectivity. Finally, silencing experiments targeting the HSPG syndecan (Sdc)1 or Sdc4 revealed that HCV entry was markedly decreased following Sdc4 silencing. This effect was not observed when HCV pseudoparticles entry was analyzed, confirming the essential role of apoE-Sdc interactions in HCV entry. Collectively, our data demonstrate that HCV-apoE-Sdc interactions mediate viral entry. Since viral entry has been shown to play a key role in acute liver graft infection and viral persistence, targeting apoE-Sdc interactions opens a new perspective to prevent HCV re-infection during transplantation and may provide novel therapeutic avenues.
The following people have nothing to disclose: Mathieu Lefevre, Daniel Felmlee, Thomas F. Baumert, Catherine Schuster
Naturally Occurring Mutations in Hepatitis C Virus E2 Conferring Resistance to Multiple Broadly Neutralizing Monoclonal Antibodies Identified by a Novel Method
Justin R. Bailey, Anna E. Snider, William O. Osburn, Stuart C. Ray
Medicine, Johns Hopkins, Baltimore, MD
Introduction: Binding epitopes of neutralizing monoclonal antibodies (mAb) against HCV are generally mapped by alanine scanning mutagenesis. These studies provide useful information on key mAb binding residues, but they do not directly test the effect of mutations on virus neutralization sensitivity, nor do they test the effect of the wide array of naturally occurring HCV envelope mutations that occur in vivo. Methods: A panel of 19 diverse genotype 1 HCV E1E2 clones was used to produce a library of HCV pseudoparticles (HCVpp). These HCVpp were tested for neutralization by 19 published monoclonal anti-HCV neutralizing antibodies (nAb). Individual HCVpp were ranked by neutralization sensitivity to each mAb, and analysis of E1E2 sequences was used to identify mutations associated with resistance. The resistance phenotypes of these mutations were confirmed by their introduction into nAb sensitive E1E2 clones. Results: We identified naturally occurring E1E2 clones that were sensitive as well as clones with 60-100% resistance to each broadly neutralizing mAb tested. To validate the HCVpp library system, we compared ranking of neutralization sensitivity of library HCVpp's to two closely related mAbs (HC33.4.10 and HC33.8) and found extremely high correlation (Spearman correlation coefficient 0.94, p<.00〇1). We subsequently compared ranking of sensitivity to two unrelated mAbs (HC33.4.10 and HC84.22) and found no correlation (correlation 0.08, p=.75). Surprisingly, we found correlation in ranking of HCVpp sensitivity to some mAbs thought to have non-overlapping binding sites (i. e. HC84.22 and AR3C, correlation 0.84, p<.0001). Through sequence analysis of resistant E1E2 clones, we identified a mutation, D431E, that could confer resistance to neutralization by many of the broadly neutralizing mAb tested, including CBH-2, AR3A, AR3B, AR3C, AR3D, and HC84.22. A second mutation, F442I, conferred resistance to mAbs HC84.22 and HC84.26. Conclusions: We have developed a novel, rapid method to identify naturally occurring mutations in E1E2 conferring resistance to neutralizing mAbs. We found unexpected correlations between ranking of HCVpp neutralization sensitivity to some mAbs thought to have non-overlapping binding sites, suggesting that some mutations or combinations of mutations may confer resistance to multiple broadly neutralizing mAbs. We have identified two such mutations, D431E and F442I. Use of this method will be critical to identify additional mutations and combinations of mutations conferring resistance to broadly neutralizing mAbs, allowing more accurate identification of mAbs most likely to be effective in vivo.
Stuart C. Ray - Advisory Committees or Review Panels: Boehringer Ingelheim, Abbott Laboratories
The following people have nothing to disclose: Justin R. Bailey, Anna E. Snider, William O. Osburn
A genome-wide functional screen identifies the let-7 family of microRNAs as host restriction factors regulating hepatitis C virus infection
Qisheng Li, Siddharth Krishnamurthy, Helen Cha, Ramy El-Diwany, Stephan Chiu, Hawwa F. Alao, T. Jake Liang
Liver Diseases Branch, NIDDK, NIH, Bethesda, MD
Host microRNAs (miRNAs), like miR-122, have previously been shown to modulate hepatitis C virus (HCV) replication, but a system-wide study to define the entire repertoire of miRNAs associated with HCV infection and their interactions with viral life cycle has yet to be conducted. Here we report an unbiased genome-wide miRNA mimic-inhibitor screen (∼1000 miRNA in miRBase Sequence 13.0) to identify cellular miRNAs involved in productive HCV infection. In the primary screen applying an infectious HCVcc system, we identified 77 miRNAs that either enhanced (proviral) or restricted (antiviral) HCV infection.23 host proviral miRNAs and 41 host antiviral miRNAs were subsequently validated by a secondary screen using a luciferase reporter virus. Taking advantage of functional genomics and various in vitro HCV models, we investigated the functions of these host miRNAs in different stages of HCV life cycle - entry, trafficking, IRES-mediated translation, RNA replication, and assembly/secretion. We further characterized several representative miRNAs for their mechanisms in modulating HCV infection. Multiple members of the let-7 family of miRNAs with conserved seed sequence were shown to restrict HCV infection at multiple stages of viral life cycle. We performed target prediction by bioinformatics and various validation assays, and demonstrated that these let-7 miRNAs target and down-regulate various host proviral factors identified in our previous small interference RNA (siRNA) screen (Li et al, PNAS 2009) at either transcriptional or translational level, potentially explaining the antiviral function of these miRNAs in HCV infection. A comprehensive investigation of cellular miRNAs modulating the complete HCV life cycle will yield critical insights into HCV pathogenesis and provide novel therapeutic targets.
The following people have nothing to disclose: Qisheng Li, Siddharth Krishnamurthy, Helen Cha, Ramy El-Diwany, Stephan Chiu, Hawwa F. Alao, T. Jake Liang
The ribosomal protein RACK1 is a specific host factor required for IRES-mediated translation of hepatitis C virus
Mohamed Lomine Hofirossou1,2, Karim Majzoub5,2/ Stefano Marzi4,2, Jean-Luc Imler5,2, Thomas F. Baumert1,3, Catherine Schuster1,2
1UMR1110 virus-host interactions and liver disease, Inserm, Strasbourg, France; 2UMR1110 virus-host interactions ond liver disease, University of Strasbourg, Strasbourg, France; 3PôIe Hépatodigestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 4UPR9002, CNRS, Strasbourg, France; 5UPR9022, CNRS, Strasbourg, France
Background: Treatment of chronic viral infection is challenged by variability of viral targets and development of resistance. Viruses depend on host factors for their life cycle, which are attractive alternative antiviral targets, provided that they are not mandatory for normal cell functions. Using a functional proteomic screen, we recently identified Receptor for Activated C Kinase 1 (RACK1) as a specific host factor required for replication of internal ribosome entry site (IRES)-containing viruses. Methods: Using state-of-the-art cell culture models for HCV infection, replication and translation, we investigated the functional impact of RACK1 as a host factor for HCV infection. Results: Silencing of RACK1 expression in Huh 7.5.1 cells resulted in a marked, specific and significant decrease in HCV Jc1 infection and infectious virion production. A similar effect was obtained when RACK1 expression was silenced in HCV replicating cells, demonstrating a crucial role of this host factor in HCV replication. In contrast, infection of non IRES-translated viruses like adenovirus or vesicular stomatitis virus remained unchanged in RACK1 silenced cells. In order to discriminate between the translation and the replication steps of the HCV life cycle, we established stable cell lines expressing either an IRESHCVluciferase reporter or a classical capped luciferase reporter, respectively. Silencing of RACK1 markedly and exclusively decreased IRESHCV-dependent translation, but not classical cap-mediated translation, demonstrating that RACK1 is specifically required for IRES-mediated translation of HCV. In agreement with these data, structural modeling indicates that RACK1 is located in close proximity to the HCV IRES on the 40S ribosomal subunit, in a region that is conformationally modified upon binding of the HCV IRES. Conclusions: Collectively, our results demonstrate that RACK1, a component of the ribosome, is a specific host factor for IRES-dependent HCV translation. Our data conceptually advance the understanding of viral translation and reveal a novel host target for the development of antivirals addressing resistance.
The following people have nothing to disclose: Mohamed Lamine Hafirassou, Karim Majzoub, Stefano Marzi, Jean-Luc Imler, Thomas F. Baumert, Catherine Schuster
CD64 (FcγRI) is a novel receptor for HCV entry into monocytes
Morven E. Cunningham, Joseph D. Wright, Joshua L. Wong, Jennifer A. Waters, Graham R. Foster
Queen Mary, University of London, London, United Kingdom
Monocytes from patients with HCV contain virus and we have shown that this virus replicates when monocytes are fused to hepatocytes. We developed a replication system in which patient-derived HCV is “captured” by the monocytic cell line THP-1 and viral replication assessed after fusion of these cells to hepatoma cells. Capture of HCV in monocytes is known to be enhanced by pretreatment with PMA and IFNy. Here we explored the receptors involved in monocyte capture/entry, specifically those involved in hepatocyte HCV entry as well as Fc receptors. Unstimulated THP-1 or cells prestimulated with PMA and IFNγ were incubated with sera from patients with chronic HCV and HCV RNA quantified by qPCR. mRNA expression of classical HCV entry receptors and FcyR was compared in stimulated and unstimulated cells and surface receptor expression analysed by FACS. Stimulated THP-1 were incubated with blocking antibodies to candidate entry receptors prior to incubation with patient sera and fusion with Huh7.5 cells. HCV RNA was quantified immediately and up to 7 days after fusion. Results are mean ± s. d. and p values were calculated using the Mann-Whitney U test. HCV associated poorly with unstimulated THP-1 cells, but this was enhanced by prestimulation with PMA and IFNγ (121 ± 62 versus 380 ± 252 HCV copies/μg total RNA after 24 hours, p = 0.026). Trypsin treatment of stimulated THP-1 after capture confirmed internalisation of HCV. Cytokine stimulation increased expression of CD64 mRNA, but not of CD81, SR-B1, LDL-R or CD32 (FcγRII). FACS analysis confirmed an increase in cell surface expression of CD64, but not of other receptors, compared to unstimulated THP-1. Claudin-1, occludin and CD16 (FcγRIII) were not expressed. CD64 blocking antibodies reduced association of patient-derived HCV with prestimulated THP-1(34 ± 16 versus 106 ± 43 copies/μg total RNA, p = 0.02), and also HCV replication after fusion of infected tHP-1 with Huh7.5 cells (19 ± 12 versus 116 ± 100 HCV copies/μg total RNA 7 days after fusion, p = 0.005). Blocking antibodies to CD81, SR-B1 or CD32 had no effect. Uptake of patient-derived HCV into THP-1 monocytes is mediated primarily through CD64. Blocking CD64 did not completely abrogate HCV uptake suggesting that other, as yet undefined receptors may also be involved but these are distinct from classical HCV entry receptors including CD81. Although we found no evidence of HCV replication in THP-1 cells, replication occurred after fusion with Huh7.5 cells suggesting that HCV internalised into THP-1 via CD64 is replication-competent. This may have implications for viral persistence and relapse after antiviral therapy.
Graham R. Foster - Advisory Committees or Review Panels: GlaxoSmithKline, Novartis, Boehringer Inqelheim, Tibotec, Chughai, Gilead, Janssen, Idenix, GlaxoSmithKline, Novartis, Roche, Tibotec, Chughai, Gilead, Merck, Janssen, Idenix, BMS; Board Membership: Boehringer Ingelheim; Grant/Research Support: Chughai, Roche, Chughai; Speaking and Teaching: Roche, Gilead, Tibotec, Merck, BMS, Boehringer Ingelheim, Gilead, Janssen
The following people have nothing to disclose: Morven E. Cunningham, Joseph D. Wright, Joshua L. Wong, Jennifer A. Waters
Loss of apolipoprotein B100 decreases viral infectivity and depletes cholesterol from HCV virions
Esperance A. Schaefer1, James Meixiong4, Daniel Motola1, Amy Deik2' Dahlene N. Fusco1, Carol Lin4, Nikolaus Jig1, Stephane Chevaliez1, Cynthia Brisac1, Pattranuch Chusri1, Wenyu Lin1, Clary B. Clish2, Kiran Musunuru3, Chod A. Cowan3, Raymond T Chung1, Lee F. Peng1
1Medicine - GI Unit, Massachusetts General Hospital, Boston, MA; 2Broad Institute, Boston, MA; 3Horvord Stem Cell Institute, Boston, MA; 4Harvard University, Boston, MA
Background and Aims: The role of apolipoprotein B100 in HCV has yet to be clearly defined. Other work has suggested that it is an important component of the HCV viral particle; however, studies using pharmacologic and/or RNAi mediated inhibition of apoB have yielded inconsistent results. We have previously demonstrated that apoB100 is required to support the HCV lifecycle and that virus generated in the absence of intracellular apoB exhibits impaired infectivity. We sought to characterize the alterations in the apoB deficient virions that contribute to this phenotype. Methods: We examined HCV and Dengue infection in a Hun-//CD81high cultured cell line with transcription activator-like effector nuclease (TALEN) mediated knockout of APOB. Dengue viral infectivity was determined using RNA viral titers assessed two hours after inoculation. For characterization of HCVcc virion, we used the JFH-1 derived JC1-E2-FLAG HCV virus, which permits affinity purification of the virus. We compared HCVcc generated in these APOB -/- cells with virion produced in APOB +/+ controls. To characterize the lipoprotein and lipid composition of the virions, we performed liquid chromatography 一 mass spectrometry (LC-MS) of the purified JC1E2-FLAG virus to characterize its lipidome. We measured apolipoprotein B and apolipoprotein E concentrations using ELISA of the purified virus. Results: We found that, unlike our previous observations in HCV, Dengue virus had no decrease in its infectivity when generated in the absence of apoB100. The APOB -/- (KO) generated dengue virus had modestly increased infectivity when compared to the wild type (WT) virus. We found that the lipidome of HCV virion generated by the KO cells was fundamentally altered from WT virus; specifically that the virus completely lacked all cholesterol esters. Further, as expected, there were undetectable levels of apoB in the KO virus. However, we also found that apoE levels were diminished in the virus, despite preserved intracellular levels. Conclusions: Loss of ApoB100 expression in vitro fundamentally alters the lipid composition of HCV, along with decreased lipoprotein content. These Kogenerated virions do not resemble human VLDL, WT virus or previously characterized HCVcc virion or lipoviral particles. These alterations are likely important contributors to the significantly impaired infectivity of HCV and the decreased ability to support HCVcc we have previously observed in APOB -/- cells. This effect on HCV by apoB appears to be virus-specific, since similar perturbations had no impact on infectious Dengue virus production.
