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Soluble ST2 Plasma Concentrations Predict Mortality in Acute-on-Chronic Hepatitis B Liver failure

Ziying Lei, Zhi Shuo Mo, Dongying Xie, Jianyun Zhu;
The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China

Background: Patients with liver failure display features of serious inflammation and immunity disorders. Immunosuppression is thought to be facilitated by negative regulators of toll-like receptors, including membrane-bound ST2. Soluble ST2 (sST2) is a decoy receptor that inhibits membrane-bound ST2 signaling. In this study, we investigated the levels of soluble ST2 in patients with acute-on-chronic hepatitis B liver failure (ACHBLF) and its correlations with disease severity and motality. Methods: The study population comprised 178 patients with ACHBLF and 68 with chronic hepatitis B (CHB) admitted to the department of infectious disease of our hospital from January 2010 to December 2011. The diagnosis criteria of ACHBLF were defined as the presence of acute hepatic insult, jaundice (total bilirubin, TB, >1OxULN (upper limit of normal)), low prothrombin activity (PTA, <40%), and complication (ascites and/or encephalopathy) within 4 weeks in a patient with previously diagnosed chronic hepatitis B. Blood was obtained from admission day. Plasma soluble ST2 were assayed by commercial ELISA kits. Results: ACHBLF patients had higher sST2 levels (24848±21318 pg/ml) than CHB (11831±16368 pg/ml, Z=6.19, P<0.0001). In ACHBLF patients, sST2 correlated with MELD score (r=0.41, P<0.001), INR (r=0.35, P<0.001), total bilirubin level (r=0.21, P<0.001) and white blood cell (WBC) count (r=0.51, P<0.001). Higher sST2 levels in ACHBLF patients with hepatic encephalopathy (HE, 37740±23155 pg/ml) and WBC count >10xl09/L (43726±21870 pg/ml) were found than non-HE (20616±18933 pg/ml, t=4.44, P<0.001) and WBC count<l 0x109/L (1 8260±16608 pg/ml, t=7.14, P<0.0001). Nonsurvivors displayed elevated sST2 levels (33873±21851 pg/ml) compared with survivors (18887±18685 pg/ml, t=4.69, P<0.001). Conclusions: Our findings demonstrate that ACHBLF results in elevation of soluble ST2. Moreover, soluble ST2 levels correlate with disease severity and mortality.

Disclosures:

The following people have nothing to disclose: Ziying Lei, Zhi Shuo Mo, Dongy-ing Xie, Jianyun Zhu

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Functionality of HBV-specific CD8 T-cells is jointly regulated by the transcription factors T-bet and Eomesoder-min

Peter Kurktschiev1,2, Bijan Raziorrouh1,2, Winfried Schraut1,2, Martin Wächtler4, Markus Backmund2,5, Michael Spannagl3, Reinhart Zachoval2, Gerald Denk2, HelmutM. Diepolder1,2, Maria-Christina Jung6, Norbert Grüner1,2;
1Institute for Immunology, LMU Munich, Munich, Germany2Medical Department II, University of Munich, Munich, Germany; 3Laboratory for Immunogenetics, LMU Munich, Munich, Germany;4Medical Department, Klinikum Schwabing, Munich, Germany; 5Praxis im Tal, Munich, Germany; 6Leberzen-trum, Munich, Germany

BACKGROUND: Recent findings have highlighted the importance of the transcription factors T-bet and Eomesodermin (Eomes) for functionality of CD8 T-cells. In chronic HBV infection functional properties are gradually lost on HBV-specific CD8 T-cells. In this study we analyzed the coexpression of T-bet and Eomes in HBV-specific CD8 T-cells of patients with chronic HBV infection and correlated them with interferon-γ and perforin production after stimulation. Our goal was to define correlates of a successful antigen-specific CD8 T-cell activation on transcriptional level. METHODS: PBMCs of patients with chronic (n= 11) and acute (n=8) HBV infection were stained ex vivo with MHC-I A0201-restricted HBV-core 18-27 (c18-27) specific pentamers. Pentamer+ cells were enriched with MACS-beads, stained intracellularly for T-bet and Eomes and analyzed by flow cytometry. For functional analysis PBMCs were either cultured for 3 days with an enhanced stimulation protocol (c18-27 peptide+costimulatory cytokines) or left unstimulated. On day 3 expression of T-bet, Eomes, interferon-γ (ifn-γ) and perforin (perf) was determined by intracellular cytokine staining. RESULTS: In acute HBV the frequency of T-bet+Eomes+ (T+/E+, mean 19,4%) and T-bet+Eomes- (T+/E-, 41,8%) c18-27 specific CD8 T-cells was significantly (p<0.001) higher than in chronic HBV (T+/E+ 0,8%, T+/E- 6,3%). Stimulated CD8 T-cells were grouped according to T-bet+/- and Eomes +/- transcriptional phenotypeand ifn-γ+/-, perf+/-functional phenotype (table1). Frequencies of ifn-γ+/ perf+,ifn-γ+/ perf- and ifn-γ/ perf+ cells were significantly (p<0.001) increased in T+/E+ and T+/E-compared to T-/E- phenotype while there was no difference in T-/E+. T+E+ cells had significantly (p<0.001) higher frequencies of ifn-γ+/perf+ and ifn-γ+/perf- cells compared to T+E-. CONCLUSIONS: We found a strong correlation between T-bet expression in stimulated HBV-specific CD8 T-cells and production of interferon-γ and perforin. T-bet+/Eomes- cells showed significantly increased production of interferon-γ and perforin compared to T-bet-/Eomes- and T-bet-/Eomes+ CD8 T-cells which expressed only low amounts of both effector molecules. The strongest induction of interferon-γ was found in T-bet+Eomes+ cell indicating an additive effect of Eomes in T-bet+ cells. Induction of T-bet and Eomes could be a promising approach towards reactivation of dysfunctional HBV-specific CD8 T-cells.

Table 1

 ifn-γ+ perf-ifn-γ+ perf-ifn-γ- perf+ifn-γ+ perf-
T-bet+ Eomes+84,57%69,44%43,89%4,38%
T-bet+ Eomes-13,44%20,13%42,15%15,09%
T-bet- Eomes+1,78%5,91%5,79%8,3%
T-bet- Eomes-0,4%4,51%8,18%72,23%

Disclosures:

Markus Backmund - Speaking and Teaching: Roche, MSD, Janssen, Sanofi-Aven-tis

The following people have nothing to disclose: Peter Kurktschiev, Bijan Raziorrouh, Winfried Schraut, Martin Wächtler, Michael Spannagl, Reinhart Zachoval, Gerald Denk, Helmut M. Diepolder, Maria-Christina Jung, Norbert Grüner

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Isolated antibody to Hepatitis B Core Antigen (anti-HBc) in HIV-1 infected patients: immunological characteristics and response to Hepatitis B vaccine (preliminary results of the ANRS HB EP03 CISOVAC Study)

Lionel Piroth1, Odile Launay2, Patrick Miailhes3, Dani Nazzal4, Faiza Ajana5, Catherine Chirouze6, David Zucman7, Fabrice Carrat8, Marie-Josée Wendling9, Christine Binquet10, Marie-Louise Michel4, David Rey11;
1CHU, Dijon, France; 2Université Paris Descartes, Inserm CIC BT505, Hôpital Cochin, Paris, France; 3Infectious Diseases Department, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France; 4Laboratoire de Pathogenèse des virus de l'hépatite B, Department of Virology, ; INSERM U845 Institut Pasteur, Paris, France; 5Infectious Diseases Department, University Hospital,, Tourcoing, France; 6Infectious Diseases Department, University Hospital, and University of Franche-Comté,, Besançon, France; 7Internal Medicine Departement, Hopital Foch,, Suresnes, France; 8Infectious Diseases Epidemiology Unit, INSERM U707, Paris, France; 9Laboratoire de Virologie, Hôpitaux Universitaires, Strasbourg, France; 10Centre d'investigation Clinique Epidémiologie clinique/Essais Cliniques, INSERM, CIE1,, Dijon, France; 11Center for HIV infection care, Hôpitaux Universitaires, Strasbourg, France

Background: An isolated anti-HBc (IAHBc) serology pattern (i.e. without HBsAg or HBsAb) is frequently observed in HIV infected patients (15-20%), and has been linked to a risk of HBV reactivation or reinfection. The underlying immunological characteristics and the interest of vaccination against HBV remain to be clarified. Methods: A prospective multicenter study was conducted in treated HIV-1 infected patients (CD4>200/mm3, undetectable HIV viral load) with IAHBc. Immunological, virological and serological characteristics were assessed and one single 20-ug anti-HBV vaccine injection was given at week 0 (W0). Patients with no anamnestic response (AR) at W4 ie HBsAb<10 mIU/mL, received 3 additional double doses (40 ug) of HBV vaccine at W5, W9 and W24. HBsAb titers were assessed for all patients at W28. Results: All 55 patients included (32 men, median age 46y, median CD4 536/mm3) had negative HBV DNA and HBeAg, whereas HBeAb and HCV RNA were positive in 16 (29%) and 7(13%), respectively. At W0, HBV preS2- and S-specific IFN-g-secreting T cells were detected in PMBC from 14% and 33% of the 43 patients with complete immunological evaluation. In parallel, a response to HBV capsid was observed in 16% of these 43 patients, contrasting with 56% to HIV Gag. Only 5% and 12% had HBs- and HBc-specific proliferative responses, compared with a response rate to recall antigens (TT and PPD) of 66%. At W4, among the 54 patients receiving the first dose of HBV vaccine, 46% experienced AR (median HBsAb titer 43 mIU/mL, IQR 20-258), whereas 54% did not. In multivariate analysis, only CD4/CD8 ratio at W0 was significantly associated with an AR (OR for +0.1: 1.32, p=0.008).AtW28, 14/24 maintained an HBsAb titer≥10 mIU/mL (median 67 mIU/mL, IQR 23-87). Though all (8/8) those with a W4HBsAb titer≥l00 mIU/mL maintained a titer≥l0 mIU/mL at W28, only 6/16 (37%) with a titer <100 did so. Of the 27 patients without AR who received the 3 double doses of vaccine, 24 (89%) had HBsAb≥10 mIU/mL at W28 (median 293 mIU/mL, IQR104-777). Only CD8 percentage at W0 was inversely correlated with W28 response in multivariate analysis (p=0.045). Conclusions: 1) Most of the HIV infected patients with IAHBc also had an at least partial specific defect in T cell response to HBV antigens, which may preclude protection against HBV reinfection and/or reactivation. 2) The AR rate following one single dose of HBV vaccine was higher than that usually reported, but with a low percentage of sustained seroprotection at W28 and low HBs Ab titers. 3) Double dose HBV vaccination gave a high rate of response with high HBsAb titers at W28, and might also be considered in patients with low AR.

Disclosures:

Fabrice Carrat - Grant/Research Support: Janssen, Merck, Gilead, BMS, Roche

Marie-Louise Michel - Consulting: Transgene, Wittycell, ITS

The following people have nothing to disclose: Lionel Piroth, Odile Launay, Patrick Miailhes, Dani Nazzal, Faiza Ajana, Catherine Chirouze, David Zucman, Marie-Josée Wendling, Christine Binquet, David Rey

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Difference of Expression Pattern of Serum Cytokines between HBeAg-positive and HBeAg-negative Chronic Hepatitis B

Dengming He1,2, Maoshi Li1, Shimin Guo1, Hongfei Huang1, Zhaoxia Tan1, Yuming Wang1;
1Institute of Infectious Disease, Southwest Hospital, Third Military Medical University, Chongqing, China; 2Liver Disease Diagnoses and Treatment Center of Chinese PLA, the 88th Hospital of Chinese PLA, Taian, China

The immune response initiated by the T-cell response to viral antigens is thought to be fundamental for viral clearance and disease pathogenesis in hepatitis B virus (HBV) infection. However, there is not clear about the role of numerous cytokines in the mechanism of clinical phenotype of chronic HBV infection. We investigated the expression pattern of 30 cytokines associated with anti-HBV immunity in chronic hepatitis B patients. Sixty treatment-naïve chronic hepatitis B patients (30 with HBeAg-positive and 30 with HBeAg-negative) were assigned.