Raymond T. Chung - Advisory Committees or Review Panels: Idenix; Consulting: Enanta; Grant/Research Support: Gilead, Merck, Mass Biologic, Gilead
The following people have nothing to disclose: Esperance A. Schaefer, James Meixiong, Daniel Motola, Amy Deik, Dahlene N. Fusco, Carol Lin, Nikolaus Jilg, Stephane Chevaliez, Cynthia Brisac, Pattranuch Chusri, Wenyu Lin, Clary B. Clish, Kiran Musunuru, Chad A. Cowan, Lee F. Peng
Type III IFN (Interferon Lambda) Induces Hepatitis Clearance In Type I (Interferon Alpha) Resistant Free Fatty Acid HCV Cell Culture
Ramazan Kurt1, Partha K. Chandra2, Fatma Aboulnasi2, Nathan J. Shores1, Rajesh Panigrahi2, Pauline Ferroris2, Srikanta Dash1'2, Luis A. Balart1
1Gastroenterology and Hepotology, Tulane Medicol School, New Orleans, LA; 2Pathology and Laboratory Medicine, Tulane Medical School, New Orleans, LA
Background: Hepatic steatosis is known as a risk factor for liver disease progression and impaired response to interferon alpha (IFN-a) plus ribavirin combination therapy in chronic HCV patients. The mechanism for this lack of response to interferon therapy is unclear. Previously we published that free fatty acids (FFA) induce endoplasmic reticulum (ER) stress and block antiviral activity of interferon alpha against hepatitis C virus in cell culture. This study was performed to compare the type I interferon (IFN-α), type II interferon (IFN-λγand type Ill interferon (IFN-γ) induced antiviral clearance in the FFA treated HCV cell culture model. Method: HCV infected Huh 7.5 cells were cultured with or without a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracyfoplasmic fat accumulation was visualized by nile red staining. Clearance of HCV in FFA cell culture after long term therapy with IFN a, IFN λ and IFN y was compared by Renilla luciferase activity and HCV core immune staining. Jak Stat signaling induced by interferons was examined by Western blot analysis. Results: FFA treatment induced dose dependent hepatocelular steatosis and lipid droplet accumulation in the HCV infected Huh 7.5 cells. FFA treatment blocked IFN-α and IFN-γ response and viral clearance by reducing the phosphorylation of Stat 1 and Stat 2. Whereas IFN-λ1(IL 29) and IFN-λ2 (IL28A) alone or in combination with IFN-α inhibited HCV replication to completion in the FFA treated cell culture. Renilla luciferase activity and HCV core immune staining confirm that HCV replication was undetectable after repeated treatment with IFNλ but not IFN α and IFN . Furthermore, IFN λ induce Stat 1, Stat 2, Stat 3 phosphorylation and nuclear translocation in IFN a resistant FFA HCV cell culture. Pretreatment of Jak1 inhibitor (Pyridone 6) prevented the antiviral activity of interferon lambda against HCV in free fatty acid culture Conclusion: IFN γclears HCV replication and overcome HCV resistance to IFN a in FFA cell culture model by initiating Stat 1, Stat 2, and Stat phosphorylation and their nuclear translocation. Our findings show that IFN is a better choice for treatment of HCV infection.
Nathan J. Shores - Advisory Committees or Review Panels: Gilead; Speaking and Teaching: Vertex, Merck, Salix
Luis A. Balart - Advisory Committees or Review Panels: Genentech, Genentech; Grant/Research Support: Merck, Genentech, Bayer, conatus, Ocera, Hyperion, Gilead Sciences, Bristol Myers Squibb, Mochida, Eisai, Vertex, Merck, Genentech, Bayer, Conatus, Ocera, Hyperion, Gilead Sciences, Bristol Myers Squibb, Mochida, Eisai, Vertex, takeda, GI Dynamics; Speaking and Teaching: Merck, Merck, Merck, Merck
The following people have nothing to disclose: Ramazan Kurt, Partha K. Chandra, Fatma Aboulnasr, Rajesh Panigrahi, Pauline Ferraris, Srikanta Dash
A Cyclic Retinoid, Peretinoin, Inhibits Hepatitis C Virus Replication in Cell Culture
Tetsuro Shimakami1, Masao Honda1, Takayoshi Shirasaki1, Stanley M. Lemon2, Shuichi Koneko1
1Kanazawa University, Kanazawa, Japan; 2University of North Carolina, Chapel Hill, NC
[Background and Aims] An oral acyclic retinoid, Peretinoin, was shown to significantly reduce the incidence of post-therapeutic hepatocellular carcinoma (HCC) recurrence and improve the survival rates of patients in a clinical trial (NEJM, 1996, 1999), and larger-scale clinical studies are ongoing in various countries to confirm its clinical efficacy. Hepatitis C Virus (HCV) is known as a major causative pathogen of HCC. Therefore, depending on the results of clinical studies, Peretinoin may be used for HCV-infected patients as a chemo preventive drug for HCC. However, the precise mechanisms of Peretinoin on HCV life cycle have not been evaluated. Here, we extensively examined the effect of retinoids including Peretinoin on HCV infection in cultured cells. [Methods] The Effects of several retinoids, such as Peretinoin, 9-cis retinoic acid (9-cis RA), 13-cis RA, and all-trans retinoic acid, on HCV RNA replication in cultured cells were examined by using different HCV genomes (genotypes 1a, 1b, and 2a) encoding the Gaussia luciferase reporter protein. The mechanisms of its inhibitory effect on HCV infection were assessed by evaluating cellular signaling pathways, such as interferon and lipid metabolism, and various aspects of HCV life cycle, such as virus translation, RNA amplification, infectious virus production including assembly, secretion, and entry. [Results] Peretinoin inhibited HCV RNA replication for all genotypes without affecting cell proliferation and showed the strongest antiviral effect among retinoids tested. Such inhibition was nearly restored by a retinoid X receptor (RXR) antagonist. Peretinoin had no effect on either HCV translation or activation of cellular IFN signaling and Peretinoin-resistant HCV mutants did not emerge after long culture of HCV-replicating cells with Peretinoin. Interestingly, Peretinoin dramatically reduced the numbers of lipid droplets (LDs), triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. As lipids including LDs and triglyceride have been reported to be important for efficient infectious virus production, Peretinoin dramatically reduced infectious virus production by specifically inhibiting HCV virus secretion without affecting either assembly or entry of virus. [Conclusions] Peretinoin alters lipid metabolism and inhibits HCV RNA replication and virus release through RXR signaling. These effects may be beneficial in addition to its potential for chemoprevention of HCC in HCV-infected patients.
Stanley M. Lemon - Advisory Committees or Review Panels: Merck, Santaris, Abbott, Gilead; Consulting: Achillion, Idenix; Grant/Research Support: Merck, Tibotec, Scynexis; Speaking and Teaching: Hoffman LaRoche
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan
The following people have nothing to disclose: Tetsuro Shimakami, Masao Honda, Takayoshi Shirasaki
Role of the PDIPI protein in the IFN response during hepatitis C virus infection: impact of PPAR nuclear receptors
Stephane Chevaliez1 , 2, Cynthia Brisac1,Esperance A. Schaefer1, Daniel Wombua1, Nikolaus Jilg1, Jay Luther1, Pattranuch Chusri1, Laurent Zablocki1, Wenyu Lin1, Lee F. Peng1, Dahlene N. Fusco1, Raymond T. Chung1
1GastrointestinaI Unit, Massachuset General Hospital, Boston, MA; 2Virology ond INSERM U955, University Hospital Henri Mondor, Creteil, France
Background: Alpha interferon (IFN), a type I cytokine, plays a major role in the antiviral treatment of chronic hepatitis C virus (HCV) infection. IFN modulates the innate immune response and exerts a direct antiviral mechanism by activating more than 500 intracellular genes. Many studies identified genes implicated in the IFN response in hepatoma cell lines infected by the HCV strain JFH1, such as PDIP1 (PPAR-gamma DNA-binding domain-interacting protein 1) (Fusco et al. Gastroenterology 2013). Objective: The aim of this study was to elucidate the role of PDIP1 in the antiviral mechanisms of IFN during HCV JFH1 infection of Huh7.5.1 cells, a human hepatoma cell line. Methods & Results: Treatment of j FH1-infected Huh7.5.1 cells with different PPAR alpha and gamma ligands including MK886, GW6471 and CP868388 showed a dose-dependent antiviral effect, in the absence of cell toxicity measured by luminescent cell viability assay (CellTiter-GIo®). These PPAR ligands were able to reduce the proportion of core-positive cells by at least 70% compared to vehicle-treated control cells. To investigate at which step(s) of the viral lifecycle PPAR alpha ligands were capable of inhibiting JFH1 infection, Huh7.5.1 cells were treated with 10 μM of MK886, 5 to 10 μM of GW6471 or 100 μM of CP868388 at various time points before, concomitantly to or after JFH1 infection. Intracellular RNA and viral production were measured in the supernatant 48 hours post-infection. The maximum inhibition of infection (−2 Log10 lU/mL in TCID50/mL compared to untreated cells or vehicletreated control cells) was observed when MK886 was present at the early and late steps of JFH1 infection, suggesting that more than one step of the JFH1 lifecycle were blocked. In contrast, CP868388 ligand inhibited only the early step of JFH1 infection (−1.10 Log10 in TCID50/mL), whereas GW6471 ligand had only a weak effect on JFH1 infection (−0.5 to 1.0 Log10 lU/mL in TCID50/mL at 5 μM and 10 μM, respectively), without targeting a specific step. Neither IFN treatment nor JFH1 infection had an effect on PPAR alpha and gamma mRNA and protein expressions. In addition, shRNA-mediated suppression of PDIP1 resulted in a significant reduction of JFH1core positive cells after PPAR alpha ligand treatment, similar to what was observed in Huh/.5.1 cells. Conclusions: PPAR alpha ligands exert antiviral activity against JFH1 infection in a hepatoma cell line. Our findings suggest that the antiviral effect of PPAR alpha ligand is PDIP1-dependent.
Raymond T. Chung - Advisory Committees or Review Panels: Idenix; Consulting: Enanta; Grant/Research Support: Gilead, Merck, Mass Biologic, Gilead
The followinq people have nothinq to disclose: Stephane Chevaliez, Cynthia Brisac, Esperance A. Schaefer, Daniel Wambua, Nikolaus Jilq, Jay Luther, Pattranuch Chusri, Laurent Zablocki, Wenyu Lin, Lee F. Peng, Dahlene N. Fusco
Hepatitis C Virus and Lipid Droplets: Role of Adipose Differentiation-Related Protein in Lipid Droplet Morphology and Viral Life Cycle
Emilie Branche1, Sophie Clement3, Pierre Levy1, Clotilde Parisot1, Stéphanie Conzelmonn1, Francesco Negro2,3
1Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland; 2Gastroenterology and hepatology, University Hospifols of Genevo, Geneva, Switzerland; 3Clinical pathology, University Hospitals of Geneva, Geneva, Switzerland
Background and aims: Hepatitis C virus (HCV) is a positivestrand RNA virus of the Flaviviridae family, whose life cycle is tightly associated with lipid metabolism. HCV assembly and maturation start at the surface of lipid droplets, while viral egress depends on very-low density lipoprotein secretion. In order to better understand the relationship between HCV and lipid metabolism, we analyzed the impact of lipid droplets on HCV life cycle with a particular focus on Adipose Differentiation-Related Protein (ADRP), a lipid droplet-associated protein. Methods: We transduced human hepatoma cells (Huh-7) with a lentiviral vector expressing ADRP and evaluated the impact of ADRP overexpression on (i) lipid droplet morphology and (ii) HCV life cycle and viral particle production in the setting of infection with a cell cultured-derived HCV (full length Jc1 construct). We assessed the effect of ADRP on HCV entry with the HCV pseudoparticles system and by measuring the expression level of HCV receptors (i. e. CD81, Low-Density Lipoprotein Receptor, Scavenger receptor class B member 1, Claudin 1, 〇ccludin, Niemann-Pick disease type C1)by quantitative realtime PCR. Results: ADRP mRNA expression level was increased by 2-fold during the course of Jc1 infection. The lentiviral-mediated overexpression of ADRP induced an increase of the lipid droplet volume without modifying the number of lipid droplets per cell, and this modification in lipid droplet morphology was accompanied by an increase of main lipid droplet components (1.5- and 5-fold increase of triglycerides and cholesterol esters, respectively). The amount of intracellular viral RNA and protein was decreased in cells overexpressing ADRP (by 50% and 30%, respectively). Moreover the infectivity of intracellular HCV particles was also decreased in these cells (by 70%), while the HCV particles production secreted and their infectivity were significantly increased by this overexpression (extracellular HCV RNA level and infectivity were respectively increased by fold and 4-fold). Interestingly, ADRP overexpression likewise increased the HCV entry (by 17-fold) probably through an increase of the entry receptor occludin by approximately 2fold. No change was observed of the expression level of other viral receptors. Conclusion: These findings indicate that the upregulation of ADRP by HCV infection may lead to an increased infectious viral particle entry, suggesting that this LDassociated protein is a critical factor for HCV life cycle.