Thirty cytokines, including IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-15, IL-17A, IL-1 7C, IL-21, IL-22, IL-23p19, IL-28A, IL-29, CCL5, CCL16, CCL20, CCL22, CXCL9, CXCL10, CXCL11, TNFRSF8, TNFRSF18, IL-6R, gp130, and TGF-β1, were measured using a human cytokine antibody array. Signal intensities were obtained by laser scanner. Protein-protein interactions were analyzed by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins). Significant correlation between cytokines levels and alanine transaminase (ALT) levels was observed for 21 cytokines in HBeAg-positive patients and 15 cytokines in HBeAg-negative patients (including negative correlations in CCL22, TGF-β1, and CCL5). The strong correlation was in CXCL1 1 (Pearson = 0.806, p = 7.659x10-8 in HBeAg-positive and Pearson = 0.884, p = 9.673x10-11 in HBeAg-negative). The significant correlation was in other chemokines, including CXCL9, CXCL10, and CCL20, and in inflammatory cytokine, including IL-23p19, IL-1β, IL-4, IL-10, IL-12p70, IL-12p40, and IL-17. The significant correlation in IFN-γ and IL-29 was only observed in HBeAg-positive patients (Pearson = 0.693, p = 2.175×10-5 for IFN-γ; Pearson = 0.726, p = 5.699×10-6 for IL-29). The correlation in IL-21 was only observed in HBeAg-negative patients (Pearson = 0.507, p = 0.004). No significant correlation in γ chain cytokines, including IL-2, IL-15, and IL-7 was observed. In cytokines with significant positive correlation, the highest intensity was in patients with ALT levels over 5 times upper limit of normal. All cytokines with significant correlation have interaction with JAK-STAT signaling pathway and up-regulate FOXP3, SOCS3 and MX1. Conclusions: CXCL1 1 is the preferred immune markers to assess the degree of active hepatitis. Defection up-regulation expression of IL-29, IL-17, IFN-γ, and CCL5 and compensatory up-regulation of IL-21 may be associated with the mechanism of HBeAg-negative chronic hepatitis B. Elevation of IL-29 and IFN-γ may be used as markers to evaluate serum immunological response for HBeAg-negative chronic hepatitis B patients.

Disclosures:

The following people have nothing to disclose: Dengming He, Maoshi Li, Shimin Guo, Hongfei Huang, Zhaoxia Tan, Yuming Wang

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Sustained restoration of innate immune responses and sAg decline in sequential NUC therapy following failed Pegylated-Interferon-α therapy in Chronic Hepatitis B

Upkar S. Gill1, Dimitra Peppa2, Lorenzo Micco2, Graham R. Foster1, Mala K. Maini2, Patrick T. Kennedy1;
1Hepatology Unit, Centre for Digestive Diseases, Blizard Institute, Barts and The London, School of Medicine & Dentistry, QMUL, London, United Kingdom; 2Division of Infection & Immunity, UCL, London, United Kingdom

INTRODUCTION: Treatment strategies in Chronic Hepatitis B (CHB) are rapidly evolving and greater consideration is being given to combination/sequential therapies. Recently a Pegylated-Interferon-α (PEG-IFN-α) switch strategy following viral suppression with a nucleos(t)ide analogue (NUC) was demonstrably superior to NUC monotherapy in sAg reduction and loss. The mechanism underpinning this effect remains ill defined. We have demonstrated boosting of NK cell responses in eAg-negative patients treated with PEG-IFN-α whereas longterm NUC's have been shown to restore T-cell responses. We tested whether it would be possible to combine these two complementary effects by investigating whether augmented NK cell responses can be maintained on sequential NUC therapy, following PEG-IFN-α failure. PATIENTS & METHODS: PBMC's from 18 eAg-positive patients during PEG-IFN-α therapy were utilised. 8/18 patients, considered PEG-IFN-α non-responders after 48 weeks therapy, progressed to sequential NUC therapy and were followed until viral suppression was achieved. Phenotypic and functional analysis of NK cell subsets was performed by multicolour flow cytometry. Changes in immune responses were correlated with simultaneous measurements of ALT, HBV DNA and sAg titres. RESULTS: PEG-IFN-α treatment expanded the CD56bright NK cell population by a mean of 3-fold (p=0.0001). Expression of the C-type lectin receptors (NKG2A, NKG2C, NKG2D) and natural cytotoxicity receptors (NKp30, NKp44, NKp46) were analysed. A 2-fold increase in NKp30 and a 1.5-fold increase in NKp46 expression on CD56bright NK cells was noted during PEG-IFN-α (p=0.03 and 0.04 respectively), sustained on sequential NUC therapy; whilst no significant change was detected in the C-type lectin receptors or NKp44 expression. An increase in the expression of IFN-Y producing CD56bright NK cells, during PEG-IFN-α therapy (p=0.05) was sustained on sequential NUC therapy; a functional restoration not achieved with NUC's alone. These changes were associated with a sAg decline of 1.22 logIU/ml from baseline (p=0.05). CONCLUSIONS: PEG-IFN-α expanded the CD56bright NK cell subset; NK cell expression of NKp30, NKp46 and IFN-γ production was augmented and sustained on sequential NUC therapy, correlating with a greater reduction in sAg levels compared to PEG-IFN-α alone or NUC monotherapy. Thus PEG-IFN-α in non-responders induces sustained innate boosting which is maintained on sequential NUC therapy, in association with sAg decline. Further work is needed to establish whether this priming effect is also present when early PEG-IFN-α stopping rules are applied.

Disclosures:

Graham R. Foster - Advisory Committees or Review Panels: GlaxoSmithKline, Novartis, Boehringer Ingelheim, 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

Mala K. Maini-Advisory Committees or Review Panels: Roche; Consulting: Transgene, ITS; Grant/Research Support: BMS; Speaking and Teaching: BMS

Patrick T. Kennedy - Grant/Research Support: Roche, Gilead; Speaking and Teaching: BMS

The following people have nothing to disclose: Upkar S. Gill, Dimitra Peppa, Lorenzo Micco

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Dynamics of the Natural Killer Cell Response to Hepatitis Delta Virus-Infection and Interferon-α Treatment

Sebastian Lunemann1,2, David F. Malone1, Jan Grabowski2, Kerstin Port2, Vivien Beziat1, Birgit Bremer2, Karl-Johan Malmberg1,3, Michael P. Manns2, Johan K. Sandberg1, Markus Cornberg2, Hans-Gustaf Ljunggren1, Heiner Wedemeyer2, Niklas K. Björkström1,4;
1Center for Infectious Medicine, Karolinska Institutet, Stockholm, Sweden; 2Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; 3Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; 4Liver Immunology Laboratory, Karolinska Institutet, Stockholm, Sweden

Infection with hepatitis delta virus (HDV) causes the most severe form of viral hepatitis. Worldwide, 15 to 20 million people are chronically infected with this pathogen. Although hepatitis delta is considered an immune-mediated disease, adaptive immune responses against HDV are hardly detectable. Thus, other immune compartments, including natural killer (NK) cells, may play a role in the immunopathogenesis of hepatitis delta. Here, we performed a high-resolution analysis of NK cells in 34 patients with chronic HDV infection. Of these, 16 patients were followed longitudinally during treatment with pegylated interferon alfa (peg-IFNα). HDV-infected patients exhibited higher baseline levels of NK cells than uninfected controls. Redistribution of NK cell subsets was observed following peg-IFNα treatment, with increased numbers of CD56bright NK cells despite a general decrease in total numbers of NK cells. Strikingly, treatment almost completely eliminated the most mature NKG2A-CD57+ CD56dim NK cell subsets from peripheral blood, whereas more immature NKG2A+CD57- subsets increased. Furthermore, NK cells from HDV-infected patients displayed impaired functionality, and this phenotype was exacerbated during treatment. In particular, the lack of response to IFNα restimulation was pronounced. Overall, our results show that HDV infection, and interferon treatment of this infection, reshapes the NK cell compartment and has consequences for the functional capacity of this compartment.

Disclosures:

Kerstin Port - Speaking and Teaching: Roche

Michael P. Manns - Consulting: Roche, BMS, Gilead, Boehringer Ingelheim, Novartis, Idenix, Achillion, GSK, Merck/MSD, Janssen, Medgenics; Grant/Research Support: Merck/MSD, Roche, Gilead, Novartis, Boehringer Ingelheim, BMS; Speaking and Teaching: Merck/MSD, Roche, BMS, Gilead, Janssen, GSK, Novartis

Markus Cornberg - Advisory Committees or Review Panels: Merck (MSD Ger-mamny), Roche, Gilead, Novartis; Grant/Research Support: Merck (MSD Ger-mamny), Roche; Speaking and Teaching: Merck (MSD Germamny), Roche, Gilead, BMS, Novartis, Falk

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, ITF

The following people have nothing to disclose: Sebastian Lunemann, David F. Malone, Jan Grabowski, Vivien Beziat, Birgit Bremer, Karl-Johan Malmberg, Johan K. Sandberg, Hans-Gustaf Ljunggren, Niklas K. Björkström

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Nature of pathogenic and protective immunity in chronic hepatitis B virus infection

Sheikh Mohammad Fazle Akbar1,3, Mamun A. Mahtab2, Yoichi Hiasa3, Julio Cesar Aguilar4;
1Department of Medical Sciences, Toshiba General Hospital, Tokyo, Japan; 2Bangbandhu Sheikh Mujib Medical University, Dhaka, Bangladesh; 3Ehime University Graduate School of Medicine, Toon, Japan; 4Center for Genetic Engineering and Biotechnology, Havana, Cuba

Background/Aims: Host immunity may be detrimental (pathogenic) or beneficial (protective) for chronic HBV-infected subjects. But the nature of host immunity with pathogenic and protective potentialities has not been explored in details. This has induced a state of confusion for development of immune therapy for chronic hepatitis B. Methods: In this preclinical study, HBV transgenic mice (TM) that express Dane particles, HBV DNA, HBsAg, and HBeAg were injected with polyclonal immune modulators (IL-2 and IFN-gamma) once a week for 6 times. HBV TM were also immunized with HBV-specific antigens (HBsAg, HBcAg, and combination of both HBsAg and HBcAg [HBsAg/HBcAg]) once a week for 6 times. The levels of SGPT and IL-2 were measured in the sera. Also, the levels of HBsAg and anti-HBs were measured in the sera. HBsAg- and HBcAg-specific T cells and IFN-gamma producing CTL were assessed in the spleen and in liver non-parenchymal cells (NPC). Results: Injections with polyclonal immune modulators did not significantly alter HBsAg levels in HBV TM. Also, anti-HBs were not detected in any HBV TM injected with IL-2 and IFN-gamma. However, these HBV TM expressed significantly higher levels of SGPT and IL-2 in sera and cultures of spleen cells and liver NPC compared to their pre-treatment levels (p<0.05). These HBV TM expressed moderate to severe infiltrations of lymphocytes in the liver. In spite of inducing an inflammatory mucosal milieu in the liver, polyclonal immunemodulators could neither induce significant levels of HBsAg or HBcAg-specific T lymphocytes or CTL in the spleen cells and liver NPC. On the other hand, immunization with HBsAg, HBcAg and HBsAg/HBcAg caused negativity of HBsAg in 60-80% HBV TM and formation of anti-HBs in 50-60% HBV TM without any evidence of liver damages. Also, significantly higher levels of HBsAg and HBcAg-specific T lymphocyte proliferation and CTL were detected in the spleen and liver of these HBV TM (p<0.05) compared to their pre-treatment levels (p<0.05). Immunization with HBsAg/HBcAg induced significantly higher immune modulation compared to immunization with only HBsAg (p<0.05). Also, HBsAg-based immunization failed to induce HBcAg-specific CTL in the liver. However, HBcAg-based immunization induced both HBcAg and HBsAg-specific CTL in liver NPC. Conclusions: This study showed that HBV antigen-related, especially HBcAg-related inducible immunity seems to be endowed with protective immunity in HBV TM. This study may aid to design an evidence-based therapeutic approach (non-antigen-specific polyclonal modulators versus HBV antigen-specific immune modulation, as well as HBsAg-based versus HBcAg-based) for chronic HBV infection.