Francesco Negro - Advisory Committees or Review Panels: Roche, MSD, Gilead, Boehringer ingelheim; Grant/Research Support: Roche, Gilead
The following people have nothing to disclose: Emilie Branche, Sophie Clement, Pierre Levy, Clotilde Parisot, Stephanie Conzelmann
Microsomal triglyceride transfer protein (MTP) inhibitors efficiently block the production of infectious hepatitis C virus (HCV) in primary human adult hepatocytes
Veronique M. Pene, Arielle R. Rosenberg
University Poris Descartes, EA4474 Hepatitis C Virology, Paris, France
Background and Aim. In the blood of patients infected with hepatitis C virus (HCV), infectivity is mainly supported by viral particles associated with triglyceride-rich lipoproteins containing apolipoprotein B (ApoB) and ApoE. These complexes are believed to assemble within the hepatocyte, which is both the primary replication site of HCV and the cell type specialized in the secretion of very-low-density lipoproteins (VLDL). The microsomal triglyceride transfer protein (MTP), which 丨ipidates ApoB, is the rate-limiting enzyme in VLDL biogenesis, and hence a candidate target for therapeutic intervention against HCV infection. However, studies with the classical HCV culture system in the hepatocarcinoma-derived cell line Huh-7 suggested that MTP inhibitors might not efficiently block HCV production unless high, cytotoxic concentrations that also inhibit ApoE secretion are used. Here we have reassessed this question using a most relevant HCV culture system in primary human adult hepatocytes (PHH), which, contrary to Huh-7 cells, secrete authentic VLDL and infectious particles. Methods. PHH were infected with the HCV strain JFH1, and then treated with increasing doses of MTP inhibitors. Cultures were evaluated for production of infectious virus (focus-formation assay), secretion of ApoB and ApoE (enzyme-linked immunosorbent assays), and cytotoxic effects (LDH release assay). Results. The pharmacological MTP inhibitor CP-346086 induced a dose-dependent decrease of infectious HCV production in PHH, reaching up to 95% inhibition at moderate concentrations that did not cause cytotoxicity. As expected, ApoB steady-state expression and secretion were concomitantly decreased, but ApoE secretion remained unaltered. PHH treatment by the grapefruit flavonoid naringenin, known to inhibit MTP activity indirectly through a PPARα-mediated mechanism, also led to a significant reduction of infectious HCV production without any detectable cytotoxic effect. Interestingly, no parallel decrease of either ApoB or ApoE secretion was observed in this case. Conclusion. These data in differentiated human hepatocytes confirm that MTP function is indeed required for production of infectious HCV, yet this requirement may not necessarily involve the role of this enzyme in ApoB lipidation. MTP inhibitors developed primarily for the treatment of lipid metabolism disorders also appear as promising host-targeting drugs to combat HCV infection in complement with directly acting antivirals.
The following people have nothing to disclose: Veronique M. Pene, Arielle R. Rosenberg
Sequence analysis and mode of swine hepatitis E virus infection and replication in the primary human hepatocytes
Yukio Oshiro1, Hiroshi Yasue2, Shouji Ideno3, Shinji Hattori3, Kaoru Sakai3, Osari Suguru4, Kaoru Takeuchi4, Kyosuke Nagata4, Nobuhiro Ohkohchi1
1Department of Surgery, Division of Gastroenterological and hepatobiliary Surgery, and Organ Transplantation, University of Tsukuba, Tsukuba, Japan; 2Animal Genome Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan; 3Japan Blood Products Organization, Kobe, Japan; 4Department of Infection Biology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan
Background and aims: Hepatitis E virus (HEV) is a major cause of epidemic and acute sporadic hepatitis in many developing countries. HEV is believed to undergo zoonotic transmission, with a reservoir in pigs, in industrialized countries. We have recently demonstrated in vitro infection and replication of swine HEV in primary-cultured human hepatocytes, using a genotype 3 HEV. Previous reports demonstrated that genetic changes were found in HEV progenies in the course of its habituation in human cancer line cells, which may bring change of HEV infection spectrum. Therefore, we investigated mutational events and mode of swine HEV infection and replication in the primary-cultured human hepatocytes. Methods: Hepatocytes were primary cultured from the resected normal liver of patients with metastic malignant tumor using collagenase treatment, and the cultured hepatocytes were infected with HEVs (genotype 3) derived from swine. Viral replication was monitored by a strand-specific reverse transcription-polymerase chain reaction assay. Viral replication sites in cells were investigated with in situ hybridization (ISH). Immunofluorescence assay was performed using antibody against HEV 〇RF2 antigen. In addition, the sequences of propagated and inoculated HEV were determined in order to examine whether propagation of swine HEV in the primary-cultured human hepatocytes generate base change in HEV sequence. Results: The HEV RNA increased in cultured medium as well as in cells after infection of HEV to primary-cultured human hepatocytes. ISH using cRNA of HEV as a probe demonstrated existence of genotype 3 HEV RNA in cytoplasm of hepatocytes. The immunofluorescent study revealed the following. The infected cells were found to form “infected cell clusters” and the number of the clusters do not change. However, the number of infected cells in each cluster was found to increase with the passage of culture time. These findings suggest that the HEV infection spread in hepatocytes were mainly attributed to cell-to-cell infection through cell membrane. Sequence analysis demonstrated that there had no sequence difference between inoculated and propagated HEVs. Conclusion: This study demonstrated that no sequence deviation is necessary for swine HEV propagation in primary-cultured human hepatocytes.
The following people have nothinq to disclose: Yukio Oshiro, Hiroshi Yasue, Shouji Ideno, Shinji Hattori, Kaoru Sakai, Osari Suquru, Kaoru Takeuchi, Kyosuke Nagata, Nobuhiro Ohkohchi
Substitution of amino acid 70/91 in the hepatitis C virus core region affects infectious virus production and cell surface expression of MHC class I
Megumi Tasaka-Fujita1, Nao Sugiyama1, Wonseok Kang2, Asako Murayama1, Yasuhiro Asahna3, Naoya Sakamoto4, Takaji Wakita1,Eui-Cheol Shin2, Takanobu Kato1
1Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; 2Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea; 3Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan; 4Department of Gastroenterology and Hepatology, Hokkaido University, Hokkaido, Japan
The amino acid (aa) substitutions at aa70 and/or aa91 in the Hepatitis C Virus (HCV) core region have been reported as a predictor for poor response to pegylated interferon (IFN) plus ribavirin combination therapy (Peg-IFN/Rbv) and hepatocarcinogenesis. However the underlying mechanisms are yet to be elucidated. To assess the effects of these substitutions to the sensitivity for IFN and the life cycle of HCV, we exploited the HCV cell culture system with genotype 1b/2a chimeric virus (TH/JFH- strain) because the clinical observations have been reported in genotype 1b patients. The amino acid substitutions (R/ Q at aa70 and L/M at aa91) were introduced into TH/JFH-1 chimeric virus and designated TH/JFH1-RL, RM, QL, QM. Full length RNA of these chimeric viruses were transfected into HuH7.5.1 cells, and core antigen (Ag) levels in cells and supernatants were measured. Although no significant difference of core Ag was observed in these strains transfected cells, core Ag in supernatants were 10 fold higher in TH/JFH1-RL and -RM than in TH/JFH1-QL and -QM. The infectivity titers in cells and supernatants were lower in TH/JFH-1-QL and -QM as compared with TH/JFH1-RL and -RM. The flow cytometry analysis revealed that the amounts of core protein in HCV positive cells were higher in TH/JFH-1-QL and -QM transfected cells than that in TH/JFH-1-RL and -RM transfected. Thus, we assumed that the infectious virus production was deteriorated in strains with Q at aa70, and, as a result, core protein was accumulated in these strains replicating cells. To assess the effects of this intracellular core protein accumulation to host's immune system, we investigated the cell-surface expression of MHC class I molecule induced by IFN-gamma treatment. The MHC class I expression was substantially attenuated in TH/JFH-1 -QL and -QM transfected cells as compared with TH/JFH-1-RL and -RM transfected cells. In conclusion, the substitution of aa70 in HCV core reduced the efficiency of infectious virus production, and this lower virus production of TH/JFH-1-QL and -QM resulted in accumulation of HCV core protein in cells and suppression for cell-surface expression of MHC class I. These observations may explain the strain-associated resistance to Peg-IFN/Rbv and hepatocarcinogenesis through suppression of antigen presentation and evasion from CD8+ T cell responses.
The following people have nothing to disclose: Megumi Tasaka-Fujita, Nao Sugiyama, Wonseok Kang, Asako Murayama, Yasuhiro Asahina, Naoya Sakamoto, Takaji Wakita, Eui-Cheol Shin, Takanobu Kato
Inhibition of calmodulin-dependent kinase II delta reduces NS5A hyperphosphorylation and HCV replication
Yi-Hung Chen, Ming-Jiun Yu
National Taiwan University, Taipei, Taiwan
Hyperphosphorylation of the non-structural protein 5A (NS5A) has been implicated in hepatitis C virus (HCV) replication. Cellular kinase responsible for NS5A hyperphosphorylation thus might be an alternative antiviral target next to enzymatic viral proteins e. g. NS3 and NS5B. We have previously identified an NS5A phosphorylation site responsible for NS5A hyperphosphorylation. Phosphorylation level of this site increased upon viral infection; In addition, abrogation of its phosphorylation by mutation completely abolished viral replication, indicating its roles in HCV replication. In the present study, we sought to identify kinases responsible for NS5A phosphorylation at this site. Our bioinformatic analysis and the existing chemical proteomics data suggested a role of calmodulin-dependent kinase (CaMKII) in NS5A phosphorylation at this site. Calmodulin inhibitor (W7) inhibited NS5A phosphorylation at this site and reduced HCV RNA levels in infected Huh7.5.1 cells in a dosedependent manner. Similarly, CaMKII specific inhibitor (KN93) reduced NS5A phosphorylation and reduced HCV RNA levels in infected Huh7.5.1 cells in a dose- and time-dependent manner. Reverse transcription plus polymerase chain reaction analysis indicated expression of CaMKII gamma and delta in the Huh7.5.1 cells. Small hairpin RNA based gene knockdown of CaMKII delta not gamma reduced HCV RNA levels in infected Huh7.5.1 cells. We conclude that CaMKII delta may be responsible for NS5A hyperphosphorylation at the identified site and that inhibition of CaMKII reduces NS5A phosphorylation and reduces HCV RNA levels in infected Huh7.5.1 cells. (This work is supported by NSC 101-2324-B-002-022 and NHRI EX10210213-BI, TAIWAN)
The following people have nothing to disclose: Yi-Hung Chen, Ming-Jiun Yu
Hepatitis C virus receptor-specific antibodies are associated with viral clearance in a single-source outbreak
Rajeevkumar G. Tawar1, Michael Roggendorf2, Helga Meisel3, Thomas Berg4, Mirjam B. Zeisel1, Thomos F. Boumert1,5
1Virologie, INSERM 1110, University of Strasbourg, Strasbourg, France; 2Virology University of Essen, Essen, Germany; 3Virology, Charite University Medicine, Berlin, Germany; 4lsektion Hepatologie, University of Leipzig, Leipzig, Germany; 5Pôle Hépato-digestif, Hopifoux Universitaires de Strasbourg, Strasbourg, France
BACKGROUND: Hepatitis C virus (HCV) causes persistent infection in the majority of infected individuals. However, the mechanisms of persistence and clearance are only partially understood. CD81-CLDN1 co-receptor complex plays a pivotal role in initiation and maintenance of infection. A monoclonal antibody targeting the co-receptor complex has been shown to confer protection against HCV infection. AIM: We aimed to study the presence of anti-receptor autoantibodies in HCV infected patients and its correlation to persistence and spontaneous viral clearance. METHODS: Because of the central role of CD81-CLDN1 co-receptor complexes in HCV infection, we used a recombinant soluble CD81/CLDN1 protein to develop a novel sensitive ELISA that could detect low nanomolar concentrations of anti-CD81/CLDN1 antibodies. Using 50 serum samples from healthy individuals as control and a well defined cohort of single-source outbreak of HCV (Pestka, Zeisel et al. Proc. Natl. Acad. Sci.2007) we measured the end-point titers of anti-CD81/CLDN1 antibodies in early phase and late phase serum samples from patients that either spontaneously cleared the virus or developed chronic hepatitis C. RESULTS: HCV infected patients exhibited significantly higher antiCD81/CLDN1 antibody titers compared to healthy individuals (p < 0.0001). Among HCV infected patients, individuals who cleared the virus had higher antibody titers during the acute phase of infection compared to individuals progressing to chronic infection (p = 0.0197). Furthermore, in the majority of patients that resolved hepatitis C, virus-neutralizing antibody titers were associated with anti-CD81/CLDN1 titers. CoNCLUSION: Our data suggest that anti-receptor autoantibodies are produced in the early phase of viral infection and that these antibodies could contribute to spontaneous viral clearance in conjunction with anti-viral responses. Characterization of these anti-receptor autoantibodies may open new avenues to prevent and treat HCV infection.
Michael Roggendorf - Speaking and Teaching: Abbott, novartis
Thomas Berg - Advisory Committees or Review Panels: Gilead, BMS, Roche, Tibotec, Vertex, Jannsen, Novartis, Abbott, Merck; Consulting: Gilead, BMS, Roche, Tibotec; Vertex, Janssen; Grant/Research Support: Gilead, BMS, Roche, īibotec; Vertex, Jannssen, Schering Plough, Boehringer ingelheim, Novartis; Speaking and Teaching: Gilead, BMS, Roche, īibotec; Vertex, Janssen, Schering Plough, Novartis, Merck, Bayer
The following people have nothing to disclose: Rajeevkumar G. Tawar, Helga Meisel, Mirjam B. Zeisel, Thomas F. Baumert
Differential microRNA expression in the liver during the advanced stage of chronic hepatitis C
Rika Horii, Honda Masao, Takayoshi Shirasaki, Hikari Okada, Tetsuro Shimakami, Mikiko Nakamura, Shuichi Kaneko
Kanazawa University Graduate School of Medicine, Kanazawa, Japan
BACKGROUND & AIMS: MicroRNAs (miRNAs) are an important class of small non-coding RNA molecules that bind to their complementary sequence on their target mRNAs, resulting in translational repression. MiRNAs play important roles in development, metabolism, infection, and cancer. In this study, we analyzed the changes of miRNA expression associated with the progression of chronic hepatitis C (CHC). METHODS: Liver biopsy samples were obtained from 54 patients with CHC and patients with a normal liver. All CHC patients were infected with genotype 1b HCV. MiRNAs were obtained from the biopsy specimens, and the expression of 328 miRNAs was determined with the ĪaqMan Real-time PCR detection system using the ĪaqMan MicroRNA Assays Human Panel. The functional relevance of fibrosis-related miRNAs was evaluated in Lx- cells, a human stellate cell line, by the overexpression or knocking down of specific miRNAs using mimic-miRNA or antimiRNA. HCV replication was evaluated in Huh-7.5 cells using the infectious genotype 1a clone pH77S.3/Gluc2A with a Gaussia reporter gene. HCV translation (HCV-IRES) activity was monitored in the stably transformed IRES reporter cell line RCF26.. RESULTS: The expression of 55 miRNAs was significantly different between patients with early stage fibrosis (F1-2) and advanced stage fibrosis (F3-4), and the prediction performance was 83% accurate according to the support vector machine algorithm. We focused on the 7 most differentially expressed miRNAs: miR-10a, −19a, −27a, −195, −199a, −214, and −218. Interestingly, the expression of these miRNAs, except miR-19a, was significantly up-regulated in Huh-7.5 cells following HCV infection. Furthermore, some of these miRNAs—miR-10a, −199a, and −214—are potential profibrogenic miRNAs. Genechip analysis showed that knocking down miR-214 significantly suppressed the expression of genes of the cytoskeleton and cell adhesion groups, while it also increased protein translation in Lx-2 cells. The overexpression of miR-10a, −27a, −195, −199, and −214 significantly repressed HCV replication in Huh-7.5 cells, while miR-19a and −218 had no effect on HCV replication. Interestingly, miR-10a, −199, and −214 significantly suppressed HCV translation. CONCLUSIONS: Expression analysis of miRNAs in the liver of advanced CHC patients identified predictive miRNAs that were related to the fibrosis stage of the liver. These miRNAs were induced by HCV infection and participate in the progression of fibrosis and the induction of hepatocyte dysfunction. Conversely, HCV replication was repressed by these miRNAs, and this may help to keep the virus load low and establish a prolonged and persistent HCV infection.