Disclosures:

The following people have nothing to disclose: Sheikh Mohammad Fazle Akbar, Mamun A. Mahtab, Yoichi Hiasa, Julio Cesar Aguilar

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The ratios of Treg/Th17 and TGF-β1 /IL-17 are associated with the survival and disease progression in patients with HBV-associated liver cirrhosis

Xueping Yu, Zhijun Su, Ruyi Guo, Desong Ming, Lvye Huang, Milong Su, Yong Deng, Zhenzhong Lin;
Department of infectious diseases, The first hosipital of Quanzhou affilliated to Fujian Medical, Quanzhou, China

Background: Many studies have suggested that regulatory T (Treg) cells and Th17 cells are mutually antagonistic in the immune response of chronic hepatitis B virus (HBV) infection. However, little is known about Treg and Th17 cells and their related cytokines in patients with HBV-associated liver cirrhosis (HBV-LC). This study aimed to evaluate whether the ratios of Treg/Th17 cells and TGF-β1/IL-17 may be associated with the survival and disease progression in patients with HBV-LC. Methods: The frequencies of Treg and Th17 cells were analyzed in 28 patients with HBV-LC (Child A: 11; Child B: 11; Child C: 6), 70 patients with CHB and 20 normal controls (NC) by flow cytometry. The levels of cytokines related to Treg/Th17 differentiation, including IL-10, TGF-β1, IL-17 and IL-23, were measured by enzyme-linked immunosorbent assay (ELISA). Results: Compared with NC, Treg cells were significantly increased in CHB patients, slightly increased in HBV-LC patients, whereas Th17 cells were significantly increased in patients with CHB and HBV-LC. HBV-LC patients, especially the non-survival ones, manifested a profound decrease in the Treg/Th17 ratio, which was negatively correlated with Child-pugh and MELD scores. For the prediction of survival in HBV-LC, the area under the curve (AUC) for the Treg/Th17 ratio was 0.78 (P=0.024); a cut-off value of 21.26 yielded the highest predictive value with a sensitivity of 85.0% and a specificity of 75.0%. In addition, serum IL-10, TGF-β1, IL-17, IL-23 levels and the TGF-β1/IL-17 ratio were significantly increased in HBV-LC patients, and the TGF-β1/IL-17 ratio was especially increased in non-survival and decompensated liver cirrhosis (DCLC) patients and positively correlated with TBil, Child-pugh and MELD scores. A cutoff TGF-β1/IL-17 ratio of 0.31 provided the best predictive value for DCLC (AUC 0.91, P=0.001) with a sensitivity of 80.0% and a specificity of 87.5%. Conclusions: The decreased ratio of Treg/Th17 and increased ratio of TGF-β1 /IL-17 may be associated with the survival and disease progression in HBV-LC patients and both ratios can be used independently to predict prognosis and disease progression. Key words: hepatitis B virus, liver cirrhosis, Treg cells, Th17 cells

Disclosures:

The following people have nothing to disclose: Xueping Yu, Zhijun Su, Ruyi Guo, Desong Ming, Lvye Huang, Milong Su, Yong Deng, Zhenzhong Lin

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The correlation between suppressor of cytokine signaling family and hepatitis B virus: possible involvement in resistance of interferon treatment

Hong Tang1,2, Ling-Yao Du1,2, Yao-Li Cui1,2, En-Qiang Chen1,2, Xing Cheng1,2, Li Liu1,2;
1 Center of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, China; 2Division of Infectious Diseases, State Key Laboratory of Biotherapy, Sichuan University, Chengdu, China

Aims: The suppressor of cytokine signaling family(SOCS) is an extremely important kind of negative regulator in JAK-STAT signaling pathway. This study was designed to explore the correlation among SOCS, Hepatits B Virus(HBV) and interferon(IFN). The relationship between SOCS and IFN therapeutic efficacy was also studied. Materials and Methods: In animal experiment, four groups of mice model were established. Group A was administered with HBV replicative plasmid pHBV4.1 and IFN inducer Poly IC while Group B with pHBV4.1, Group C with Poly IC and Group D with saline. Liver tissues were then harvest to analyze the SOCS expression. In clinical trial, CHB patients enrolled were required to receive IFNα-2b treatment for 24-48 weeks. The baseline SOCS expression was tested with liver tissues from biopsy. The serum assays were implemented at baseline, week 12, 24 and 48 for efficacy evalution and correlation analysis. Results: In animal experiment, The expression levels of SOCS-1 and SOCS-3 were highest in Group B, then Group A, Group C and Group D sequentially. There was no difference in the SOCS-2 expression among the four groups. In clinical trial, the expression of SOCS-1, SOCS-3 was higher in 24 enrolled CHB patients compared to normal control. SOCS-2 was observed in none of them. The baseline HBV-DNA level was in positive correlation with SOCS-1 and SOCS-3 expression. The age, viral genotype, HBVDNA, SOCS-1 and SOCS-3 were related to IFN efficacy. Conclusions: HBV could induce SOCS-1 and SOCS-3 expression no matter internal IFN was up-regulated or not. The internal IFN up-regulation would directly up-regulate SOCS-1 and SOCS-3 via negative feedback as well as indirectly down-regulate SOCS-1 and SOCS-3 via inhibiting HBV. The HBV contributed more than IFN in SOCS up-regulation. It might account for the reseason why patients with high HBV viral load would encounter poor efficacy in IFN treatment.

Disclosures:

The following people have nothing to disclose: Hong Tang, Ling-Yao Du, Yao-Li Cui, En-Qiang Chen, Xing Cheng, Li Liu

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Successful immunological responses against hepatitis B virus in human peripheral blood mononuclear cells-engrafted mice

Satoshi Aono1, Tomohide Tatsumi1, Hayato Hikita1, Seiichi Tawara1, Akira Nishio1, Takatoshi Nawa1, Yoshiki Onishi1, Satoshi Shimizu1, Ryotaro Sakamori1, Takuya Miyagi1, Naoki Hiramatsu1, Hiroshi Suemizu2, Takeshi Takahashi2, Tetsuo Takehara1;
1Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan; 2Central Institute for Experimental Animals, Kawasaki, Japan

Background&Aims: Reconstitution of human immune cells is warranted in liver chimeric mice (which is immune deficient) to examine the mechanism of inflammation and hepatocarcino genesis by hepatitis B virus (HBV). Injection of human peripheral blood mononuclear cells (PBMC) into severely immunodeficient NOG mice resulted in being lethal due to severe graft-versus-host disease (GVHD). Recently, we generated NOG mice with double knockout of both MHC class I and II (DKO-NOG mice). In this study, we evaluated immune responses against HBV in PBMC-engrafted DKO-NOG mice. Methods: We used NOG (NODShi.Cg-PrkdcscidIl12rgtm 1sug) mice and DKO-NOG (NOG-Iaβ/β2m double KO) mice which are deficient in both MHC class I and II. Human PBMC were injected into NOG and DKO-NOG mice via tail vein. We evaluated liver histology and serum ALT levels. To detect the engrafted human immune cells in mice, liver mononuclear cells (MNCs) isolated from these mice were subjected to flow cytometry. Next, we evaluated the production of anti-HBs antibody (anti-HBs) in sera of human PBMC-engrafted DKO-NOG mice after inoculation of recombinant hepatitis B vaccine. We also evaluated the induction of HBc-derived peptide-specific cytotoxic T lymphocytes (CTL) by using specific tetramer after vaccination of HBc-derived peptide-pulsed dendritic cells (DCs). Results: After inoculation of human immune cells, both infiltration into the liver of human immune cells and hepatocyte damage were observed in NOG mice, but not in DKO-NOG mice. Serum ALT levels were severely elevated in NOG mice, but not in DKO-NOG mice (510±299 IU/l vs, 14.5±4.12 IU/L at day 15, respectively). These demonstrated that GVHD was prevented in PBMC-engrafted DKO-NOG mice. In consistent with this, 7 of 8 NOG mice died within 2 month after injection of human PBMC whereas all DKO-NOG mice survived more than 70 days. On day 28 after injection of human PBMC, replacement rates of human immune cells in the liver increased up to 85% and the frequencies of human CD8+ T cells and B cells was 40% and 10% respectively in DKO-NOG mice. Inoculations of recombinant hepatitis B vaccine resulted in the production of anti-HBs in 2 of six vaccinated mice. DC vaccination resulted in significant increase of HBc-derived peptide-specific human CTL in DKO-NOG mice, but not in control mice. Conclusion: The present study demonstrates that human PBMC-engrafted DKO-NOG mice allowed long survival of human mature immune cells without liver damage and produced immunological responses against HBV. This system would provide us with a new promising model evaluating immunological responses against HBV in human chimeric livers.

Disclosures:

Tetsuo Takehara - Grant/Research Support: Chugai Pharmaceutical Co., MSD K.K.

The following people have nothing to disclose: Satoshi Aono, Tomohide Tatsumi, Hayato Hikita, Seiichi Tawara, Akira Nishio, Takatoshi Nawa, Yoshiki Onishi, Satoshi Shimizu, Ryotaro Sakamori, Takuya Miyagi, Naoki Hiramatsu, Hiroshi Suemizu, Takeshi Takahashi

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miR-155 regulates dendritic cell activation in chronic hepatitis B virus (CHBV) infected patients on Tenofovir plus Peg Interferon Therapy

Nirupma Trehanpati1, Ritu Khosla1, Paul David1, Rashi Sehgal1, Ashish Vyas1, Arshi Khanam1, Anupama Prashar1, Syed Hissar1, Gayatri Ramakrishna1, ShivK. Sarin1,2;
1Research, Institute of Liver and Biliary Sciences, New Delhi, India; 2Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India

Background: Viral load reduction with antiviral agents followed by immune modulation with interferon (sequential therapy) is a promising new option to achieve higher response rates. Dendritic Cell (DC) maturation and proliferation is important for initiating antiviral immunity, efficient immune response and viral clearance during antiviral therapy. Aim: To assess the expression of miRNAs specific for activation, maturation and proliferation of DCs; miR- 222, miR 221, miR 146a and miR 155. Patients and Methods: CHB Patients ( n=10) (Age (Yrs) mean ± SD; 30.2 ± 11, M:F 7:3) with mean HBV DNA levels 7.0 ± 0.2 log 10 copies/ml, received tenfovir for 8 weeks followed by a combination of tenfovir plus PEG IFN as sequential therapy. 20 ml of blood was collected from patients at baseline and after 8th weeks of sequential therapy. Pan DCs were isolated from the whole blood with magnet assisted cell sorting using Pan DC enrichment kit (Stem Cell Technologies). Total RNA was isolated from DC+ve fraction using miRVANA kit (Ambion). cDNAs were prepared with miR specific stem loop primers and using miR CURY LNA Universal RT and microRNA PCR Universal cDNA synthesis kit. Quantitative RT-PCR was performed in duplicate in Roche light cycler 480 using syber-Green. 5srRNA was used as the control for normalization. Results: With more than 2 log viral decline, functionality of DCs were increased more than fivefold (detected by phagocytic assay) in patients undergoing Tenefovir and PEG interferon sequential anti-viral therapy compared to baseline. Relative expression of specific miR 146a, miR 221 and miR 222 did not show any significant difference in DCs before and after therapy. However, miRNA 155 which is particularly playing important role in maturation and proliferation of immune cells was significantly upregulated in DCs. Conclusion: miR 155 expression was significantly increased in DCs of CHBV patients undergoing Tenefovir plus PEG interferon therapy. miR 155 can be used as a marker for efficient immune response after Tenefovir plus PEG interferon therapy.

Disclosures:

The following people have nothing to disclose: Nirupma Trehanpati, Ritu Khosla, Paul David, Rashi Sehgal, Ashish Vyas, Arshi Khanam, Anupama Prashar, Syed Hissar, Gayatri Ramakrishna, Shiv K. Sarin

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Toll-Like Receptors 2 and 4, and Quantitative Hepatitis B Antigen Levels in Chronic Hepatitis B

Erdem Akbal1, Erdem Kocak1, Omer Basar2, Bilal Ergül1, Seyfettin Köklü3, Selma Uysal4, Sema Uysal4;
1Gastroenterology, Ankara Education and Research Hospital, Ankara, Turkey; 2Gastroenterology, Akdeniz University, School of Medicine, Antalya, Turkey; 3Gastroenterology, Hacettepe University, School of Medicine, Ankara, Turkey; 4Biochemistry, Ankara Numune Education and Research Hospital, Ankara, Turkey

Background and aims: Hepatitis B is the most common liver infection which can lead to cirrhosis and liver cancer. Knowledge on the mechanism how hepatitis-B infection increases the risk of chronic infection is still lacking. Toll-like receptors (TLR) was shown to play an important role in the pathogenesis of chronic liver disease. However, little is known whether TLR effects pathogenesis and progression of different status of chronic Hepatitis B. The aim of this study was to investigate serum TLR2, TLR4 and HbsAg levels in different stages of the patients with chronic hepatitis B. Material-methods: A total 38 naive HbeAg negative chronic hepatitis-B, 22 HbsAg inactive carriers and 20 healthy control subjects were recruited in this study. Liver tests, HBV DNA, TLR2, TLR4 and quantitative HbsAg levels were evaluated among all groups. The relationship between TLR2, TLR4, quantitative HbsAg levels and liver tests and liver histological findings were investigated with correlation analysis. Results: TLR2 and TLR4 levels in HbeAg negative chronic hepatitis-B patients and in inactive carriers were higher than in control group (TLR2: 1.35±1.12, 1.2±0.85, 0.88±0.82 ng/ml; respectively. TLR4: 0.51 ±1.2, 0.22±0.13, 0.14±0.06 ng/ml; respectively). Quantitative HbsAg levels in HbeAg negative chronic hepatitis-B patients were significantly higher compared to inactive carriers (5972, 3372 IU/ml; respectively). Although TLR2 and TLR4 levels were higher in chronically infected patients compared with inactive carriers, the difference was not statistically significant (p:0.785). Indeed, a positive correlation between TLR2, TLR4, and HBV DNA and ALT levels were observed (ForTLR2 and HBV DNA r:0.254, p:0.026; For TLR4 and HBV DNA r:0.281, p:0.013 and For TLR2 and ALT r:0.262, p:0.021; For TLR4 and ALT r:0.232, p:0.04). Similarly, while quantitative HbsAg levels were correlated with HBV DNA (r:0.805, p<0.001), no correlation between quantitative HbsAg levels and TLR 2 and TLR 4 levels was found (For TLR2 and HBsAg r:0.237, p:0.118; For TLR4 and HBsAg r:-0.275, p:0.067). Conclusion: TLR can have an important role in hepatitis B pathogenesis. Also, this is the first study showing the relation of TLR in inactive Hepatitis B carriers. Liver injury in chronic hepatitis B may cause elevated TLR 2 and TLR 4.