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan
The following people have nothing to disclose: Rika Horii, Honda Masao, Takayoshi Shirasaki, Hikari Okada, Tetsuro Shimakami, Mikiko Nakamura
Rs117648444 nonsynonymous variant in INFL4 gene impacts on HCV RNA decline during the first 4 weeks of Pegylated Interferon and Ribavirin therapy in HCV-1 IL28B rs12979860 CT/TT patients
Enrico Galmozzi, Alessio Aghemo, Elisabetta Degasperi, Roberta D'Ambrosio, Roberta Soffredini, Eleonora Grassi, Stella De Nicola, Massimo Colombo
Fondazione IRCCS Cá Granda Ospedole Moggiore Policlinico, Universita degli Studi di Milano, Milan, Italy
Background and aim. Chromosome 19q13.13 contains a transiently induced gene region harboring a dinucleotide variant ss469415590 (TT or ΔAG) in high linkage disequilibrium with rs12979860, a genetic marker of outcome to interferon (IFN)-based therapy of hepatitis C virus (HCV). In presence of the ss469415590[ΔG] frameshift variant, this region encodes the novel interferon-^4 protein (INFL4) which is moderately similar to IFNL3 (IL28B). on the other hand the ss469415590[TT] allele eliminates INFL4 production. Since three nonsynonymous variants found within IFNL4 gene (rs73555604, rs142981501 and rs11/648444) could potentially modulate virological responses in carriers of the ss469415590[G] IFNL4-generating allele, we sequenced IFNL4 in a well characterized cohort of chronic hepatitis C (CHC) patients. Methods. Direct sequences of IFNL4 gene was performed by Sanger method in 103 HCV-1 patients treated with pegylated (peg)IFN and Ribavirin (Rbv) for 48 week. Results. The distribution of the ss469415590 genotype (28% for TT/TT, 58% for TT/AG and 14% for AG/AG) matched that of the rs12979860 variant (28% for CC, 59% for CT and 13% for TT) confirming their high linkage disequilibrium (r2=0.94). As 30% of subjects carrying the unfavorable ss469415590[ΔG] allele included the minor allele of rs117648444 nonsynonymous variant (p. Pro70Ser), we stratified the 74 carriers of IFNL4-generating allele into wildtype (WT) and mutated (MUT) patients. MUT patients had a more pronounced mean HCV RNA decline at week 4 of therapy compared to WT (2.2 Log10 lU/mL vs 1.69 Log10 lU/mL, p=0.02), that translated in more patients achieving a rapid virological response (MUT:14% vs WT: 0%, p=0.02) and fewer patients experiencing a less than 1 Log10 IU/ml decline (MUT: 5% vs WT: 21%, p=0.1). However, the sustained virological response rates between MUT and WT carriers did not reach the limit of statistical significance (55% vs 41% p=0.3). Conclusion. Our findings confirm the strong linkage between rs12979860 and ss469415590 variants, and show that the minor allele of the rs117648444 nonsynonymous variant (p. Pro70Ser) in IFNL4 defines a subset of IL28B unfavorable carriers (rs12979860 CT/TT) with a faster HCV RNA decline in the first 4 weeks of PeglFN/Rbv therapy.
Alessio Aghemo - Advisory Committees or Review Panels: Roche, Janssen; Grant/Research Support: Gilead Sciences, Roche; Speaking and Teaching: MSD, Roche, Janssen
Massimo Colombo - Advisory Committees or Review Panels: BRISTOL-MEYERSSQUIBB, SCHERING-PLOUGH, ROCHE, GILEAD, BRISTOL-MEYERS-SQUIBB, SCHERING-PLOUGH, ROCHE, GILEAD, Janssen Cilag, Achillion; Grant/Research Support: BRISTOL-MEYERS-SQUIBB, ROCHE, GILEAD, BRISTOLMEYERS-SQUIBB, ROCHE, GILEAD; Speaking and Teaching: Glaxo Smith-Kline, BRISTOL-MEYERS-SQUIBB, SCHERING-PLOUGH, ROCHE, NOVARTIS, GILEAD, VERTEX, Glaxo Smith-Kline, BTISTOL-MEYETS-SQUIBB, SCHERING-PLOUGH, ROCHE, NOVARTIS, GILEAD, VERTEX
The followinq people have nothing to disclose: Enrico Galmozzi, Elisabetta Deqasperi, Roberta D'Ambrosio, Roberta Soffredini, Eleonora Grassi, Stella De Nicola
Overexpression of GRIM19 suppresses hepatitis C virus replication through the alteration of lipid metabolism by enhancing AMPK activity
Jung-Hee Kim1, Wonhee Hur1, Jung Eun Choi1, Eun Byul Lee1, Tian Zhu Li1, Sung Woo Kim1, Sung Woo Hong1, Young Ki Lee1, Sung Min Kim1, Joon Ho Lee1, Sung Won Lee1, Pil Soo Sung2, Eui-Cheol Shin2' Seung Kew Yoon1
1Catholic Liver Research Center, Depart ment of Internal Medicine, The Catholic University of Korea, Seoul, Republic of Korea; 2Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea
Background/Aims: It is well known that many host factors are involved in the life cycle of hepatitis C virus (HCV). One of them is signal transducer and activator of transcription 3 (STAT3) as a pro-viral factor. It has been reported that STAT3 is activated in HCV replicating cells by interacting with HCV core protein. Continuously activated STAT3 is related to viral pathogenesis by playing the important roles in cell growth, anti-apoptosis and cell transformation. Recent studies have shown that gene associated with retinoic-interferon-induced mortality 19 (GRIM19), mitochondria-resident protein, both interacts with and negatively regulates STAT3. In this study, we investigated the inhibitory effect of GRIM19 overexpression on HCV replication and its related molecular mechanism. Methods: The expression level of GRIM19 was measured in Huh7 cells harboring HCV replicon (FR1 and SR1) or tissues from patients with chronic HCV infection by Western blot analysis. To define the effect of GRIM19 overexpression on inhibiting HCV replication, the level of HCV RNA was determined by quantitative real-time RT PCR in GRIM19-transfected FR1 or SR1 cells. In addition, in order to confirm the change of lipid accumulation in GRIM19-transfected cells, the intracellular lipid levels were quantitatively measured by Nile Red staining, and the distribution of lipid droplets were visualized by transmission electron microscopy. The expression of cellular proteins modulated by GRIM19 overexpression was tested by Western blot analysis. Results: In the present study, GRIM19 expression was down-regulated not only in FR1 and SR1 cells but also in tissues of the patients with chronic HCV infection. Furthermore, our results showed that GRIM19 overexpression significantly decreased lipid accumulation in oleic acid-treated cells and reduced HCV RNA replication in FR1 and SR1 cells to 40∼60 %. Ectopically expressed GRIM19 in HCV replicating cells enhanced the activity of AMPactivated protein kinase (AMPK), a key regulator of lipid metab-olism. Conclusion: Our results demonstrated that HCV downregulated the level of GRIM19 to maintain the suitable microenvironment for its replication. Also, overexpression of GRIM19 reduced HCV replication by abrogation of lipid accumulation though regulation of AMPK pathway. In conclusion, these results suggest that GRIM19 might be exploited for the development of novel antiviral agents.
The following people have nothing to disclose: Jung-Hee Kim, Wonhee Hur, Jung Eun Choi, Eun Byul Lee, Tian Zhu Li, Sung Woo Kim, Sung Woo Hong, Young Ki Lee, Sung Min Kim, Joon Ho Lee, Sung Won Lee, Pil Soo Sung, Eui-Cheol Shin, Seung Kew Yoon
Cross-sectional Assessment of 1500 Clinical Samples Submitted for HCV NS3/4A Protease Inhibitor Drug Resistance Testing in the US
Sunny S. Choe1, Joseph M. Volpe1, Jacqueline D. Reeves2, Wei Huang2, Mojgan Haddad3, Christos J. Petropoulos2,1, Charles M. Walworth1
1Medical Affairs and Education, Monogram Biosciences, South San Francisco, CA; 2Virology R&D, Monogram Biosciences, South San Francisco, CA; 3Bioinformatics/Biostatistics, Monogram Biosciences, South San Francisco, CA
Purpose: Attributes of the first 500 patient samples tested in a commercially available genotypic NS3/4A protease inhibitor (PI) resistance assay for HCV genotype 1(GT1) were previously reported. This study compares telaprevir (TVR) and boceprevir (BOC) resistance trends in the first 1500 samples to prior results and examines the prevalence of Q80 substitutions, which are not associated with resistance to TVR or BOC, but are associated with resistance to simeprevir (SMV), a second generation HCV PI. Methods: HCV GT1a or GT1b patient samples with viral loads > 2000 lU/mL were sent to Monogram Biosciences for PI resistance analysis using the HCV GenoSure® NS3/4A resistance assay. Briefly, the entire nonstructural protein 3 (NS3) and 4A (NS4A) region of HCV was amplified by RT-PCR using GT1 a or GT1 b specific primers, analyzed by population sequencing and compared to either the H77 (GT1a) or Con 1 (GT1b) reference sequence. Resistance-associated variants (RAVs) were identified and a prediction of drug susceptibility was derived using a rules-based algorithm. The HCV genotype of the NS3/4A region was also determined. Results: The trends observed in the initial 500 samples remained consistent with the addition of 1000 more samples. of the 1500 samples analyzed, 77% were GT1a and 23% were GT1 b. Overall predicted resistance to both TVR and BoC was 22%; 20% and 21% for TVR and BOC, respectively, in GT1a patient samples, but only 2% and 3%, respectively, in GT1b samples. The most commonly observed RAVs for both drugs were R155K (14.7%), V36M (13.4%) and T54S (4.9%). These variants were often present in combinations, with V36M+R155K (10.4%), T54S+R155K (2.2%) and V36M+T54S+R155K (1.2%) occurring most often. The combination of V36M+T54S was seen only in the triple variant, V36M+T54S+R155K. Q80 substitutions were seen in 34.2% of GT1a patients and 1.3% of GT1b patients. Q80K, the most frequent substitution, was seen in 32.3% of GT1a patient samples, but only 0.1% of GT1b samples. The combination of Q80K+R155K was seen in GT1 a samples only, at a frequency of 6.0%. Conclusions: The analysis of TVR and BoC RAVs among the first 1500 samples tested is consistent with that of the first 500. Our findings demonstrate a higher prevalence of HCV PI RAVs among GT1a versus GT1b samples. For TPV and BOC RAVs this is consistent with a higher genetic barrier for GT1b viruses. Q80K substitutions were frequently observed, which may significantly impact treatment decisions utilizing SMV. These RAVs were also observed during clinical trials. These findings support the consideration of baseline NS3/4A resistance testing prior to the initiation of SMVcontaining regimens.
Sunny S. Choe - Employment: Monogram Biosciences Joseph M. Volpe - Employment: Monogram Biosciences Jacqueline D. Reeves - Employment: Monogram Biosciences Wei Huang - Employment: monogram biosciences Mojgan Haddad - Employment: Monogram Biosciences
Christos J. Petropoulos - Employment: Monogram Biosciences, LabCorp; Management Position: Monogram Biosciences, LabCorp; Patent Held/Filed: Monogram Biosciences, LabCorp; Stock Shareholder: LabCorp
Charles M. Walworth - Employment: Monogram Biosciences
IFNL4 genotype is in strong linkage disequilibrium with IL28B genotype and predicts peg-interferon plus ribavirin treatment outcomes: an independent validation study
Jacinta A. Holmes1,2, Mario Congiu2, Sara Bonanzinga3, Manjeet K. Sandhu1, Sally Bell1, Tin Nguyen1, David M. Iser1, Kumar Visvanathan2,4, William Sievert5, Scott Bowden3, Paul V. Desmond1,2, Alexander J. Thompson16
1Gastroenterology, St Vincent's Hospital, Fitzroy, VIC, Australia; 2Medicine, St Vincent's Hospital, Universify of Melbourne, Melbourne, VIC, Australia; 3Moleculor Microbiology, Victorian Infectious Diseases Reference Laboratory, Melbourne' VIC, Australia; 4lnfecfious Diseases, St Vincents Hospital, Melbourne, VIC, Australia; 5Gastroenterology Monosh Medical Centre, Monash University, Melbourne, VIC, Australia; 6Gastroenterology, Duke University Medical Centre, Duke Clinical Research Institute, Durham, NC
BACKGROUND: In 2009, IL28B genotype (gt) was identified as the strongest baseline predictor of peginterferon+ribavirin (PR) response in HCV1. In 2013, a novel dinucleotide variant in interferon-lambda-4 (IFNL4, ss469415590, ΔG/TT), in high linkage disequilibrium (LD) with IL28B polymorphism, was proposed to be the causal variant. IFNL4 gt was a better predictor of sustained virological response (SVR). We have performed the first independent validation study of the association between IFNL4 gt, IL28B gt, and PR treatment outcomes in Australian HCV1/3 patients. METHODS: HCV1/3 patients who received PR were included. IL28B (rs12979860) and IFNL4 (ss469415590) gts were determined (ĪaqMan allelic discrimination kit, custom designed primers where testing unsuccessful). IFNL4 gt was correlated with rapid virological response (RVR) and SVR, and compared to IL28B gt using logistic regression modeling and LD calculation. RESULTS: 270 PR treatment patients were included: 56% HCV1, 44% HCV3. Self-reported ethnicity for HCV1 was 79% Caucasian and 20% Asian, and for HCV3 was 90% Caucasian and 3% Asian. Overall SVR rates were 50% (HCV1) and 82% (HCV3). IFNL4 gt could not be determined in 31 patients, and DNA re-extraction +/- concentration was required. For HCV1, IFNL4 gt frequency was 45%, 43% and 13% for TT/TT, TT/ΔG and ΔG/ΔG, and LD with rs12979860 was very high (0.98). The TT/TT IFNL4 gt was strongly associated with RVR (TT/TT 46% vs TT/ΔG 11% vs ΔG/ΔG 0%, p<0.001) and SVR (TT/TT 78% vs TT/ΔG 28% vs ΔG/ΔG 21%, p<0.001). In HCV3, IFNL4 gt distribution was 42%, 43% and 15% for TT/TT, TT/ΔG and ΔG/AG, respectively, and LD with rs12979860 was high (0.98). RVR was highest in TT/TT IFNL4 gt and lowest in AG/aG IFNL4 gt patients (74% vs 59% vs 50%, p=0.085). Similarly, SVR rates were highest in TT/TT patients (90%) and lower in TT/AG (77%) and ΔG/ΔG (72%) patients (p=0.117), similar to IL28B gt observations. Only 8 patients had discordant IL28B and IFNL4 gts (Table). In these patients, IFNL4 gt more accurately predicted treatment outcome. In a logistic regression model, IFNL4 gt, HCV gt, HCV RNA and ALT were independent predictors of SVR. CONCLUSIONS: This is the first independent validation study to confirm the strong association between IFNL4 gt and PR response in HCV1. Our data confirms that IFNL4 and IL28B gts are in strong LD. The clinical utility of IFNL4 gt for predicting SVR was comparable to that of IL28B gt.
Sally Bell - Speaking and Teaching: MSD, Roche, BMS
William Sievert - Advisory Committees or Review Panels: Merck, Janssen, AbbVie, Gilead; Speaking and Teaching: Bristol-Myers Squibb, Merck
Paul V. Desmond - Advisory Committees or Review Panels: Jansen, Roche, BristolMyers Squibb, Merk, Giliad, Jansen, Roche, Bristol-Myers Squibb, Merk, Giliad; Speaking and Teaching: Roche, Roche
Alexander J. Thompson - Advisory Committees or Review Panels: Merck, Inc, Roche, Janssen (Johnson & Johnson), BMS, GSK Australia, Novartis, GILEAD Sciences, Inc; Consulting: GILEAD Sciences, Inc; Grant/Research Support: Merck, Inc, Roche, GILEAD Sciences, Inc; Speaking and Teaching: Merck, Inc, Roche,
The following people have nothing to disclose: Jacinta A. Holmes, Mario Congiu, Sara Bonanzinqa, Manjeet K. Sandhu, Tin Nguyen, David M. Iser, Kumar Visvanathan, Scott Bowden
Kinetics of miR-122 expression in the liver during acute HCV infection
Youkyung Choi1, Hans P. Dienes2, Kris Krawczynski1
1Division of Viral Hepatitis, NCHHSTP, Centers for Disease Control and Prevention, Atlanta, GA; 2Institute of Pathology, Univeristy of Cologne, Cologne, Germany
The relationships among micro RNA 122 (miR-122) expression in the liver, hepatitis C virus (HCV) replication and hepatic damage were analyzed in three chimpanzees observed for 180 days after inoculation with HCV genotype 1a. Levels of miR-122 in the liver and serum were measured by real-time RT PCR in serial liver biopsies and serum samples. Hepatic miR-i22 levels were normalized separately for each of three chimpanzees with small RNAs and microRNAs that are endogenous to and stably expressed in the liver. A two- to 4-fold rise in hepatic miR-122 levels was observed in all three chimpanzees during the first 4 weeks of HCV infection when HCV titers in the liver and serum increased rapidly, in concordance with in vitro data indicating the miR-122 significance for HCV replication. Between 10 to 14 weeks after inoculation, when hepatic and serum HCV RNA titres exceeded 3 logs and serum alanine aminotransferase (ALT) activity was elevated, hepatic miR-122 levels were in decline. Cumulative data derived from all three chimpanzees from 180 days of observation documented an inverse (negative) correlation between hepatic miR-122 and HCV RNA in the liver and serum and positive correlation between level of serum miR-122 and HCV replication. Thereafter, rise of miR-122 levels during HCV clearance and serum ALT normalization occurred. These data suggest a tri-phasic relationship among hepatic miR-122 expression, HCV replication and hepatic destruction. It was particularly apparent in one chimpanzee. The findings imply complexities in the virus-host interaction during the acute phase of HCV infection.
The following people have nothing to disclose: Youkyung Choi, Hans P. Dienes, Kris Krawczynski
HMG-CoA Reductase Inhibition Alters the Lipidome and Infectious Properties of HCV Virus Particles
Lee F. Peng1, James Meixiong1, Amy Deik2, Esperance A. Schaefer1/ Nikolous Jiig1, Pattanuch Chusri1, Cynthia Brisoc1, Stephane Chevaliez1, Chuanlong Zhu1, Jay Luther1, Daniel Wambua1, Dahlene N. Fusco1, Wenyu Lin1, Clary B. Clish2, Raymond T. Chung1
1GI Unit, Massachusetts General Hospital, Boston, MA; 2The Broad Institute, Cambridge, MA
Background: Hepatitis C virus (HCV) chronically infects over 170 million people worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. The dependence of HCV on host lipid metabolism is extensive. We have previously reported that inhibition of HMG-CoA reductase suppresses HCV replication. It is not known whether HMG-CoA reductase inhibition also alters overall viral infectivity or changes the lipid composition of the virion particle. We sought to assess the effect of HMG-CoA reductase inhibition on other steps of the HCV lifecycle and on the lipid composition of HCV particles. Methods: Using liquid chromatography tandem mass spectrometry (LCMS), we performed lipidomic analyses of HCV particles. We also assessed the effect of HMG-CoA reductase inhibition on non-replicative HCV lifecycle steps. Results: In addition to decreasing HCV replication, inhibition of HMG-CoA reductase leads to the formation of HCV particles with impaired overall infectivity. These particles also exhibit decreased entry into hepatocytes. The lipidome of HCV particles is altered by HMGCoA reductase inhibition, resulting in lower cholesterol content with compensatory increases in other lipid species, including triacylglycerols, sphingomyelins, and phosphatidylcholines. Conclusions: HMG-CoA reductase inhibition not only inhibits viral replication, but also alters the functional and physical properties of HCV particles. The decreased cholesterol content of the virions is the likely basis for their altered functional properties. These findings offer additional rationale for use of HMGR inhibitors as adjunctive, host targeted antivirals.
Raymond T. Chung - Advisory Committees or Review Panels: Idenix; Consulting: Enanta; Grant/Research Support: Gilead, Merck, Mass Biologic, Gilead
The following people have nothing to disclose: Lee F. Peng, James Meixiong, Amy Deik, Esperance A. Schaefer, Nikolaus Jilq, Pattranuch Chusri, Cynthia Brisac, Stephane Chevaliez, Chuanlonq Zhu, Jay Luther, Daniel Wambua, Dahlene N. Fusco, Wenyu Lin, Clary B. Clish
Did Ultra-Deep pyrosequencing of the NS3 region can help to predict sustained virological response to pegylated interferon-ribavirine-anti-protease triple therapy in previously treated chronic HCV genotype 1 infection?
Sylvie Larrat1, Om Kulkarni3, Jean-Baptiste Claude4, Rejane Beugnot1, Mickaël Blum3, Katia Fusillier1, Agnes Plages2, Alice Marlu2, Anne Signori-Schmuck1, Olivie Francois3, Jean-Pierre H. Zarski2, Patrice Morand1, Vincent Leroy2
1Laboratoire de Virologie - UMI 3265 UJF-EMBL-CNRS, CHU de Grenoble, Grenoble, France; 2Clinique Universitaire d'Hépato-Gastroentérologie/Inserm U823' CHU de Grenoble, Grenoble, France; 3Equipe Biologie Comp: fa tionnelle et Mathématique, Laboratoire TIMC IMAG, Grenoble, France; 4Genostar - Bioinformatics solutions, Grenoble, France
Background & Aims: Despite sustained viral response (SVR) gain provided by the use of NS3/4A protease inhibitors telaprevir (TPV) and boceprevir (BoC), failures occur and are mostly associated with emergence of resistant strains. The aim of this study was to determine if baseline analysis of the NS3 viral region using ultra-deep pyrosequencing (UDPS) could help to predict SVR to triple therapy. Methods: Forty genotype 1 patients failing to achieve a SVR with Peg-IFNa + Ribavirin combination (null responders: n=18; partial responders: n=14, relapsers: n=8 and retreated with triple therapy adding BOC or TPV were included. Their main characteristics were: mean age 55+/-8 years, 47.5% subtype 1a, 77.5% F3-F4. Baseline UDPS of the NS3-protease viral gene was performed on plasma and peripheral blood mononuclear cells (PBMC). Sequences obtained were analyzed in terms of resistance mutations with a threshold of 1% determined by using a control transcript. Heterogeneity of quasispecies was evaluated by the calculation of Shannon Entropy (SE). Results: Baseline mutations were found in 4 patients who achieved SVR with triple therapy and in 4 patients who did not. For these last patients, mutations were already major in three patients and persisted until viral breakthrough. In the fourth patient, the mutated population accounted for only 1.4% of the total viral population at baseline but dramatically rose upon failure. In two patients, minor mutations were found in PBMC while not in plasma, and corresponded to mutations observed at the viral rebound. Compartmentalization between plasma and PBMC was confirmed with the analysis of the obtained sequences. More broadly, the NS3 quasipecies heterogeneity expressed as SE was significantly lower at baseline in patients achieving SVR compared to nonSVR (SE= 26.98016.64 x 10-3 vs 44.93 ± 19.58 x 10-3, p=0, 0049). By multivariate analysis, independent predictors of SVR were F0F2 fibrosis stage (OR =13.3, CI95% 1.25141.096, p<0.03) and SE below median value (oR=5.4, CI95% 1.22-23.87, p<0.03). Conclusion: More than the presence of baseline minor mutations in plasma or in PBMC, NS3 viral heterogeneity determined by UDPS is an independent factor of SVR in previously treated patients receiving a triple therapy with an anti-protease drug. This parameter could be included in a score predicting response to therapy.
Jean-Pierre H. Zarski - Advisory Committees or Review Panels: BMS, Gilead, Janssen Cilag, BMS, Gilead, Janssen Cilag; Consulting: Roche, Scherring Plough, Novartis, Roche, Scherring Plough, Novartis; Speaking and Teaching: Siemens
Vincent Leroy - Board Membership: roche, merck, gilead, bms, roche, merck, gilead, bms, roche, merck, gilead, bms, roche, merck, gilead, bms; Consulting: jansen, jansen, jansen, jansen; Grant/Research Support: roche, gilead, bms, roche, gilead, bms, roche, gilead, bms, roche, gilead, bms; Speaking and Teaching: bms, merck, gilead, roche, bms, merck, gilead, roche, bms, merck, gilead, roche, bms, merck, gilead, roche
The following people have nothing to disclose: Sylvie Larrat, Om Kulkarni, JeanBaptiste Claude, Rejane Beugnot, Mickael Blum, Katia Fusillier, Agnes Plages, Alice Marlu, Anne Signori-Schmuck, Olivier Francois, Patrice Morand
Hepatitis C virus alternate reading frame suppresses type I interferon response mediated by RIG-I/MDA-5 in hepatocytes
Seung Bum Park, Bhargav Koduru, Scott Seronello, Wasima Mayer, David M. Ojcius, Jinah Choi
University of California, Merced, Merced, CA
Hepatitis C virus (HCV) actively evades the host type I interferon (IFN) response through proteolytic inactivation of pattern recognition receptor (PRR) signaling proteins by NS3/4A protease, among other mechanisms. However, the mechanisms of how HCV evades host innate immune response are not completely understood. In this study, we show that another HCV element that can suppress PRR signaling independently of NS3/4A. Point mutations in the HCV core protein-coding sequence that disrupt the −2/+1 frame coding for frameshift/alternate reading frame protein (F/ARFP) without affecting the core protein sequence of the zero frame or substantially altering RNA stem loops V and VI, enhanced type I IFN induction by HCV. This effect was diminished in Huh7.5 cells that have defective retinoic-acid-inducible gene inhibitor (RIG-I), by decreasing RIGI expression with siRNA in Huh7 cells, and by adding F/ARFP back through trans-complementation. Furthermore, comparison of HCV RNA pathogen-associated molecular pattern and poly(IC)stimulated type I IFN responses suggested that the suppression can occur independently of and in combination with HCV NS3/4A, and that the viral element involved in the suppression is likely to be F/ARFP. The −2/+1 frame mutants, on the other hand, were not resistant to exogenous IFNalpha. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress the type I IFN response occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of PRR modulation by HCV and suggests a biological function of the HCV alternate frame in the modulation of host innate immunity.