Disclosures:

The following people have nothing to disclose: Erdem Akbal, Erdem Kocak, Omer Basar, Bilal Ergül, Seyfettin Köklü, Selma Uysal, Sema Uysal

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Regulation of TLR2, IRAK1 and IRAK4 expression on human monocyte-derived dendritic cells by hepatitis B virus E antigen

Jun Li, Yuying Wu, Longfeng Jiang, Zuhu Huang;
Department of Infectious Diseases, The First Affiliated Hospital with Nanjing Medical University, Nanjing, China

Background: The hepatitis B e-antigen (HBeAg) is a nonparticulate secretory protein expressed by all viruses within the family Hepadnaviridae. It is not essential for viral assembly or replication but is important for establishment of persistent infection in vivo. The aim of this study was detect the effect of HBeAg on TLR2 signal pathway of dendritic cell,to further explore the mechanism of HBeAg on mDCs in chronic hepatitis B virus infection process. Method: Firstly, Monocytes were isolated from normal peripheral blood mononuclear cells (PBMCs), cultured with cytokines including recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF), recombinant human interleukin 4 (rhIL-4) to induce mDCs. Secondly, on Day 7 the cells were divided into 8 groups, add RPMI 1640, OVA, recombinant HBeAg, HBsAg, complexes of HBeAg and anti-HBe, anti-HBe, Pam3CSK4, HBeAg+Pam3CSK4(adding Pam3CSK4 after 3h of HBeAg stimulation) respectively, γ-globulin (final concentration of intervention of each group is of 10 ng/ml) as intervention. Each group was incubated for 9 h. We set 1640 as blank control, OVA as Unrelated protein control. Then each group respectively was examed the expression of related surface molecules of mDCs by flow cytometry, and detect the secretion of IL-12 by ELISA. Detect the alternations of expression of TLR 2, IRAK 1and IRAK 4 by SYBR Green I realtime quantitative PCR of OVA group, control group and recombinant HBeAg group. Results: The expression of CD11 c+TLR 2+ of HBeAg Group was 65.94% ± 3.022, the unstimulated group was 79.70 % ± 1.795, the OVA group was 79.16 % ± 1.759. We can see the expression of HBeAg group is significantly lower than the expression of unstimulated group (t=3.915, P<0.01, n=10) and OVA group (t=3.167, P<0.01, n=10).The relative quantity of mRNA of TLR 2 in HBeAg group compare to which in the unstimulated group is 0.727, The relative quantity of mRNA of IRAK 1 in HBeAg group compare to which in the unstimulated group is 0.345, The relative quantity of mRNA of IRAK 4 in HBeAg group compare to which in the unstimulated group is 0.645. The secretion of IL-12p70 of HBeAg group is lower than control group and OVA group. The expression of CD11c+TLR2+ of HBeAg/anti-HBe group and HBeAg+Pam3CSK4 group was 78.02%±3.524, 80.71 %±2.674, which is higher than HBeAg group, but we can see no obviously different than control group; the secretion of IL-12p70 of HBeAg/anti-HBe group and HBeAg+Pam3CSK4 group were both higher than HBeAg group. Conclusion: HBeAg can inhibit TLR 2 signaling pathway of mDCs, which is one of the mechanism of cellular immunity deficiency in chronic HBV infection.

Disclosures:

The following people have nothing to disclose: Jun Li, Yuying Wu, Longfeng Jiang, Zuhu Huang

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Lack of support of association of the KIF1B rs 17401966 variant and hepatocellular carcinoma

Ping An1, Zheng Zeng2, Cheryl A. Winkler1;
1SAIC-Frederick, Inc, Frederick, MD; 2Peking University First Hospital, Beijing, China

Background: A recent genome-wide association study (GWAS) performed in the Chinese chronic hepatitis B virus (HBV) carriers with or without hepatocellular carcinoma (HCC) revealed a strong association between SNP rs17401966 in the KIF 1 B gene and the risk of HCC (Odds ratio, OR = 0.62; Nature Genetics, 2010). However, this association was not replicated in other studies. We therefore attempted to replicate this association for HCC and cirrhosis in a case-control study. Methods: We investigated the associated KIF 1 b rs17401966 in a well characterized case-control study of Northern Chinese comprising the spectrum of HBV infection stages comprising a control group of HBV chronic carriers (n= 540) and two case groups: cirrhosis (n = 346); and HCC (n = 239). Genotyping was determined using a Taqman assay and analysis was performed by logistic regression for additive, dominant, and recessive models. Results: Genotype frequencies for rs17401966 were in Hardy-Weinberg equilibrium in the cases and control groups. The allele and genotype frequencies were similar in chronic hepatitis patients with HCC (30.0%) or without HCC (G allele frequency, 28.0%), similar to that in the reference HapMap and 1000 genome Chinese populations (24-28%). Under an additive genetic model as used in the original discovery publication, we found no associations between rs17401966 and HCC [OR: 0.92, 95% confidence interval (CI): 0.73,1.14; p = 0.44].; further adjustment with sex and age made no difference (p = 0.53), with age being a significant risk factor of HCC (OR:2.32, p < 0.0001). No association was observed in dominant, and recessive models. Furthermore, we found no association with development of cirrhosis (OR: 1.03, 95%CI: 0.82,1.25, p = 0.91). Conclusion: Although we had 98% power to detect an association with OR of 0.62/per allele as previously reported, we found no significant associations with liver cirrhosis or HCC. However, as the possibility of a smaller effect of HIF1b rs17401966 cannot be excluded, the role of HIF1 B warrants further investigation with greater power.

Disclosures:

The following people have nothing to disclose: Ping An, Zheng Zeng, Cheryl A. Winkler

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“Hepatitis Delta virus and autoimmune hepatitis: A spurious association?”

Sandra Beinhardt1, Judith Stift2, Robert Paul Strassl3, Karoline Rutter1,
Albert Stättermayer1, Hans Peter Dienes2, Heidemarie Holzmann4, Fritz Wrba2, Peter Ferenci1, Harald Hofer1; 1Internal Medicine III; Department of Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria; 2Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria; 3Depart-ment of Laboratory Medicine, Medical University of Vienna, Vienna, Austria; 4Department of Virology, Medical University of Vienna, Vienna, Austria

Background & Aims: Hepatitis Delta virus (HDV) infection is known to induce autoimmune epiphenomena. However, the presence of autoimmune features in patients with chronic HDV infection may cause a diagnostic and moreover a therapeutic dilemma as interferon-treatment may deteriorate autoimmune induced liver injury. The aim of the present study was to investigate presence of serologic and histologic autoimmune features in HDV infected patients. Patients & Methods: Data of 53 HBs-Ag/anti-HDV serum-positive patients (male:36; age:43.4±13.4[21-73]years;mean±SD[range]) were retrospectively analyzed. HBe-Ag-status was available in 37(70%) patients; all of them were HBe-Ag negative. HBV-DNA was either under limit of detection (<LoD) or low (range:-822 IU/ml). Liver-biopsies were performed in 32(60%) patients. Histologic assessment was conducted by three blinded board-certified pathologists, unaware of clinical or laboratory data. Presence of antinuclear antibodies (ANA), anti smooth muscle antibodies (SMA), antibodies against liver-kidney microsomal type1 (LKM-1) as well as soluble liver antigen (SLA) was evaluated. Results: 11/53 (21%) HDV-infected patients had autoantibodies (10[19%]; ANA 1:80 in 7[13%]; 1:320 in 1 [2%], 1:640 in 1[2%], ASMA 1:160 in 1 [2%] patient(s)) and/or elevated immunoglobulin G-levels (IgG; 6[11%], mean 2490±351 (mg/dl;[±SD]). Histo-pathologic findings in concordance to pathognomonic features of autoimmune hepatitis were found in 28% of patients (9/32; see table). Cirrhosis was present in 15(47%) patients at diagnosis. Autoimmune-thyroiditis and -thrombocytopenia was seen in one patient. Majority of HDV-patients were immigrants (Africa: 10[19%]; Asia:7[13%], Eastern Europe/GUS:29[55%]), 7(13%) were of Austrian origin. Co-infection with hepatitis C virus was found in 12(23%) patients. Conclusion: In HDV infected patients serologic as well as histologic findings, mimicking autoimmune hepatitis, are frequent. Further investigations in larger patient cohorts are needed to confirm this observation.

Patient-#PortaltractsPlasmacellsEmperi-polesisRosettesPericentral InfiltrationBile duct alterationANAIgG quant.
111scatteredyesyesyesno1:80-
24groupsnoyesnono1:6402210
31groupsyesyesyesno1:3203020
412groupsnoyesnono1:802600
5cirrhosisgroupsyesyescirrhosisyes--
623groupsnoyesnono1:802480
712groupsyesyesnoyes1:802140
819groupsnoyesyesyes--
917groupsyesyesnoyes--

Disclosures:

Peter Ferenci - Advisory Committees or Review Panels: Roche, Idenix, Roche, MSD, Vertex, Salix, Madaus Rottapharm, Tibotec, EWdhringer Ingelheim, Achilleon, GSK; Grant/Research Support: MSD, Vertex, Madaus Rottapharm; Patent Held/Filed: Madaus Rottapharm; Speaking and Teaching: Roche, Gilead, Roche, Gilead, Salix

Harald Hofer - Speaking and Teaching: Janssen, Roche, MSD

The following people have nothing to disclose: Sandra Beinhardt, Judith Stift, Robert Paul Strassl, Karoline Rutter, Albert Stättermayer, Hans Peter Dienes, Heidemarie Holzmann, Fritz Wrba

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Monitoring HBV-specific immunity in patients with chronic HBV infection during pegylated interferon α-2a therapy and upon a novel pDC-based immunotherapeutic strategy : ANRS HB06 PEGAN study

Caroline Aspord1, Juliana Bruder1, Tania Dufeu-Duchesne4,5, Noelle Pouget2, Fabrice Carrat2, Marc Bourlière3, Joel Plumas1, Vincent Leroy4,5;
1EFS, Grenoble, France; 2Hopital Saint-Antoine, Paris, France; 3Hopital Sant-Joseph, Marseille, France; 4CHU de Grenoble, Grenoble, France; 5IAB - INSERM U823 - IAPC, Grenoble, France

The immune control of HBV infection is essential for viral clearance. Restoring functional anti HBV immunity is therefore a promising immunotherapeutic approach for treatment of Chronic Hepatitis B (CHB) infection. Plasmacytoid dendritic cells (pDC) play a crucial role in triggering antiviral immunity through their ability to secrete large amounts of type I IFN, and their potential to capture and process viral antigens to subsequently induce adaptive immune responses. In line with these concepts, pegylated interferon α2a (PEG IFN) therapy represents an alternative to current antiviral approaches. However the mechanisms leading to a positive clinical outcome remain not fully understood. We therefore investigated the immunomodulatory effects of PEG IFN on HBV specific T cell responses. First, we developed a novel immunotherapeutic strategy based on a HLA A*0201+ pDC line loaded with HLA A*0201 restricted peptides to elicit specific and functional T cells. We demonstrated the ability of the pDC-based strategy to amplify functional HBV-specific CD8 T cells ex vivo from PBMC and TIL from chronic HBV patients in 45.8% of cases. The specific T cells from the “responder” group secreted IFNγ, expressed CD107 upon restimulation and efficiently lysed HBV antigen expressing hepatocytes. Circulating HBeAg was found to distinguish the group of patients not responding to the pDC stimulation and appears to be critical in determining the outcome of immunotherapies in chronic HBV patients. We then assessed the ability of patients with CHB infection treated with nucleos(t)ide analogue alone or together with PEG IFN to respond to the pDC-based strategy. Notably, patients initially unable to respond to a specific immune stimulation based on HBV derived-peptide loaded pDCs became responsive during the course of PEG IFN therapy. More widely, we analysed before and during PEG IFN treatment the frequency of HBc-, HBs-, HBx and pol specific T cell responses by intracellular cytokine staining upon stimulation with overlapping peptide pools derived from the corresponding HBV antigens. We observed a potent increase in the frequency of TNFα and/or IFNγ producing HBc, HBs and HBx specific T cells during the course of PEG IFN therapy. Thus, a pDC based immunotherapeutic approach could be of interest in attempts to restore functional antiviral immunity, which is critical for the control of the virus in chronic HBV patients. PEG IFN therapy drives a potent improvement in the frequencies of HBV specific T cell responses and is able to convert non-responders into responders to a specific immune stimulation.