The following people have nothing to disclose: Seung Bum Park, Bhargav Koduru, Scott Seronello, Wasima Mayer, David M. Ojcius, Jinah Choi
IFN-α and RBV Combination Synergistically Inhibit HCV IRES Translation at the level of polyribosome formation
Rajesh Panigrahi1, Sidhartha Hazari4, Sruti Chandra1, Partha K. Chandra1, Craig E. Cameron2, Zhuhui Huang3, Luis A. Balart1, Srikanta Dash1
1Pathology and Laboratory Medicine, TULANE MEDICAL SCHOOL, NEW ORLEANS, LA; 2Deporfmenf of Biochemistry ond Molecular Biology, Penn State University, Pennsylvanio, PA; 3Hepatitis Research Program,, Southern Research Institute, Frederick, MD; 4Department of Pathology and Laboratory Medicine, UC Davis Medical Center, California, CA
Background: Although chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFNa) and ribavirin (RBV) over a decade but the synergy antiviral mechanism is not understood. Aim: To determine the synergy antiviral mechanisms of IFN-a and RBV combination treatment using HCV genomic and sub-genomic replication model in cell culture. Methods: Persistently infected Huh-7.5 cells and subgenomic replicon cell line were treated with IFN-a, RBV alone and in combination. The antiviral efficacy in the combination treatment was quantitatively measured by Renilla Luciferase and Green Fluorescence Protein (GFP) expression. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was also investigated. The synergy antiviral effect of IFN-a and RBV combination treatment was verified using both the median effect principle using CalcuSyn Software and three-dimensional modeling using MacSynergy II Software. Results: RBV combination with IFN-α efficiently inhibits HCV replication in a replicon cell line and in an infected cell culture. Our results demonstrate that IFN-α, interferon-lambda (IFN-入)and RBV each inhibits the expression of HCV-IRES GFP and they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. IFN-a and RBV treatment resulted in an arrest of the majority of HCV IRES-GFP mRNA in the monosome peaks and reduction in the polysome fractions. The combination treatment of RBV along with IFN-a or IFN-λ was highly synergistic with combination index <1. We show that IFN-a treatment induced the levels of PKR and eIF2a phosphorylation that prevented ribosome loading to the HCV IRES GFP mRNA in Huh-7 cells. Silencing of PKR expression in Huh-7 cells prevented inhibitory effect IFN-a on HCV IRES-GFP expression. on the other hand, RBV also blocks polyribosome loading of HCV-
IRES GFP mRNA through the inhibition of cellular IMPDH activity, and induction PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented ribavirin induced HCV IRES mediated GFP translation. Conclusions: We demonstrated that the synergy antiviral mechanisms of IFN-α and RBV inhibit HCV IRES mediated translation through prevention of polyribosome formation. IFN-α and RBV combination treatment inhibits HCV IRES translation by using two different mechanisms involves PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH. Acknowledgment: This work was supported from NIH grant CA127481, CA089121.
Craig E. Cameron - Consulting: Gilead, Alios; Grant/Research Support: Bristol Myers Squib, Indigo Biosciences
Luis A. Balart - Advisory Committees or Review Panels: Genentech, Genentech; Grant/Research Support: Merck, Genentech, Bayer, conatus, Ocera, Hyperion, Gilead Sciences, Bristol Myers Squibb, Mochida, Eisai, Vertex, Merck, Genentech, Bayer, Conatus, Ocera, Hyperion, Gilead Sciences, Bristol Myers Squibb, Mochida, Eisai, Vertex, takeda, GI Dynamics; Speaking and Teaching: Merck, Merck, Merck, Merck
The following people have nothing to disclose: Tajesh Paniqrahi, Sidhartha Hazari, Sruti Chandra, Partha K. Chandra, Zhuhui Huang, Srikanta Dash
Antiviral Toxicity and PK Studies of Novel Picomolar
Zneng-Yun J. Zhan1,2, Xudong Wu2, Hua Yan1
1AB Pharma Ltd., Shanghai, China; 2Zannan SciTech Co. Ltd. Shanghai, China
BACKGROUND: NS5A of hepatitis C virus (HCV) is a nonstructural protein that is considered essential for viral replication and infectivity. It has been intensively studied for globally urgent need of new effective HCV inhibitors since 2002, and we have developed several of novel antiviral compounds highly potent and selective as an NS5A inhibitor. RESULTS: This presentation discloses development of a novel optimized antiviral compound as one of the most competitive HCV NS5A inhibitors. It was found that a novel HCV inhibitor ZN6168 was not only highly potent (EC50: picomolar potency, 1-50pM for GT-Ia, GT-Ib and GT-IIa, respectively) but also showed excellent PK and TK in all rats and monkeys. There was no test-article related side effects determined in combination of ZN6168 with different kinds of potential targets such as hERG, Cytochrome P450, etc, respectively. The metabolic stability in human liver and plasma is very good (T1/2: >120min). Regarding the safety issue of ZN6168, there was no any death, no any serious drug-related toxicity and side effects observed during different toxicity studies in rats and monkeys with oral dosing levels 50-1000mg/kg/day, respectively. Moreover, there were no any adverse events observed in the blood examination, clinical biochemistry, organ coefficient, electrocardiogram (ECG), pathological and tissue microscopic examinations in rats and/or monkeys, respectively. CONCLUSIONS: All antiviral toxicity and PK results indicated that ZN6168 had not only picomolar potency, but also excellent safety and PK. Its metabolic stability in human liver microsomes was very good and its half-life time was more than two hours, so ZN6168 could be formulated as once-daily dosing tablet. Overall, ZN6168 is selected as a lead compound for more preclinical studies, and it is ongoing for us to develop a leading anti-HCV therapy with not only an NS5A inhibitor but also our another best-in-class NS3 protease inhibitor ZN2007.
Zhenq-Yun J. Zhan - Grant/Research Support: Company
The following people have nothing to disclose: Xudong Wu, Hua Yan
Development of a Panel of Genotypic Specific Resistance Assays for the Detection of Drug Resistance Associated Amino Acid Changes in Domain 1 of HCV NS5a
Adele L. McCormick, Lawrence T. Wang, Ana Garcia-Diaz, Malcolm J. Macartney, Tim C. Conibear, Clare L. Booth, Dianne N. Irish, Daniel P. Webster, Tanzina Haque
Virology, Royal Free Hospital, London, United Kingdom
Background: NS5a, a membrane associated phosphoprotein, plays a crucial role in regulating viral replication and host cell interactions. NS5a inhibitors which target Domain I of HCV NS5a protein, have demonstrated promising antiviral activity against a variety of HCV genotypes, and provide an interferonfree treatment regimen. We have developed a series of NS5a genotypic resistance assays specific for HCV genotypes 1a, 1b, 2, 3 and 4 to assess prevalence of NS5a amino acid (aa) changes in Domain I at positions M28T, Q30H/R, L31F/M/V, P32L and Y93C/H/N, known to confer reduced susceptibility to certain NS5a inhibitors. Methods: HCV RNA was quantified using Abbott real-time HCV quantitative assay and genotyped using Abbott real-time HCV genotyping assay and primers directed towards 5'UTR or NS5b of HCV. The first 213 amino acids of domain I of NS5a was amplified from plasma viral RNA, using reverse transcription and PCR amplification in a one-step RT-PCR system (Qiagen), followed by a Hot Star Taq nested PCR (Qiagen) incorporating genotype/subtype specific primers for HCV 1a; 1b, 2; 3 and 4 in both PCRs. Annealing temperature gradients, magnesium, primers and dNTPs were titrated to provide optimal PCR conditions. PCR amplicons were purified, sequenced and consensus sequences aligned in SeqScape v2.5, submitted to NCBI Blast for identification and translated using BioEdit. Results: PCR amplicons were generated from plasma derived HCV viral RNA loads of 25 IU/ml for notypes 2 and 3; 50 IU/ml for genotypes 1a and 4 and 250 ml for genotype 1b. HCV genotyping based on sixty NS5a sequences were comparable with Abbott real-time, except for one discordant, which Abbott genotyped as 1b but based on NS5a sequence data, genotyped as 1 a. Preliminary experiments of aa changes associated with resistance to NS5A inhibitors, showed 2/29 (6.9%) genotype 1a HCV infected patients harboured changes at either codon 30 (Q30R) or 93 (Y93H/Y) respectively and 12.5% individuals infected with HCV 1b harboured the mutation Y93H. None of NS5a genotype 3 showed changes at the relevant aa positions and genotype 2 specimens harboured L31M in certain patients. Conclusions: Utilisation of our validated genotypic specific NS5a resistance assays, we were able to identify albeit at low frequencies, natural polymorphisms which are known to confer resistance to certain NS5a inhibitors. In addition, we observed a low discordance rate regarding genotyping when NS5a, 5'UTR and NS5b sequences were compared.
Daniel P. Webster - Grant/Research Support: ViiV; Speaking and Teaching: Janssen, BMS
The following people have nothing to disclose: Adele L. McCormick, Lawrence T. Wang, Ana Garcia-Diaz, Malcolm J. Macartney, Tim C. Conibear, Clare L. Booth, Dianne N. Irish, Tanzina Haque
Biphasic kinetics of HCV-RNA decay and quasispecies evolution during DAA-treatment are associated with risk of virological failure
Valeria Cento1, Velio Chiara Si Maio1, Fabrizio Valenti2, Monica Tornodonati3, Soniele Armenia1, Maria Concetta Bellocchi1, Luco Carioti1, Francesco Paolo Antonucci1, Francesca Trove4, Pierluigi Cacciatore4, Francesca De Luca1, Alessandra F. Monunta5, Ado Bertoli6, Mario Angelico7, Eligio Pizzigallo3, Sergio Babudieri5, Giustino Parrujti4, Francesca Ceccherini-Silberstein1, Carlo Federico Perno1,6
1Experimental Medicine and Surgery, University of Rome “Tor Vergoto”, Rome, Italy; 2Economics, Institutions and Law, Uni versity of Rome “Tor Vergoto”, Rome, Italy; 3Infectious Disease Clinic/ “S. S. Annunzioto” Hospital, Chieti' Italy; 4! nfect'ious D/sease Unit, Pescara General Hospital, Pescara, Italy; 5Clinical and Experimental Medicine, University of Sassari, Sassari, Italy; 6Mol eculor Virology Unit, University Hospital of Rome “Tor Vergoto”, Rome, Italy; 7HepatoIogy Unit, University Hospital of Rome “Tor Vergoto”, Rome, Italy
Introduction: Aim of this study was to examine early kinetics of HCV-RNA decay and quasispecies rearrangements during telaprevir (TVR)-based triple therapy in treatment experienced and/or cirrhotic HCV-patients, providing insights into viral dynamics underlying viral failure. Methods: HCV-RNA decay (detection limit=15 IU/ml) was assessed per protocol and at early time points (1h-2h-3h-4h-5h-6h-8h-12h-24h-28h-48h-1w2w), and modeled according to Neumann et al., Science 1998. NS3-protease sequences were obtained during TVRtreatment (baseline-8h-24h-48h) by both population sequencing and ultradeep 454-pyrosequencing (UDPS). Results: Sixteen HCV-infected patients (GT1 b=11; GT1 a=5) received TVR+peglFN/RBV after previous failure to peglFN/RBV-treatment (non-responders=9; relapsers=5) or as first-line regimen (N=2). Both naīve patients and 4/9(44.4%) previous nonresponders were cirrhotic. HCV-RNA decay was biphasic, starting after 6h since first-dose (median[IQR] decay=0.6[0.4-1.0] logIU/ml). In all patients, independently from previous treatment experience or HCV-genotype, phase I decay was rapid, with a median[IQR] of 2.4[2.2-2.7] loglU/ml decrease in the first 24h and 2.8[2.6-3.2] loglU/ml in 48h. The median virion clearance rate (c) was 9.8 day-1 (8.0 day-1 with IFNa2b+RBV), and virion half-life was 2.0 h. Phase II decay was characterized by a median cell clearance rate (5) of 0.30 day1(0.14 day-1 with IFN-a2b+RBV) and a median[IQR] 2w HCVrNa drop of 4.3[3.6-4.6] LoglU/ml. At 2w, a cut-off value of 100 IU/ml divided patients into two groups, with a significantly different median[IQR] HCV-RNA decay from baseline: 3.3[2.0-LoglU/ml in the 4 patients with >100 IU/ml vs.4.5[4.2-LoglU/ml in the 10 patients with <100 IU/ml (p<0.01). Notably, 3/3 failing patients had HCV-RNA>100 IU/ml vs.0/5 patients who reached End Of Treatment (EOT) (p=0.02). By UDPS, failing patients showed an increase in nucleotide NS3 quasispecies variability after 24h of treatment (baseline mean[0SD] evolutionary divergence=0.012±0.003 nsubs/site vs.0. 025±0.004 nsubs/site at 24h; p<0.01), less seen in EOT patients (baseline=0.010±0.002 vs.24h=0.012±0.002 nsubs/site; p=0.70). At the amino acid level, the dominant strain remained invariant in all patients (prevalence>98%). Conclusions: Triple therapy administration affects viral dynamics with an extent of first phase decline and an acceleration of second phase, mediated by the higher effectiveness of DAAs. A slower kinetic is accompanied by an increase in quasispecies variability, indirectly reflecting viral ability to replicate under drug-pressure.
Francesca Ceccherini-Silberstein - Consulting: Gilead; Grant/Research Support: Merck Sharp & Dohme, Janssen
The following people have nothinq to disclose: Valeria Cento, Velia Chiara Di Maio, Fabrizio Valenti, Monica Tontodonati, Daniele Armenia, Maria Concetta Bellocchi, Luca Carioti, Francesco Paolo Antonucci, Francesca Trave, Pierluigi Cacciatore, Francesca De Luca, Aiessandra F. Manunta, Ada Bertoli, Mario Angelico, Eligio Pizzigallo, Sergio Babudieri, Giustino Parruti, Carlo Federico Perno
Post-Treatment Viral Evolution Assessment Using UltraDeep Pyrosequencing in Samples from Patients with Hepatitis C Virus Infection: The EXTEND Study
Inge Dierynck1, Kim Thys1, Doug J. Bartels2, Anne Ghys1,Elizabeth Van Rossem1, Eileen Z. Zhang2, Andrew Davis2, Mark I. Friedman2, Tara L. Kieffer2, Jeroen Aerssens1,Sandra Se Meyer1 , James C. Sullivan2
1Jonssen Infectious Diseases BVBA, Beerse, Belgium; 2Vertex Pharmaceuticals Incorporated, Cambridge, MA
In previous interim analyses of EXTEND, a 3-year virology follow-up study in patients previously treated with telaprevir, including patients who achieved SVR and treatment failures, population sequencing (PS, minority variant LOD=20%) demonstrated that telaprevir-resistant hepatitis C virus (HCV) variants tend to be lost from viral populations over time. The objective of this sub-study was to assess whether ultra-deep pyrosequencing (UDPS; LoD=1%) could detect telaprevir-resistant variants at a lower level. Samples collected at the last available visit from 1/4 treatment-failure patients in the EXTEND study were used to evaluate whether UDPS could detect low-level minority HCV variants that were not detected by PS. Libraries for UDPS were prepared from amplicons spanning NS3-4A and were sequenced using the lllumina platform. Processing was successful for 169/174 samples, with a median coverage of 33, 816 reads per position and per sample across the first 181 amino acids of the NS3 protease. There was a median interval of 3.0 years between the time of treatment failure and the sample used for UDPS (Table). The number of samples determined to be WT by PS and UDPS are shown in the Table. Twenty-five samples had resistant variants detected by PS; 24 of these also had resistant variants detected by UDPS. Overall, among the 144 patients with only WT virus detected by PS, 89% (n=128) also had only WT virus detected by UDPS, with 87/103 genotype 1a and 41/41 genotype 1b samples WT by UDPS. The remaining 16 genotype 1a samples had low levels (median 5%, Q1, Q3: 3%, 11%) of telaprevir-resistant variants V36A, V36m, o54S, R155K or combinations thereof by UDPS. These samples came from patients who had a shorter median follow-up time (2.1 years; Q1, Q3: 1.8, 3.8) relative to those with WT virus by UDPS (3.1 years; Q1, Q3: 2.5, 4.2). Consistent with previous analyses of the EXTEND study, these results using a more sensitive UDPS technique suggest that telaprevir-resistant variants dissipate after removal of the drug selective pressure.
|All Samples||n (%) WT2|
|Genotype||N||Median time (Q1, Q3)1||PS||UDPS|
|La||124||2.9 (2.3, 4.2)||103 (83)||883 (71)|
|Total||169||3.0 (2.3, 4.2)||144 (85)||129 (76)|
Inge Dierynck - Employment: Janssen Infectious Diseases, Johnson & Johnson; Stock Shareholder: Janssen Infectious Diseases, Johnson & Johnson
Kim Thys - Employment: Janssen Infectious Disease
Doug J. Bartels - Employment: Vertex Pharmaceuticals
Eileen Z. Zhang - Employment: Vertex; Stock Shareholder: Vertex
Andrew Davis - Employment: Vertex
Mark I. Friedman - Employment: Vertex Pharmaceuticals Incorporated; Stock Shareholder: Vertex Pharmaceuticals Incorporated
Tara L. Kieffer - Employment: Vertex; Stock Shareholder: Vertex
Jeroen Aerssens - Employment: Janssen Infectious Diseases bvba; Stock Shareholder: Johnson & Johnson
Sandra De Meyer - Employment: Janssen Infectious Diseases bvba (J&J); Stock Shareholder: J&J
James C. Sullivan - Employment: Vertex Pharmaceuticals Incorporated; Stock Shareholder: Vertex Pharmaceuticals Incorporated
The following people have nothing to disclose: Anne Ghys, Elizabeth Van Rossem
Neutralizing antibody dynamics during and following PEG-interferon induced clearance of acute HCV genotype 1 infection in HIV-infected MSM
Xiomara V Thomas1, Sylvie M. Koekkoek1, Jan T. van der Meer2, Maria Prins2,3, Sabrina Merat4, Tim Beaumont4, Richard Molenkamp1, Janke Schinkel1
1Medical Microbiology Amsterdam Medical Center, Amsterdam, Netherlands; 2Department of Internal Medicine, Division of Infectious Diseases, Tropical Medicine and AIDS, Amsterdam Medical Center, Amsterdam, Netherlands; 3Department of Research, Cluster Infectious Diseases, Public Health Service of Amsterdam, Amsterdam, Netherlands; 4Research ond Development, AIMM Therapeutics, Amsterdam, Netherlands
Background: The reported high frequency of hepatitis C virus (HCV) reinfection in pegIFN-treated HIV-infected men who have sex with men (MSM) suggests that treatment-induced viral clearance does not lead to sterilizing immunity against HCV. We longitudinally investigated the presence, breadth and persistence of neutralizing antibody (nAb) responses in HIV-infected MSM with acute HCV infection and sustained viral response (SVR) following treatment. Methods: Eleven peglFN-treated HIV-infected MSM with documented primary HCV-1a infection were selected for this study. All patients received peglFN and ribavirin treatment for 24 weeks, achieved SVR and remained HCV RNA negative for a median of 2 years (IQR, 1-5). Presence of nAbs against different genotypes was tested using a panel of 12 HCVpseudo-particles (HCVpp) consisting of genotypes 1b, −2a, 2b, −3a, −4a, - 4d and 6 HCV-1a strains, of which 5 were derived from locally circulating viruses. The presence of nAbs was investigated at the first RNA positive timepoint, start of treatment, last RNA positive sample and at 3 and 6 months after last RNA positive sample. Results: NAbs against one or more HCVpps were present in 8 of 11 patients during follow up. In 7 of 8 patients nAbs were already present at the first RNA positive timepoint. Breadth of response, indicated by the number of HCVpps neutralized, peaked at 3 months after viral clearance. Six months after viral clearance the number of HCVpps neutralized was strongly reduced. The number of HCVpps neutralized was strongly correlated with CD4+ count at the time of HCV acquisition. Interestingly, we observed a strong trend for the presence of nAbs against HCV-genotype 1 compared to non-1 genotypes (p = 0, 056). Conclusion: Although limited by small numbers, our results suggest the presence of partial immunity against HCV, mainly directed at the primary infecting genotype. In addition, breadth of nAb responses in HIV-infected individuals is correlated with the CD4+ count. We are currently investigating the titres of the nAbs in more detail and in more patients.
Maria Prins - Speaking and Teaching: msd, roche
Tim Beaumont - Employment: AIMM Therapeutics
Richard Molenkamp - Independent Contractor: Roche Diagnostics
The following people have nothing to disclose: Xiomara V. Thomas, Sylvie M. Koekkoek, Jan T. van der Meer, Sabrina Merat, Janke Schinkel
Differential kinetic of HCV-RNA clearance in plasma and PBMCs from patients in triple therapy with telaprevir
Antonio Madejon1,2, Miriam Romero1,2, Araceli G. Sánchez1,2, Irene Martin1,2, Jorge Carbo1, Roberta Bogoi1, Marta SanchezCarrillo 1,2, Javier Garcia-Samaniego1,2
1Hepatology Unit. Hospital Carlos IIl, Madrid, Spain; 2CIBERehd, Madrid, Spain
Background. The confirmation of serum HCV-RNA undetectability in several critical points during treatment (weeks 4,and 24) is crucial for monitoring antiviral response in patients with chronic hepatitis C (CHC) treated with peglFN + Ribavirin + Telaprevir. However, there are few data on the kinetic of HCV-RNA negativization in peripheral blood mononuclear cells (PBMCs), an extrahepatic HCV infection target of unclear clinical significance. Aim. To compare the kinetic of HCV-RNA negativization in plasma and PBMCs of patients with CHC under telaprevir-based triple therapy. Patients. We included 15 Caucasian patients (4 naīve, 8 relapsers, 2 partial responders and 1 null responder to previous dual PeglFN+Ribavirin treatment) who completed the treatment period with triple therapy. Eight (53%) completed the follow-up period. Serum HCV-RNA titers were tested according the treatment protocol. HCV-RNA in PBMCs was tested using an in house RT-nested PCR with a ĪaqMan probe at 0, 4, and 12 weeks after treatment in all patients and at the end of treatment in 6 patients. Results. Extended rapid virological response (eRVR) was achieved in 11/15 (73%) patients and serum HCV-RNA became negative at week 12 in 3 (20%) aditional patients. Only one patient discontinued the treatment due to an HCVRNA titer of 1687 IU/ml at week 4 (stopping rule). No breakthrough was observed and 14 (93%) patients were HCV-RNA negative at the end of treatment. In addition, all 8 patients with follow-up achieved SVR24. In PBMCs HCV RNA was detected in 11/15 (73%) patients at baseline, in 7/15 (47%) at week 4, in 4/14 (28%) at week 12 and in 1/6 (16%) at week 24 (12 weeks after serum HCV-RNA negativization). Persistence of HCV-RNA in PBMCs was significantly higher in patients without eRVR than in responders (positivity at week 4: 4/4(100) vs 3/11 (27%); p=0.02). There was no differences among patients with and without eRVR in the IL28B polymorphisms, baseline HCV-RNA and/or HCV-1 subtypes Conclusions: HCVRNA levels decrease sharply in PBMCs during telaprevir-based therapy but with a slower kinetic than that observed in plasma. The persistence of viral sequences in PBMCs is associated with the lack of eRVR.
Javier Garda-Samaniego - Consulting: Boehringer-Ingelheim
The following people have nothing to disclose: Antonio Madejon, Miriam Romero, Araceli G. Sanchez, Irene Martin, Jorge Carbo, Roberta Bogoi, Marta Sanchez-Carrillo
Baseline characteristics and treatment response of HCV genotype 1 patients with previous null response that are retreated with TVR or BOC triple therapy in real world setting
Wolf P. Hofmann1, Stefan Mauss2, Axel Baumgarten3, Klaus H. Boeker4, Ralph Link5, Uwe Naumann6, Peter R. Geyer7, Dietrich Hueppe8, Albrecht Stoehr9, Hanns-Friedrich F. Loehr10, Andreas Schober1, Gero Moog12, Stefanie Holm13, Renate Heyne14' Christoph Eisenbach15' Thomas Lutz16, Ulrich Alshuth17, Marcus Schuchmann18
1Polikum Institut, Berlin, Germany; 2Center for HIV and Hepatogastroenterology, Duesseldorf, Germany; 3mib Dienstleistung GmbH, Berlin, Germany; 4Center for Hepatology, Honnover, Germany; 5MZV Offenburg, Offenburg, Germany; 6Center for Addiction-Medicine, Hepatology and HIV, Proxiszenfrum Kaiserdomm, Berlin, Germany; 7Center of Gastroenterology, Fulda, Germany; 8Center of Gastroenterology, Herne, Germany; 9ifi-lnstitute for Interdisciplinary Medicine, Hamburg, Germany; 10Center of Gosfroenferology, Wiesbaden, Germany; 11Center of Gosfroenterology, Goettingen, Germany; 12Center of Gastroenterology, Kossel, Germany; 13Private Practice Dres. Kuhlmann/Holm/Heiken' Hannover, Germany; 14Liver and study cenfer Checkpoint, Berlin, Germany; 15Dept. of Gastroenterology, University of Heidelberg, Heidelberg, Germany; 16lnfektiologikum, Frankfurf/M, Germany; 17Virology, Roche Pharma AG, Grenzach-Wyhlen, Germany; 18Depf Med 1, Gastroenterology and Hepofology Johannes Gufenberg Univ Mainz, Mainz, Germany
Introduction: In HCV genotype 1 null responder to a previous course of peginterferon alfa (PEG) and ribavirin (RBV), retreatment with telaprevir (TVR) or boceprevir (BOC) as part of a triple therapy is the only licensed therapeutic option today. However, recent phase Ill clinical trials indicate low antiviral response rates in these difficult-to-treat patients and physicians are challenged to give the best treatment recommendation. Methods: The PAN study is a non-interventional study conducted at multiple sites in Germany and includes treatment naive and treatment experienced patients with HCV genotype 1 infection who receive triple therapy with TVR or BoC at the physicians discretion. In the present analysis, all consecutive retreated patients with HCV genotype 1 infection and well defined previous non response to PEG and RBV were included. Baseline characteristics and treatment efficacy data were analysed. Results: There were 65 Patients with previous null response (63, 1% male, mean age 50, 5 years, BMI 26, 1 kg/m2, 49, 2% with HCV genotype 1b) and 121 Patients with previous partial response (59, 5% male, mean age 50, 0, BMI 26, 7 kg/m2, 56, 2% with HCV genotype 1b) who received retreatment with PEG and RBV plus TVR (n = 155) or BOC (n=31). Liver cirrhosis (at least one result of ultrasound, transient elastography, histology or clinical signs) was present in 41, 5% and 21, 5% of patients with previous null response and partial response, respectively. High baseline viral load (>400.000 IU/ml HCV RNA) was detected in 87, 7% and 81, 0% of the two groups, respectively. An extended rapid virologic response (eRVR, adjusted for TVR at weeks 4 and 12 and BOC at weeks and 24 of therapy) was achieved in 29, 5% of previous null responders and 56, 8% of previous partial responders. Preliminary SVR rates (available for a total of 68 patients) were 11, 8% (4/34) in previous null responders and 23, 5% (8/34) in previous partial responders. Discontinuation rates due to virologic failure or adverse events were 55, 0% and 24, 8% for the two groups, respectively. Conclusion: The baseline characteristics of non responder patients in the PAN study demonstrate the high proportion of cirrhotic patients especially among previous null responders in Germany. In the real world setting, virologic response rates are poor and discontinuation rates are high and strongly confirm the medical need for new therapeutic options in this difficult-to-treat population.
Stefan Mauss - Advisory Committees or Review Panels: Roche, Gilead, BMS, Janssen, Boehringer ingelheim; Speaking and Teaching: Janssen, Roche, MSD, Gilead, BMS, Boehringer Ingelheim
Klaus H. Boeker - Speaking and Teaching: MSD, Roche, Janssen, Gilead, BMS, Falk Foundation, Novartis
Uwe Naumann - Speaking and Teaching: MSD, Roche, BMS, Abbott, VIIV, Janssen
Dietrich Hueppe - Advisory Committees or Review Panels: Roche, MSD, Novatis,
Albrecht Stoehr - Board Membership: MSD, BVdhringer ingelheim; Speaking and Teaching: Janssen, MSD, Gilead, Abbvie, BVdhringer ingelheim
Christoph Eisenbach - Advisory Committees or Review Panels: Roche; Speaking and Teaching: Bristol Myers Squibb, Roche, Gilead
Thomas Lutz - Advisory Committees or Review Panels: Gilead, MSD, Abbott, BMS, Janssen Cilag; Grant/Research Support: Boehringer ingelheim, Gilead, GlaxoSmithKline, Schering-Plough, Roche, MSD, Abbott, Janssen Cilag; Speaking and Teaching: Boehringer Ingelheim, GlaxoSmithKline, Roche, BMS
Ulrich Alshuth - Employment: Roche
Marcus Schuchmann - Advisory Committees or Review Panels: Roche, BMS, Norgine, Boehringer; Speaking and Teaching: Gilead, Merck, Falk
The following people have nothing to disclose: Wolf P. Hofmann, Axel Baumgarten, Ralph Link, Peter R. Geyer, Hanns-Friedrich F. Loehr, Andreas Schober, Gero Moog, Stefanie Holm, Renate Heyne
One-Fourth of Liver Biopsies Can Be Avoided Using a Simple Non-Invasive Bedside Blood Test (Fibrofast Score)
Gamal Shiha1,2, Waleed Samir2, Khaled R. Zalata3, Amira Elbeeh2, Ammal Metwally4
1Internal medicine, faculty of medicine-monsouro university, Monsouro, Egypt; 2Egyptian Liver Research Institute And Hospifol (ELRIAH)' Mansora, Egypt; 3Pothology department- Faculty of medicine-Monsouro university, Monsoro, Egypt; 4Communify Medicine Research depf., National Research Cernter, Cairo, Egypt
Introduction: A simple non invasive score (Fibrofast) was developed using five routine laboratory tests (ALT, AST, Alkaline phosphatase, Albumin and Platelets count) for the detection of significant hepatic fibrosis in patients with chronic hepatitis C (CHC) (Attallah et al., Hepatology Research (2006) 34: 163-169). Accordingly, we validated the accuracy of Fibrofast score on 1067 cases from several international centers, which revealed a sensitivity of 61.5 %, specificity of 81.1%, positive predictive value of 59% and negative predictive value of 82.6%. This indicated that the performance of the test is not enough as a suggestive alternative to liver biopsy. The aim of this study was to develop a new cut off score of the test that allows the diagnosis of established cirrhosis (F4) and (F0-F3) ensuring accuracy (more than 95%). Method: Subjects were 1 873 patients with CHC. All biopsies were scored using METAVIR system. Our fibrosis score (Fibrofast) were measured and the performance of the new cut off score were done using ROC curve. Results: Liver biopsy showed that 1646 cases (F0F3) and 227 cases established cirrhosis (F4). Using the ROC curve we develop new 2 cut off scores. The positive one is for diagnosis of (F0-F3) and the negative one is for diagnosis of established cirrhosis (F4).145 out of 227 cases (63.87%) were established cirrhosis and 328 out of 1646 cases (19.9%) (F0F3) i. e.463 out 1873 cases (24.7%) was positively correlated with liver biopsy (r = 0.393, P= 0.0〇1), sensitivity 95%, specificity 95%. Conclusion: Fibrofast score with the new two cut off scores could be an alternative to liver biopsy in about onefourth of the patients with sensitivity 95% and specificity 95%. Being non invasive, it could be an ideal marker for follow up during and after treatment in many patients.