Disclosures:

Fabrice Carrat - Grant/Research Support: Janssen, Merck, Gilead, BMS, Roche

Marc Bourlière - Advisory Committees or Review Panels: Schering-Plough, Bohringer inghelmein, Merck, Schering-Plough, Bohringer inghelmein, Merck ; Board Membership: Bristol-Myers Squibb, Gilead, Bristol-Myers Squibb, Gilead; Consulting: Roche, Novartis, Tibotec, Abott, glaxo smith kline, Roche, Novartis, Tibotec, Abott, glaxo smith kline

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: Caroline Aspord, Juliana Bruder, Tania Dufeu-Duchesne, Noelle Pouget, Joel Plumas

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Association of Toll-like receptor 9 genetic polymorphism with spontaneous viral clearance of hepatitis B virus infection and reactivity of liver disease

Xuan Zhou1, De-Ming Tan1, Xu-Wen Xu1, Xiao-Yu Fu1, Li Chen2, Cui-Mei Liu1, Jian-Ping Xie1;
1Department of Infectious Diseases, Xiangya Hospital, Central South University, Changsha, Hunan, China; 2Department of Infectious Diseases, The Third Affiliated Xiangya Hospital, Central South University, Changsha, Hunan, China

Objective: To explore the correlation between the single nucleotide polymorphisms(SNPs) of Toll-like receptor 9(TLR9) gene and clinical status of Hepatitis B virus infection. Methods: Case-controlled study was performed in 422 patients with per-sisitent chronic HBV infections which included 149 asymptomatic carriers(ASC), 178 chronic hepatitis B(CHB) patients and 95 patients associated acute on chronic liver failure(ACLF), and 174 cases of HBV spontaneous clearance persons The four TLR9 gene SNPs at positions rs 187084, rs352139, rs352140 and rs5743836 were analyzed by the Taqman probe and realtime polymerase chain reaction and the genotypic distribution was analyzed according disease progression of HBV infeciton, inflammation degree of the liver and serum HBV-DNA loads. Results: TLR9 rs187084, rs352139, rs352140 genotyper and allele distribution were different between ASC group and HBV spontaneous clearance persons (P<0.05). The frequency of rs187084 genotype TT, TC and allele T of HBV spontaneous clearance persons were significantly higher than those of chronic HBV infection (P<0.05). In the chronic HBV infection group, The frequency of rs187084 genotype TT, TC and allele T of CHB and ACLF group were significantly higher than those of ASC group (P<0.05), genotype AA, allele A of rs352139 and genotype CC, allel C of rs352140 of CHB group were obviously higher than those of ASC group. The three SNPs analysis of rs187084, rs352139, rs352140 has no obvious correlation with the inflammation degree of the liver and serum HBV-DNA loads. The study also found that rs5743836 may not exist gene polymorphism in this crowd of this study . And there was a relationship of Linkage disequilibrium between rs187084, rs352139 and rs352140. Conclusion: TLR9 gene rs187084, rs352139, rs352140 polymorphism is associated with the human immune clearance of HBV infection. The T allele of TLR9 genen rs187084, the A allele of rs352139 and the C allele of rs352140 may promote the reactivity of chronic hepatitis B infection.

Disclosures:

The following people have nothing to disclose: Xuan Zhou, De-Ming Tan, Xu-Wen Xu, Xiao-Yu Fu, Li Chen, Cui-Mei Liu, Jian-Ping Xie

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Enhanced expression of potassium ion channel gene KCNJ15 is associated with Acute-on-chronic Hepatitis B liver failure

LinZhu1, Tao Chen1, Li Song1, Xiaoping Luo2, Qin Ning1;
1Institute and Department of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

Increased KCNJ15 expression has been found in patients with hepatitis B virus (HBV)-induced Acute-on-chronic liver failure (HBV-ACLF) in our previous gene microarray study. However, the mechanism underlying the enhanced expression of KCNJ15 in patients with HBV-ACLF remains unclear. In this study, we further investigate the up-regulated KCNJ15 and its possible regulatory mechanisms in HBV induced liver injury. The expression of KCNJ15 was studied in patients with HBV-ACLF on both mRNAand protein levels in peripheral blood mononuclear cells (PBMC) and liver tissue, while the function of KCNJ15 was further determined in Jurkat cells in vitro by transfection of a human KCNJ15 expression plasmid. The results showed that KCNJ15 was over-expressed on PBMCs and hepatic infiltrating lymphocytes from HBV-ACLF patients. Moreover, KCNJ15 had an abundant expression in peripheral and hepatic CD4+T cells in patients with HBV-ACLF. In the supernatant of KCNJ15 over expressed Jurkat cells, increased IL-17A secretion was observed, which was involved with elevated intracellular calcium concentration. Our data suggested KCNJ15 may contribute to liver injury through IL-17A production in HBV induced liver failure. Furthermore, we also provide a potential susceptible gene for patients with HBV-ACLF.

Disclosures:

Qin Ning - Advisory Committees or Review Panels: ROCHE, NOVARTIS, BMS, MSD, GSK; Consulting: ROCHE, NOVARTIS, BMS, MSD, GSK; Grant/Research Support: ROCHE, NOVARTIS, BMS; Speaking and Teaching: ROCHE, NOVARTIS, BMS, MSD, GSK

The following people have nothing to disclose: Lin Zhu, Tao Chen, Li Song, Xiaoping Luo

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Cooperative effects of Hepatitis B virus and TNF might play important roles in hepatocarcinogenesis through activation of NF-κB, metabolic and ER stress signaling

Shuang Wu, Tatsuo Kanda, Tatsuo Miyamura, Xia Jiang, Shingo Nakamoto, Fumio Imazeki, Osamu Yokosuka;
Department of Gas-troenterology and Nephrology, Chiba University,Graduate School of Medicine, Chiba, Japan

Background: Elevations of inflammatory cytokines such as TNF and IL-1 β are often seen in sera of hepatitis B virus (HBV)-infected patients. It is well known that these cytokines activate NF-κB signaling, and are associated with ER stress. We investigated whether HBV or HBx enhanced NF-κB activation in the presence of TNF/IL-1β, and whether endoplasmic reticulum (ER) stress was up-regulated in HepG2 and HepG2.2.15 with or without TNF. Methods: We examined whether HBV or HBx enhanced cytokine-induced NF-κB activation in hepatocytes using reporter assay. Human ER stress-related genes were quantified by real-time PCR between HepG2 and HepG2.2.15 with or without TNF. Expressions of TNFR1, TNFR2, IL1R1, IL1 R2, MICA, NF-κB, pNF-κBp65 (Ser536), IRE1 and XBP1 were determined by Western blot. Results: HepG2, HepG2.2.15 and Huh7 cells expressed TNFR1, TNFR2, IL1 R1, IL1 R2 and MICA which are reported to be involved in hepatocarcinogenesis. In Huh7 cells, PBS, TNF, IL1β and TNF plus IL1β activated NF-κB signaling (1.0, 1.9, 1.7, and 2.2-fold, respectively) and in the addition of HBV/HBx, TNF, IL1β and TNF plus IL1β more activated NF-κB signaling (3.2/8.8, 8.5/18, 8.0/16, and 7.4/17-fold, respectively; P<0.05 compared to control) although intracellular MICA was not altered in hepatocytes after the addition of cytokines. These activation were inhibited with 1 μM MG132, suggesting proteasome pathways were involved in HBV/HBx-mediated NF-κB activation. We also observed phosphorylation of NF-κB in HepG2 and HepG2.2.15. The result of real-time PCR showed metabolic genes APOA1, DGAT2, GK and IGFBP1, and XBP1 were up-regulated in the existence of HBV with or without TNF (100< or 100<, 10 or 33, 15 or 8, and 22 or 128, and 7.5 or 4.6-fold, respectively; P<0.05). Cooperative effects of HBV and TNF were also observed in the enhancement of IRE1 and XBP1 expression at protein levels. Conclusion: We observed cooperative effects of HBV/HBx, and TNF enhanced the activation of NF-κB as well as up-regulated metabolic genes and ER stress related genes in hepatocytes. These effects might be important in HBV-associated hepatocarcinogenesis.

Disclosures:

Tatsuo Kanda - Speaking and Teaching: Chugai Pharmaceutical, MSD, Ajinomoto, GlaxoSmithKlein, Mitsubishi Tanabe Pharma, Bristol-Myers Squibb

The following people have nothing to disclose: Shuang Wu, Tatsuo Miyamura, Xia Jiang, Shingo Nakamoto, Fumio Imazeki, Osamu Yokosuka

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T cell characteristics during acute hepatitis B infection

Annikki de Niet1,2, Sophie Willemse1,2, Femke Stelma1,2, Marjan J. Tempelmans Plat-Sinnige2, Ester Remmerswaal2, Hendrik W. Reesink1, Rene van Lier3, Ester M. van Leeuwen2;
1Hepatology, Academical Medical Center, Amsterdam, Netherlands; 2Experi-mental Immunology, Academic Medical Center, Amsterdam, Netherlands; 3Blood Supply, Sanquin, Amsterdam, Netherlands

Introduction: One of the major issues in hepatitis B virus (HBV) infection is the question why some people become chronically infected whereas other people clear the virus and form protective antibodies against the virus. In this study we investigated T cell characteristics in acute hepatitis B (AHB) patients that resolve viral infection or become chronically infected. Material and Methods: Four patients with AHB (defined by HBsAg/IgM anti-HBc positivity, ALT>10X ULN and a risk factor for AHB) were evaluated and followed until the clearance of HBsAg or the development of chronic infection. Serially obtained PBMC's were analyzed by FACS. Results: Three of the 4 patients cleared hepatitis B and developed anti-HBs antibodies within 6 months after presentation (genotype D,B and E). One patient (genotype A) established a chronic infection and had to be treated with nucleotide analogue. Mean peak ALT was 2395 U/L (range 1631-2877) and mean peak total serum bilirubin was 220 umol/L (range 30-552). Mean peak HBV DNA levels were log 6.06 IU/mL (SD 0.85; range 5.33-7.23). Two-4 weeks after onset of symptoms mean % of HLA-DR/CD38++ CD8+ T cells, representing activation of T cells, for the three patients that resolved infection was 26.0 (range 18.9-33.1). In these patients the decline of activated CD8+ T cells coincided with the decline in ALT levels. In contrast, in the patient with persistent infection, ALT levels remained high and very low amounts of HLA-DR/CD38++ CD8+ T cells of 2.8% were present. Similar findings were observed for the percentage of Ki-67+ CD8+ T cells that represent the amount of proliferated cells: 9.25% (range 7.8-10.7) in patients with resolving infection versus 0.7% in the patient with persistent virus. Characteristics of HBV specific T cells and cytotoxic potential, in relation to T cell activation, are currently under investigation. Conclusion: Currently, simple surrogate markers for the outcome of acute hepatitis B at onset of disease are not available. In this study we analyzed in AHB how the HBV-specific CD8+ T cell response is modulated. Our study suggest that at least some activation and proliferation of T cells is required to establish a full immune response to clear AHB virus infection. If studies with larger patient numbers confirm these findings additional strategies may be developed to optimize T cell function to enhance viral clearance.

Disclosures:

Hendrik W. Reesink - Consulting: Abbott, Gilead, Astex, Merck, Roche, Janssen Cilag, GlaxoSmithKline, Tibotec/JJ, PRA-International; Grant/Research Support: Vertex, Boehringer Ingelheim, Anadys, Phenomix, Chugai, Japan Tobacco, Santaris, SGS, Idenix, BMS

The following people have nothing to disclose: Annikki de Niet, Sophie Willemse, Femke Stelma, Marjan J. Tempelmans Plat-Sinnige, Ester Remmerswaal, Rene van Lier, Ester M. van Leeuwen

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Differential Hepatitis B virus core nuclear/cytoplasmic staining is a marker of longevity of chronic infection but not disease phase

Antony Chen1,2, Upkar S. Gill1, Cameron R. Ikin2, Jo-Anne Chin Aleong3, Graham R. Foster1, Patrick T. Kennedy1;
1Hepatology Unit, Centre for Digestive Diseases, Blizard Institute, Barts and The London SMD, QMUL, London, United Kingdom; 2Division of Infection & Immunity, UCL, London, United Kingdom; 3Cellular Pathology, Barts Health NHS Trust, London, United Kingdom

INTRODUCTION: The cytoplasmic processing and presentation of viral antigen is a key driver of the T-cell response in Chronic Hepatitis B (CHB). Hepatitis B virus (HBV) core protein localises in the cell nucleus and/or cytoplasm depending on the viral life cycle and timing of infection, and thus may vary with patient age. We evaluated the distribution of the core protein in liver tissue in a cohort of young adults and compared it to older patients to elucidate the relationship between staining pattern and disease phase and to determine any correlation with duration of viral infection PATIENTS & METHODS: 71 paraffin embedded sections of liver tissue from young adult patients (<30 years), median age 24 (range 11-29) were stained with Hepatitis B core antibody (HBcAb). 21 older patients, median age 39 (range 30-58) were also analysed. The percentage of hepatocytes staining positive for HBV core protein, (nuclear and/or cytoplasmic) were quantified. Data on eAg status, longitudinal ALT, HBV DNA, Ishak fibrosis stage and necroinflam-matory (NI) scores were documented to establish clinical correlations. RESULTS: 76/92 liver sections stained positive with HBcAb. The median percentage of hepatocytes positive for nuclear HBV core was 4.85% (range=0.01-32.5) and 23.7% (range=0.15-88.9) for cytoplasmic HBV core. eAg positive patients (46/92) showed a trend towards a higher affinity of nuclear HBV core positivity, but this did not meet statistical significance (p=0.06). HBV DNA viral load >7 logIU/ml correlated with a higher percentage of nuclear HBV core expression (p=0.03), more marked in patients with viral loads >8 logIU/ml (p=0.006). No correlation was identified with ALT levels and core staining. Higher NI scores were associated with increased core positive hepatocytes in the cytoplasm (p=0.05), while lower NI scores showed increased nuclear core positivity (p=0.04). Considering patient age; older patients (>30 years) showed evidence of increased cytoplasmic core staining compared to younger patients (31.5% vs. 11.5%; p=0.003), who demonstrated increased hepatocytes with nuclear core positivity (p=0.05). CONCLUSIONS: We demonstrate that younger patients, who have lower NI scores and higher levels of HBV DNA, typical of an immune tolerant profile, show increased nuclear HBV core protein staining. Conversely, older patients with longer duration of CHB infection had demonstrably higher cytoplasmic core staining. However, no correlation between serum ALT and HBV core staining was noted to confer immune activity. Immunohistochemical HBV core staining may provide additional information about longevity of CHB infection, but does not distinguish disease phase.

Disclosures:

Graham R. Foster - Advisory Committees or Review Panels: GlaxoSmithKline, Novartis, Boehringer Ingelheim, 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

Patrick T. Kennedy - Grant/Research Support: Roche, Gilead; Speaking and Teaching: BMS

The following people have nothing to disclose: Antony Chen, Upkar S. Gill, Cameron R. Ikin, Jo-Anne Chin Aleong

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Human Immunodeficiency Virus Type-1 (HIV-1) Coinfection Modulates Hepatitis B Virus (HBV) Lymphotropism

Zengina Lee1,2, Sandi Nishikawa1,2, Bertus Eksteen1,2, John Gill2, Guido van Marle2, Carla S. Coffin1,2;
1Medicine, Liver Unit, Division of Gastroenterology and Hepatology, Calgary, AB, Canada; 2Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, Canada

Approximately 3.3 million persons worldwide are HBV/HIV-1 coinfected and although both HBV and HIV can infect immune (lymphoid) cells, it is unknown whether HIV-1 coinfection impacts HBV lymphotropism. The aim of this study is to characterize the presence of HBV DNA and HBV replicative intermediates in immune cell subsets (i.e., CD4+ T cells, CD8+ T cells, CD14+ monocytes, CD19+ B cells, and CD56+ natural killer [NK] cells) in HBV monoinfected compared to HBV/HIV-1 coinfected patients. Whole blood was collect from 14 HBV monoinfected and 6 HBV/HIV-1 coinfected patients. Peripheral blood mononuclear cells were isolated on Ficoll gradient and immune cell subsets purified using magnetic beads (Miltenyi®). Cell subsets verified to have >80% purity by flow cytometry were treated with DNase/Trypsin to remove extracellular viral particles. Following total nucleic acid isolation, subsets were tested via nested PCR/nucleic hybridization assay (sensitivity of ∼10λl-10λ2 virus genome copies/μg of total DNA) for HBV DNA using HBV-specific surface (S), core (C), and polymerase (P) gene primers. Subsets which tested positive with at least one HBV gene primer were evaluated for HBV cccDNA and/or mRNA. Pearson Xλ2 test was used to calculate statistical significance between groups. In 11 HBV monoinfected patients tested (all treatment naïve and highly viremic), HBV DNA was detected in all immune cell subsets and especially in CD4+ T cells (6/9) and CD19+ B cells (4/8); as well as in CD56+ NK cells (3/8), CD8+ T cells (3/10) and CD14+ monocytes (3/10). In HBV monoinfected patients tested, mRNA or cccDNA was detected in CD14+ monocytes (1/4) and CD56+ NK cells (1/3). In 6 HBV/HIV-1 coinfected patients tested (5/6 on dual active HBV/HIV therapy with fully suppressed HBV and HIV-1 viral load), HBV DNA was not detected in CD4+ T cells (0/4) but found in all other subsets (i.e., CD8+ T cells [2/6], CD14+ monocytes [3/5], CD19+ B cells [1/4], and CD56+ NK cells [3/4]). In HBV/HIV coinfected patients tested, mRNA or cccDNA was found in all cell subsets (i.e., CD4+ T cells [1/3], CD8+ T cells [3/4], CD14+ monocytes[1/4], CD19+ B cells [1/4], and CD56+ NK cells [1/4]). HBV genomes and replicative intermediates are detectable in immune cells of viremic HBV monoinfected patients and in HBV/HIV-1 patients on therapy with suppressed plasma HBV DNA. Immune cells are significantly less likely to carry HBV replicative intermediates in HBV monoinfection compared to HBV/HIV coinfection (18% [2/11] versus 80% [4/5], P<0.026) and HBV genomes were not detected in CD4+ T cells in HIV/HBV coinfection. This study indicates that coinfection with HIV-1 modulates HBV lymphotropism.

Disclosures:

Carla S. Coffin - Grant/Research Support: BMS, Gilead Sciences, Roche

The following people have nothing to disclose: Zengina Lee, Sandi Nishikawa, Bertus Eksteen, John Gill, Guido van Marle

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Gene cloning and transgenic expression of T cell receptor specific for HBV envelope 335-343 epitope

Jing Wu1, Lin Wang1, Yan Liu1, Haiyan Ye2, Ning Ding1, Yongming Liu3, Dongping Xu1;
1Institute of Infectious Diseases and Liver Failure Research Center, Beijing 302 Hospital, Beijing, China; 2Liver disease Department, Guilin Third People's Hospital, Guilin, China; 3Guilin Medical University, Guilin, China

Background/Aims: HBV-specific cytotoxic T lymphocytes (CTL) play a key role in eliminating intracellular virus; while the mechanism study is largely hindered by the difficulty of obtaining sufficient amount of HBV-specific CTL from patients. The study aimed to circumvent the obstacle by expressing T cell receptor (TCR) of HBV-specific CTL through gene transduction. Methods: HBsAg-derived HLA-A2-restricted epitope S335-343 was selected as the study interest due to its relative high frequency in Chinese patients with acute hepatitis B (AHB). S335-343-specific CTL were induced from peripheral blood mononuclear cells of HLA-A2+ AHB patients using the epitopic peptide, purified by flow cytometric sorting, seeded into 96-well plate with a fixed cell number per well, and proliferated with feeding cells under stimulation of anti-CD3 + IL-12. The CTL RNA was extracted. The encoding genes of TCR's α and β chains were obtained using RT-PCR, 5'RACE, 3'RACE, and over-lap PCR. TCR gene recombinant retrovirus vectors were constructed. The TCR was expressed on Jurkat cells and CD8 positive T cells of HLA-A2+ healthy people through gene transduction. Results: Two pairs of TCR Vα and Vβ chains, named as TCRα21β13 and TCRα15β13, were obtained from a HLA-A2+ AHB patient. The titer of packaged recombinant retrovirus was 1.5×10λ5-5.0×10λ5 genome/mL. The introduced TCRs were expressed on recipient T cell, which was evidenced by positive staining in flow cytometric analysis of type-specific Vβ chain (Vβ13) and HLA-A2-epitope-pentamer. The expression levels were 2.25% for type-specific Vβ chain on Jurkat cells, 2.06% for type-specific Vβ chain on CD8 positive T cells, and 1.12% for HLA-A2-epitope-pentamer on CD8 positive T cells, respectively. The effort to increase TCR-retrovirus titer is being made for a higher yield of the TCR-redirected CTL. Conclusion: HBV-specific CTL TCRα/β-encoding gene with right pairing could be obtained from short-term-cultured CTL. The S335-343-targeting TCR could be artificially expressed with binding activity to its ligand.

Disclosures:

The following people have nothing to disclose: Jing Wu, Lin Wang, Yan Liu, Haiyan Ye, Ning Ding, Yongming Liu, Dongping Xu

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Trans-ethnic analyses of HLA-DPA1, DPB1 haplotypes to be associated with hepatitis B virus infection

Nao Nishida1,2, Hiromi Sawai2, Kouichi Kashiwase3, Mutsuhiko Minami3, Masaya Sugiyama1, Wai-Kay Seto4, Man-Fung Yuen4, Yong Poovorawan5, Sang Hoon Ahn6, Kwang-Hyub Han6, Kentaro Matsuura7, Yasuhito Tanaka7, Masayuki Kurosaki8, Yasuhiro Asahina8, Namiki Izumi8, Jong-Hon Kang9, Shuhei Hige10, Tatsuya Ide11, Kazuhide Yamamoto12, Isao Sakaida13, Yoshikazu Murawaki14, Yoshito Itoh15, Akihiro Tamori16, Etsuro Orito17, Yoichi Hiasa18, Masao Honda19, Shuichi Kaneko19, Eiji Mita20, Kazuyuki Suzuki21, Keisuke Hino22, Eiji Tanaka23, Satoshi Mochida24, Masaaki Watanabe25, Yuichiro Eguchi26, Masaaki Korenaga1, Yoriko Mawatari1, Minae Kawashima2, Katsushi Tokunaga2, Masashi Mizokami1;
1Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan; 2Department of Human Genetics, University of Tokyo, Bunkyo-ku, Japan; 3HLA Laboratory, Japanese Red Cross kanto-Koshinetsu Block Blood Center, Koto-ku, Japan; 4Department of Medicine, The University of Hong Kong, Pok Fu Lam, Hong Kong; 5Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 6Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea; 7Department of Virology & Liver unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 8Division of Gastroenterology and Hepatology, Musashino Red Cross Hospital, Musashino, Japan; 9Department of Internal Medicine, Teine Keijinkai Hospital, Sapporo, Japan; 10Department of Internal Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 11Department of Medicine, Kurume University School of Medicine, Kurume, Japan; 12Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan; 13Gastroenterology and Hepatology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan; 14Second Department of Internal Medicine, Faculty of Medicine, Tottori University, Tottori, Japan; 15Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Japan; 16Department of Hepatology, Osaka City University Graduate School of Medicine, Osaka, Japan; 17Department of Gastroenterology, Nagoya Daini Red Cross Hospital, Nagoya, Japan; 18Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Touon, Japan; 19Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan; 20Department of Gastroenterology and Hepatology, National Hospital Organization Osaka National Hospital, Osaka, Japan; 21 Department of Gastroenterology and Hepatology, Iwate Medical University, Morioka, Japan;22Division of Hepatology and Pancreatology, Kawasaki Medical College, Kurashiki, Japan; 23Department of Medicine, Shinshu University School of Medicine, Matsumoto, Japan; 24Division of Gastroenterology and Hepatology, Saitama Medical University, Irumagun, Japan; 25Department of Gastroenterology, Kitasato University School of Medicine, Sagamihara, Japan; 26Department of Internal Medicine, Saga Medical School, Saga, Japan