The following people have nothing to disclose: Gamal Shiha, Waleed Samir, Khaled T. Zalata, Amira Elbeeh, Ammal Metwally
Establishment of Interferon resistant genotype 2b/JFH-1 chimeric Hepatitis C virus cell culture system
Goki Suda, Yoko Tsukuda, Mitsuteru Natsuizaka, Makoto Chuma, Naoya Sakamoto
hokkaido university, Sapporo, Japan
(Background and Aim) Interferon response is an important component for the virus elimination even in the DAA-based interferon-free regimen. We established stable culture system of chimeric viruses between HCV-TMD1(G-2b) and JFH1 (G-2a). Here we have established interferon -resistant HCV clone by long-term culture in the presence of interferon-α and analyze viral components that confer interferon resistance. (Method) Long-cultured HCV-2b/JFH1 chimeric virus (C3) was cultured with or without interferon-α for 8 weeks and compared virus kinetics between the two groups, and tried to extract IFN resistant clone from C3 cultured with interferon-α. (Result) Supernatant HCV titer of C3 decreased immediately after addition of interferon and reached the lower limit of measurement sensitivity after 2 weeks. However, 6 weeks after interferon treatment, replication of one C3 clone increased in the presence of interferon-a. Next we inoculated this supernatant onto naīve Huh7.5.1 cells and ensured that the virus was replication competent. Next we compared interferon sensitivity between HCV from long-cultured C3 with interferon-α and C3 that was newly transfected into naīve Huh 7.5.1 cells (1st-C3).1st-C3 showed higher responses to interferon than long cultured C3 with interferon-a. Comparison of amino acid sequences between virus before interferon treatment and that after 6-week interferon treatment revealed two sequence differences in structure region (envelope1 and 2 respectively). Next we constructed these two sequence differences substituted C3 clone (IFNrC3) and compared interferon sensitivity between IFNrC3 and C3 by transfection into Huh7.5.1 cells and subsequently interferon treatment. The newly constructed IFNrC3 showed resistance to interferon compare with C3. (Conclusion) We succeeded to establish interferon-resistant HCV cell culture system from long cultured C3 chimeric virus. Analysis of this virus may be useful to understand the mechanism of interferon resistance.
The following people have nothing to disclose: Goki Suda, Yoko Tsukuda, Mitsuteru Natsuizaka, Makoto Chuma, Naoya Sakamoto
Management and outcomes of anemia in 1587 patients with hepatitis C genotype 1 infection from the International Telaprevir Early Access Program
Simone I. Strasser1, Heiner Wedemeyer2, Petr Urbanek3, Paulo R. Abrão Ferreia4, Christophe Moreno5, Inmaculada Fernãndez6, Djamal Abdurakhmanov7, Adrian Streinu-Cercel8, Giovanni B. Gaeta9, Filip Beeldens10, Wafae Iraqi11, Ralph DeMasi12, Andrew Hill13, Joerg M. Läuffer14' Isabelle Lonjon-Domonec11, Massimo Colombo15
1Royal Prince Alfred Hospital, University of Sydney, Sydney, NSW, Australia; 2Medizinische Hochschule Hannover, Hannover, Germany; 3Department of Internal Medicine, First Medical Faculty, Charles University, and Central Military Hospital Prague, Prague, Czech Republic; 4Outpatient Clinic to HIV and Viral Hepatitis Division of Infectious Disease, Federal University of São Paulo, São Paulo, Brazil; 5Liver Unit, Department of Gastroenterology Hepatopancreatology and Digestive Oncology, Erosme University Hospital, Université Libre de Bruxellesand Viral Hepatitis Division of Infectious Disease, Federal University of So Paulo, Brussels' Belgium; 6HospiaI Universitario 12 de Octubre' Sección de Aparato Digestivo, Madrid, Spain; 7I. M. Sechenov First Moscow State Medical University, E. M. Tareev Clinic for Nephrology, Intern nal and Occupational Medicine, Moscow, Russian Federation; 8Carol Davila University of Medicine and Pharmacy, National Institute for Infectious Diseases “Prof. Dr. Matei Bals”, Bucharest, Romanio; 9ViraI Hepatitis Unit, Department of Infectious Diseases, Second University, Naples, Italy; 10Janssen Pharmaceutica, Beerse, Belgium; 11Jonssen Pharmaceuticals, Paris, France; 12Janssen Research & Development LLC, Titusville, NJ; 13MetaVirology Ltd, London, United Kingdom; 14Janssen-CiIag AG, Zug, Switzerland; 15Department of Medicine, Division of Gastroenterology, Fondozione IRCCS Ca, Granda Ospedole Maggiore Policlinico, Universia, degli Studi di Milano, Milan, ltaly
Background and aims: Anemia is a common adverse event during standard treatment for HCV infection. HEP3002 is an ongoing, open-label, early access program of telaprevir (TVR) in 16 countries, for patients with genotype 1 hepatitis C infection with severe fibrosis or compensated cirrhosis. Methods: Liver biopsy or non-invasive tests (Fibroscan in 1040 patients) showing severe fibrosis or cirrhosis were required at entry.1587 patients were treated with TVR, pegylated interferon-alpha (P) and ribavirin (R, RBV) for 12 weeks, followed by PR. Use of iron supplements, erythropoietin (EPO) and blood transfusions was permitted. Grade 1-4 anemia included the clinically significant adverse event terms (SSC) of anemia or hemoglobin (Hb) reduction. All analyses were on the intent-to-treat (ITT) population, using 16 weeks of data. Continuous variables were used in the multivariate analyses to predict anaemia. Results: Mean age was 53 years and mean weight 78kg; 64% were male and 98% Caucasian, 66% had HCV RNA levels >800, 000 lU/mL, 47%/53% had severe fibrosis/cirrhosis, 22% had genotype 1a. Up to Week 16, 59% of patients developed grade 1-4 anemia, with 3 1% grade 3-4 cases; 630 patients (40%) dosereduced RBV, 332 (21%) received EPo157 (10%) were transfused, 14 (1%) received iron-based products and 45 (3%) discontinued treatment because of anemia. Results by baseline disease stage are shown below. There were reductions in Hb below 10 g/dL in 48% of patients. In a multivariate analysis, the four strongest predictors of Hb <10 g/dL at any time on treatment were female sex (OR=1.69, 95% Cl 1.27-2.27, p=0.0004), age >65 years (OR=2.31, 95% Cl 1.46-3.65, p=0.0003), low baseline Hb (OR=1.08, 95% Cl 1.06-1.09, p<0.0001) and higher weight-based dosing of RBV (OR=1.13, 95% Cl 1. 05-1.21, p=0.0005). Baseline Fibroscan test results were not a significant predictor of anemia. Conclusions: In this TVR early access program for patients with severe fibrosis or compensated cirrhosis, grade 3 or 4 anemia was reported in 31% of patients, but discontinuation of TVR for anemia was rare (3%). Anemia was normally managed by RBV dose reduction, transfusion or the use of EPo.
|Type of anemia||Definition||F3 patients (n=746)||F4 patients|
|Grade 1||Hb 10-10.9 or 2.5-3.4g/dL decrease||81(11%)||76 (9%)|
|Grade 2||Hb 9-9.9 g/dL or 3.5-4.4 g/dL decrease||116(16%)||169 (20%)|
|Grade 3||Hb 7-8.9 or >4.5 g/dL decrease||188 (25%)||238 (29%)|
|Grade 4||Hb <7 g/dL||26 (3%)||35 (4%)|
|Discontinued TVR for anemia||14(2%)||31(4%)|
|Anemia as serious AE||32 (4%)||43 (5%)|
Simone I. Strasser - Advisory Committees or Review Panels: Janssen, AbbVie, Roche Products Australia, MSD, Bristol-Myers Squibb, Gilead, Norgine, Bayer Healthcare; Speaking and Teaching: Bayer Healthcare, Bristol-Myers Squibb, MSD, Roche Products Australia, Gilead, Janssen
Heiner Wedemeyer - Advisory Committees or Review Panels: Transgene, MSD, Roche, Gilead, Abbott, BMS, Falk; Grant/Research Support: MSD, Novartis, Gilead, Roche, Abbott; Speaking and Teaching: BMS, MSD, Novartis, IĪF
Paulo R. Abrao Ferreira - Grant/Research Support: ABBOTT, ROCHE, BMS, JANSSEN; Speaking and Teaching: ROCHE, BMS, JANSSEN
Djamal Abdurakhmanov - Grant/Research Support: Roche; Speaking and Teaching: BMS, Jansenn, MSD, Novartis
Giovanni B. Gaeta - Advisory Committees or Review Panels: Janssen, Merk, BMS, Novartis, Gilead, Roche, Janssen, Merk, BMS, Novartis, Gilead, Roche, Janssen, Merk, BMS, Novartis, Gilead, Roche, Janssen, Merk, BMS, Novartis, Gilead, Roche
Filip Beeldens - Employment: Janssen Research and Development Wafae Iraqi - Employment: Janssen
Ralph DeMasi - Management Position: Johnson and Johnson Andrew Hill - Consulting: Janssen
Joerg M. Lauffer - Employment: Janssen; Stock Shareholder: Janssen Isabelle Lonjon-Domanec - Employment: Janssen
Massimo Colombo - Advisory Committees or Review Panels: BRISTOL-MEYERS-
SCHERING-PLOUGH, ROCHE, GIlEaD, Ja'nssen Cilag, Achillion; Grant/Research Support: BRISTOL-MEYERS-SQUIBB, ROCHE, GILEAD, BRISTOLMEYERS-SQUIBB, ROCHE, GILEAD; Speaking and Teaching: Glaxo Smith-Kline, BRISTOL-MEYERS-SQUIBB, SCHERING-PLOUGH, ROCHE, NOVARTIS, GILEAD, VERTEX, Glaxo Smith-Kline, BRISTOL-MEYERS-SQUIBB, SCHERING-PLOUGH, ROCHE, NOVARTIS, GILEAD, VERTEX
The following people have nothing to disclose: Petr Urbanek, Christophe Moreno, Inmaculada Fernandez, Adrian Streinu-Cercel
Intensive mapping of HCV quasispecies change provides novel insights into the natural history of chronic infection at a molecular level
Daniel Schmidt-Martin, Liam J. Fanning, Elizabeth Kenny-Walshe, Orio M. Crosbie
Medicine, University College Cork, Cork, Ireland
Hepatitis C virus (HCV) exists as a quasispecies (QS) of related genetic variants. QS are thought to be an important factor in the evasion of the host immune response and the maintenance of chronic infection. Furthermore, a number of studies have demonstrated associations between QS complexity and diversity in the hypervariable region 1(HVR1) and sustained viral response to treatment. Many of these studies have either been retrospective, focused on acute infection, or post transplant changes and most have used variable sampling intervals of many months if not years. We recruited and sampled the HCV HVR1 QS in 20 chronically infected individual at fortnightly for a total of 16 weeks. We analysed QS diversity, complexity, and divergence for a per sample mean of 16 (12-24) HVR1 clones which had been created using nested PCR. QS change was visualized using both phyelogenetic trees and median joining networks. We examined the samples for evidence of selection at both HVR1 wide and codon level. Finally, we investigated for evidence of multiple subpopulations. We demonstrate statistically significant less QS diversity and complexity in HVR1 QS in patients with cirrhosis (p<0.01). A number of cirrhotic patients maintain a homogenous QS profile for the entire study period which contrasts with non cirrhotic patients where marked change is found. QS diversity and complexity changes markedly over periods as short as two weeks, with one subject demonstrating a complete collapse in both with the emergence of an entirely new HVR QS far removed from the original master sequence four weeks later. We demonstrate time ordered phyelogenetic change in five subjects, suggesting strong underlying immune mediated clearance. The recently described phenomenon of QS subpopulations is demonstrated, but only in non cirrhotic patients but not in cirrhotic patients. Using MJN analysis, we are able to demonstrate the oscillating prevalence of different subpopulations over the study period. The effect of multiple subpopulations on the calculation of sequence divergence and diversity is highlighted. Our unique dataset permits the description of HCV QS change over shorter intervals than has previously been undertaken and provides novel insights into the natural history of HCV during chronic infection. The findings have important implications for the understanding of the emergence of treatment resistant variants. It is clear that single timepoint analysis as had been used to find associations between diversity and complexity and treatment outcome is insufficient to understand the future patterns of HCV QS change.
The following people have nothing to disclose: Daniel Schmidt-Martin, Liam J. Fanning, Elizabeth Kenny-Walshe, Orla M. Crosbie