BACKGROUND/AIM: Genome-wide association studies (GWAS) revealed the association of HLA-DP alleles and haplotypes with chronic hepatitis B virus (HBV) infection, mainly in Asian populations. The individuals with European ancestry indeed showed no association of HLA-DPB1 (rs9277535) with HBV infection. HLA-DPA1 *01:03-DPB1 *04:02 and HLA- DPA1*02:02-DPB1*05:01 showed significant associations with HBV infection in Japanese as a protective and a risk haplotype, respectively. Moreover, there have been no reports of HLA-DP genes to be associated with progressive chronic hepatitis B (CHB) infection. PATIENTS/METHODS: We conducted HLA-DP genotyping using a total of 3,167 samples, including Japanese, Korean, Chinese (Hong Kong) and Thais. We performed a high resolution (4-digit) genotyping of HLA-DPA1 and HLA-DPB1 alleles for HBV patients (including CHB, LC and HCC), healthy controls, and, resolved individuals (HBsAg-negative and anti-HBc-positive), using LABType SSO HLA DPA1/DPB1 kit (One Lambda, CA) and a Luminex Multi-Analyte Profiling system (xMAP; Luminex, Austin, TX), according to manufacturer's protocol. The Fisher's exact test in a two-by-two cross table was applied to acquire exact P values. For a metaanalysis in four populations, we used the DerSimonian-Laird method, which assumes a random-effects model to calculate the pooled effect estimate. RESULTS: A total of 2,895 samples were successfully genotyped. There were a total of 272 dropped samples in HLA-DP genotyping, a large part of which were belonging to Korean HBV patients and Chinese (Hong Kong) resolved individuals, presumably due to low concentration or low quality of DNA samples. Consequently, we additionally identified one high-risk haplotype and one protective haplotype to be associated with chronic HBV infection over the previously reported HLA-DP haplotypes in Asian populations including Japanese, Korean, Chinese (Hong Kong), and Thais. CONCLUSIONS: We identified new HLA-DP alleles and haplotypes to be associated with chronic hepatitis B infection in Asian populations. To determine all the HBV associated haplotypes including risk haplotypes and protective haplotypes would enable HBV infected individuals to divide into two groups who need treatment or not. Moreover, the risk and protective haplotypes to HBV infection, identified in this study by means of transethnic association analyses, would be key host factors to recognize HBV derived antigen peptides, which will lead the following functional studies of HLA-DP molecules in the future.

Disclosures:

Wai-Kay Seto - Advisory Committees or Review Panels: Gilead Science; Speaking and Teaching: Gilead Science

Man-Fung Yuen - Advisory Committees or Review Panels: GlaxoSmithKline, Bristol-Myers Squibb, Pfizer, GlaxoSmithKline, Bristol-Myers Squibb, Pfizer, GlaxoSmithKline, Bristol-Myers Squibb, Pfizer, GlaxoSmithKline, Bristol-Myers Squibb, Pfizer; Grant/Research Support: Roche, Bristol-Myers Squibb, GlaxoSmithKline, Gilead Science, Roche, Bristol-Myers Squibb, GlaxoSmithKline, Gilead Science, Roche, Bristol-Myers Squibb, GlaxoSmithKline, Gilead Science, Roche, Bristol-Myers Squibb, GlaxoSmithKline, Gilead Science

Yasuhito Tanaka - Advisory Committees or Review Panels: Nippon Boehringer Ingelheim Co., Ltd.; Grant/Research Support: Chugai Pharmaceutical CO., LTD., MSD, Mitsubishi Tanabe Pharma Corporation, Dainippon Sumitomo Pharma Co., Ltd., DAIICHI SANKYO COMPANY, LIMITED, Bristol-Myers Squibb

Namiki Izumi - Speaking and Teaching: MSD Co., Chugai Co., Daiichi Sankyo Co., Bayer Co., Bristol Meyers Co.

Shuhei Hige - Grant/Research Support: MSD, MSD, MSD, MSD

Kazuhide Yamamoto - Advisory Committees or Review Panels: Shionogi Pharmaceutical Co; Grant/Research Support: Tanabe Mitsubishi Co, MSD, Chugai Pharmaceutical Co, Esai Co

Yoshito Itoh - Grant/Research Support: MSD KK, Bristol-Meyers Squibb, Dainippon Sumitomo Pharm. Co., Ltd., GlaxoSmithkline, Chugai Pharm Co., Ltd, Mitsubish iTanabe Pharm. Co.,Ltd.

Akihiro Tamori - Grant/Research Support: MSD

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

Eiji Tanaka - Grant/Research Support: MSD pharmaceutical company, Chugai pharmaceutical company, Glaxo Smith Kline Chugai pharmaceutical company, Bristol Mayers Chugai pharmaceutical company, Sumitomo pharmaceutical company, Banyu pharmaceutical company, Takeda pharmaceutical company, Ohtsuka pharmaceutical company, Daiichi Sankyo Ohtsuka pharmaceutical company, Tanabe Mitsubishi Ohtsuka pharmaceutical company, Ajinomoto pharmaceutical company; Speaking and Teaching: MSD pharmaceutical company, Chugai pharmaceutical company, Bristol Mayers Chugai pharmaceutical company, Sumitomo pharmaceutical company, Ohtsuka pharmaceutical company, Tanabe Mitsubishi Ohtsuka pharmaceutical company, Ajinomoto pharmaceutical company

Satoshi Mochida -Grant/Research Support: Chugai, MSD, Tioray Medical, BMS; Speaking and Teaching: MSD, Toray Medical, BMS, Tanabe Mitsubishi

The following people have nothing to disclose: Nao Nishida, Hiromi Sawai, Kouichi Kashiwase, Mutsuhiko Minami, Masaya Sugiyama, Yong Poovorawan, Sang Hoon Ahn, Kwang-Hyub Han, Kentaro Matsuura, Masayuki Kurosaki, Yasuhiro Asahina, Jong-Hon Kang, Tatsuya Ide, Isao Sakaida, Yoshikazu Murawaki, Etsuro Orito, Yoichi Hiasa, Masao Honda, Eiji Mita, Kazuyuki Suzuki, Keisuke Hino, Masaaki Watanabe, Yuichiro Eguchi, Masaaki Korenaga, Yoriko Mawatari, Minae Kawashima, Katsushi Tokunaga, Masashi Mizokami

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Dynamic Changes of interleukin-17-producing helper cells and levels of interleukin-21 correlate with hepatitis B e antigen seroconversion in chronic hepatitis B patients undergoing pegylated interferon treatment

Peng Hu, Min Chen, Yu Lei, Huaidong Hu, Dazhi Zhang, Hong Ren;
Department of Infectious Diseases, Institute for Viral Hepatitis, Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education, the Second Affiliated Hospital of Chongqing Medica, Chongqing, China

Background: Pegylated interferon is associated with higher rates of HBeAg seroconversion. The dynamic changes of Th cell subsets may have an important impact to the efficacy of treatment by pegylated interferon. The aims of this study are to investigated whether the HBeAg seroconversion correlate with the expression of Th1, Th2, Th 17 and Treg cells in chronic hepatitis B patients undergoing pegylated interferon treatment. Methods: HbeAg positive chronic hepatitis B patients received pegylated interferon treatment for 48 weeks. And the frequency of Th 1, Th2, Th 17, Treg cells as well as Th 1-, Th2-, Th17-, Tregrelated cytokines in their patients were longitudinally analyzed. Results: The frequency of Th17 cells increased during the first 12 weeks treatment in all patients. But from week 12 to week 24, the frequency of Th17 cells decreased quickely in HBeAg seroconversion patients. Th1 cells decreased at the first 4 weeks treatment in all patients, but after 12 weeks, HBeAg seroconversion patients showed a significant increased in the frequency of Th1 cells. Treg cells decreased in all patients, there was no significant difference between HBeAg and non-HBeAg seroconversion patients. The serum levels of IL-2 and IL-21 significant increased from week 24 in HBeAg seroconversion patients. IL-17 increased at the first 4 weeks in all patients, and then decreased in HBeAg seroconversion patients. Conclusion: The dynamic changes of Th cell subsets and their related cytokines exhibit different trends in HBeAg and non-HBeAg seroconversion patients. Among them, Th17 and IL-21 have the most prominent changes in HBeAg seroconversion patients. Acknowledgments This research was supported by the National Natural Science Foundation of China (30930082, 81171561, 30972584), the National Science and Technology Major Project of China (2008ZX10002-006, 2012ZX1002007001), The National High Technology Research and Development Program ofChina(2011AA020111).

Disclosures:

The following people have nothing to disclose: Peng Hu, Min Chen, Yu Lei, Huaidong Hu, Dazhi Zhang, Hong Ren

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Genetic variation of IL28B and serum levels of IFN-λ 3 does not affect clinical outcome of hepatitis B virus infection

Tsutomu Takeda1, Kazumoto Murata1, Masaya Sugiyama1, Tatsuji Kimura2, Sachiyo Yoshio1, Yoshihiko Aoki1, Yoko Yamagiwa1, Masaaki Korenaga1, Masatoshi Imamura1, Tatsuya Kanto1, Naohiko Masaki1, Masashi Mizokami1;
1National Center for Global Health and Medicine, Tokyo, Japan; 2Institute of Immunology Co., Ltd, Tokyo, Japan

Background&Aims: Recent genome-wide association studies have revealed that IL28B polymorphisms were associated with interferon-induced or natural viral clearance in chronic hepatitis C patients. However, the association of genetic variation in IL28B with outcome of hepatitis B virus (HBV) infection and development of cirrhosis or hepatocellular carcinoma (HCC) is still controversial. Therefore, we examined the single nucleotide polymorphisms (SNP) of the IL28B gene (rs8099917) and serum levels of IFN-λ3 to determine these associations with clinical outcome of HBV infection. Methods: We enrolled 59 patients with HBV infection such as acute hepatitis (n = 2), asymptomatic carriers (ASC: n = 26), chronic hepatitis (CHB: n = 21), and cirrhosis with or without HCC (n = 10). Serum from healthy volunteers (n = 24) was used as controls. We quantified serum IFN-λ3 levels by our newly developed chemilumines-cence enzyme-linked immnosorbent assay (CLEIA), which specifically detects IFN-λ3 protein. Frozen stock sera taken before treatment were used for the IFN-λ3 quantifications. SNP near IL28B (rs8099917; TT is a favorable genotype for Peg-IFN/RBV therapy in chronic hepatitis C) were evaluated by InvaderPlus assay. Genomic DNA of samples were extracted with Qiagen Blood mini kit. Results: The frequency of TT homozygosity was 68.0%, 75.0% and 60.0% in ASC, CHB, cirrhosis, respectively, with no statistical significance. Serum levels of IFN-λ3 in ASC, CH and cirrhosis were 1.7 ± 1.5 pg/ml, 1.7 ± 1.5 pg/ml, and 5.1 ± 3.9 pg/ml, respectively. Serum IFN-λ3 tended to be higher in cirrhosis than ASC, but no statistical differences were observed. Serum ALT levels, HBV DNA or HBsAg titer were not correlated with serum IFN-λ3 levels, and no differences were observed in these factors between TT and non-TT. HBeAg seroconversion rate was not significant among IL28B genotypes, and no differences were observed in serum IFN-λ3 levels in HBeAb positivity as well. Serum levels of IFN-λ3 tended to be higher in non-TT genotype without statistical differences. Of 2 patients with acute hepatitis B, series serum samples were determined by CLEIA. Serum levels of IFN-λ3 were 2.1,3.1 in acute phase with high ALT levels. No significant changes were observed in serum levels of IFN-λ3 during clinical courses. Conclusions: No differences were observed in IL28B genotypes as well as serum levels of IFN-λ3 in progression of diseases, ALT levels and viral factors. Taken together with these results, the IL28B single nucleotide polymorphisms seems not to affect the immune response to HBV and clinical outcome of HBV infection.

Disclosures:

Tatsuji Kimura - Employment: Institute of Immunology, Co., Ltd.

The following people have nothing to disclose: Tsutomu Takeda, Kazumoto Murata, Masaya Sugiyama, Sachiyo Yoshio, Yoshihiko Aoki, Yoko Yamagiwa, Masaaki Korenaga, Masatoshi Imamura, Tatsuya Kanto, Naohiko Masaki, Masashi Mizokami

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Hepatic toll-like receptor 3, -7 and -9 activation is not impaired in HBV-transgenic mice lacking the HBs-antigen

Catherine I. Real1, Ruth Broering1, Kathrin Kleinehr1, Sabrina Drift-mann1, Reinhold Schirmbeck3, Mengji Lu2, Guido Gerken1, Joerg F. Schlaak1;
1Dept. of Gastroenterology and Hepatology, University Duisburg-Essen, Essen, Germany; 2Institute of Virology, University Duisburg-Essen, Essen, Germany; 3Dept. of Internal Medicine, University of Ulm, Ulm, Germany

INTRODUCTION: Chronic infection with the hepatitis B virus (HBV) is a major cause of liver-related morbidity and mortality world-wide. Previously, it has been shown that toll-like receptor (TLR) signalling is impaired by HBsAg leading to an attenuation of innate and adaptive immune responses which may possibly facilitate chronicity of infection. Here, we have studied TLR signaling in a HBV transgenic mouse model lacking the HBs-anti-gen (1.4 tg HBV-S-mut). METHODS: The ligands forTLR3 (poly I:C), TLR7 (ssRNA) orTLR9 (ODN) were injected intravenously into HBV-S-mut mice and their HBV-negative littermates. After 6h or 24h liver tissue was prepared and viral replication as well as expression of interferon sensitive gene 15 (ISG15), Interferon gamma-induced protein 10 (IP-10), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 10 (IL-10) were determined by qtRT-PCR. RESULTS: TLR3 activation significantly suppressed HBV replication 6h after application, whereas TLR7 and TLR9 agonists had only marginal suppressive effects. However, exposure to all three ligands induced the expression of ISG15, IP-10, IL-10, Il-6 and TNF-α in tgHBV mice as well as in HBV-negative littermates, while TGF-β expression was suppressed by all three ligands. The expression of poly I:C-induced IL-10 was significantly inhibited in tgHBV mice compared to negative littermates. Basal ISG15 expression was enhanced in tgHBV mice. In case of TLR9, ligand binding mediated a significant higher TGF-β expression in tgHBV mice compared to HBV-negative controls. The hepatic expression of TLR3, -7 and -9 receptors did not differ between tgHBV mice and HBV-negative littermates. CONCLUSIONS: In contrast to data from HBV-infected patients, hepatic TLR signalling is not totally abrogated in HBV transgenic mice that lack HBsAg. The present data indicated that HBV tg mice, that lack the HBsAg, consistently express IFN and ISGs. The TLR 9-induced signalling was delayed in these animals, whereas TLR 3 or TLR 7-mediated signals seemed to be slightly suppressed in HBV tg mice. Therefore, we hypothesize that HBsAg is a major component of HBV that attenuates TLR signalling thus leading to impairment of innate and adaptive immune responses in the liver.

Disclosures:

The following people have nothing to disclose: Catherine I. Real, Ruth Broering, Kathrin Kleinehr, Sabrina Driftmann, Reinhold Schirmbeck, Mengji Lu, Guido Gerken, Joerg F. Schlaak

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IL-21 -producing Tfh cells induces activation of B cells in patients with chronic hepatitis B

Xiangsheng Xu, Zheng Zhang, Weimin Nie, Fu-Sheng Wang;
Institute of Translational Hepatology, Research Center for Biological Therapy, Beijing 302 Hospital, Beijing, China

Patients with chronic hepatitis B has a tendency to autoimmune diseases such as arthritis, glomerulonephritis, extrahepatic manifestations. However the underlying mechanism remains unknown. So we intend to study the immunological characteristics of B cells in patients with chronic hepatitis B and the mechanism that causes activation of B cells. In this cross-sectional

study, we comprehensively investigated the frequency, phenotype and function of peripheral B cell subsets from 68 HBV-infected subjects, including 12 immune tolerant (IT) patients, 32 immune activated (IA) patients, 11 hepatitis B e antigen negative (ENH) patients and 13 responders (RP) with HBsAg sero-conversion induced by interferon therapy, as well as 22 HBsAg-vaccinated healthy controls (HC). The IA and ENH patients displayed lower percentages of peripheral blood memory B cells versus other groups. An overall polyclonal activation of B cells, indicated by higher levels of activation markers and total serum IgG and IgM, was also observed in IA and ENH patients relative to other groups. The level of serum IL-21 were increased in IA and ENH patients and also led to the significant general activation of their B cells in vitro. Notably, expression of the co-stimulatory molecule CD80 on B cells and the frequency of HBsAg-specific B cells were significantly decreased in IT, IA and ENH patients versus HC subjects. Notably, activation markers and IgG/IgM production were increased in cocultures of B cells and CD4+CXCR5+ Tfh cells. Furthermore the activation were inhibited when IL-21 were blocked. Enhanced activation of B cells in patients with chronic hepatits B were promoted by IL-21 from CD4+CXCR5+ Tfh cells, which maybe contributed to the tendency to autoimmune disease in CHB patients.

Disclosures:

The following people have nothing to disclose: Xiangsheng Xu, Zheng Zhang, Weimin Nie, Fu-Sheng Wang

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Collagen proportionate area but not Ishak fibrosis stage on liver biopsy correlates with age in Chronic Hepatitis B infected young adults

Upkar S. Gill1, Chandni Patel1, Sandhia Naik2, Janet Dearden3, Yiannis N. Kallis3, Paul Kooner3, Richard Marley3, Jo-Anne Chin Aleong4, Robert D. Goldin5, Graham R. Foster1, Patrick T. Kennedy1;
1Hepatology Unit, Centre for Digestive Diseases, Blizard Institute, Barts and The London SMD, QMUL, London, United Kingdom; 2Paediatric Gastroenterology & Hepatology, Barts Health NHS Trust, London, United Kingdom; 3Hepatology, Barts Health NHS Trust, London, United Kingdom; 4Cellular Pathology, Barts Health NHS Trust, London, United Kingdom; 5Liver & GI Pathology, Imperial College, London, United Kingdom

INTRODUCTION: The timing and benefit of early therapeutic intervention in young adults with Chronic Hepatitis B (CHB) remains a subject of debate. We recently demonstrated evidence of immune activity in CHB infected young adults comparable to that found in older subjects. The most appropriate modality to assess disease activity in young adults remains controversial. Liver damage is typically evaluated on liver biopsy by visual estimation, which can be subject to inter/intraobserver variations generating bias. Digital image analysis (DIA) measuring the collagen proportionate area (CPA)/percentage fibrosis may provide a more accurate indication of liver scarring and remove bias. Here, we compare the use of Ishak fibrosis stage and DIA in a cohort of young adult patients with CHB to assess disease activity and liver damage. PATIENTS & METHODS: Young adult patients followed in a dedicated clinic who underwent liver biopsy were recruited to the study; n=92 (male=60), median age 24 (range 12-30). DIA was performed on Sirius red stained sections to evaluate the CPA/percentage fibrosis. Data on eAg status, longitudinal ALT, HBV DNA and quantitative (q)HBsAg titres were documented over a 12-month period preceding liver biopsy. RESULTS: The overall median Ishak fibrosis stage was 2 (range 0-6), necroinflammatory (NI) score 3/18 (range 0-8) and median CPA/percentage fibrosis was 2.83% (range 0.55-9.98). We probed whether the CPA using DIA was more informative in determining the degree of scarring secondary to immune activity and predicting clinical outcomes over the Ishak fibrosis stage. We noted that although the CPA increased with increasing Ishak fibrosis stage, the correlation was not significant (p=n.s). There was no correlation between the NI score and Ishak fibrosis stage or CPA (p=n.s). When comparing other parameters; eAg status, HBV DNA levels, qHBsAg titres and ALT levels (based on Prati criteria), we did not notice any difference between Ishak fibrosis stages and CPA. When taking age of the young adult cohort into account we noticed there was a significant difference with the CPA which increased with advancing age (p=0.007). No correlation with age and the Ishak fibrosis stage was found (p=n.s) CONCLUSIONS: A proportion of young adults with CHB have significant liver disease histologically and warrant earlier therapeutic intervention. A more considered approach to the assessment of liver disease in this cohort is indicated and this study highlights the limitations of the Ishak fibrosis stage alone. Our data demonstrates that DIA provides a more accurate assessment of liver fibrosis which correlates with underlying immune activity.

Disclosures:

Graham R. Foster - Advisory Committees or Review Panels: GlaxoSmithKline, Novartis, Boehringer Ingelheim, 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

Patrick T. Kennedy - Grant/Research Support: Roche, Gilead; Speaking and Teaching: BMS

The following people have nothing to disclose: Upkar S. Gill, Chandni Patel, Sandhia Naik, Janet Dearden, Yiannis N. Kallis, Paul Kooner, Richard Marley, Jo-Anne Chin Aleong, Robert D. Goldin

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Interferon and interferon stimulated genes are up-regulated during viral replication in HBV-transgenic mice lacking the HBs-antigen

Catherine I. Real1, Ruth Broering1, Martin Trippler1, Kathrin Kleinehr1, Sabrina Driftmann1, Thekla Kemper2, Hans-Peter Vorn-locher3, Reinhold Schirmbeck4, Mengji Lu2, Guido Gerken1, Joerg F. Schlaak1;
1Dept. of Gastroenterology and Hepatology, University Duisburg-Essen, Essen, Germany; 2Institute of Virology, University Duisburg-Essen, Essen, Germany; 3Axolabs GmbH, Kulmbach, Germany; 4Dept. of Internal Medicine, University of Ulm, Ulm, Germany

INTRODUCTION: Chronic infection with the hepatitis B virus (HBV) is a major cause of liver-related morbidity and mortality world-wide. Previously, it has been suggested that chronicity of infection may be facilitated by an attenuation of innate and adaptive immune responses through HBsAg. Here, we have studied the immunological effects of HBV replication in a transgenic mouse model, lacking the HBsAg (1.4tgHBV-S-mut). We also characterized the siRNA-mediated suppression of HBV in this system. METHODS: Different nanolipid-formulated siRNAs targeting the HBxAg mRNA, preferentially entering hepatocytes, were injected intravenously into HBV-S-mut mice. HBV negative littermates were used as controls. After 48h and 10d tissue (liver, heart, spleen, kidney) was prepared and expression of HBV-RNA, interferon beta (IFN-β), interferon sensitive gene 15 (ISG15), interferon-induced protein with tetratri-copeptide repeats 1 (IFI-T1), Interferon gamma-induced protein 10 (IP-10), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 10 (IL-10) was determined by qtRT-PCR. HBV DNA in liver tissue was determined by Southern blot. ELISA and western blot were performed to detect HBeAg and HBcAg, respectively. RESULTS: Single injection of siRNAs against HBV led to suppression of HBV DNA after 48h which was sustained for more than 10 days. The HBeAg serum levels were also reduced about 90% after treatment with HBxAg-specific siRNA. The expression of IFN-β, IFI-T1 and ISG15 was up-regulated in HBV positive animals compared to control animals, which could be normalized by treatment with HBxAg-targeting siRNAs. CONCLUSIONS: In contrast to human liver from HBV patients, in HBV transgenic mouse model lacking HBsAg viral replication could be associated with expression of IFNs and ISGs. Thus, we hypothesize that HBsAg is suppressing the IFN induction and ISG response in vivo. This hypothesis will be challenged in future experiments using transgenic mice that additionally express the HBsAg.

Disclosures:

Hans-Peter Vornlocher - Management Position: Axolabs GmbH

The following people have nothing to disclose: Catherine I. Real, Ruth Broering, Martin Trippler, Kathrin Kleinehr, Sabrina Driftmann, Thekla Kemper, Reinhold Schirmbeck, Mengji Lu, Guido Gerken, Joerg F. Schlaak

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Expression of DNA vaccine encoding hepatitis B virus core antigen with mutation in vitro

Yiping Xing, Jun Li, Zuhu Huang;
Department of infectious diseases, the first affiliated hospital of Nanjing medical university, Nanjing, China

Objective: To observe the effect on expression of DNA vaccine encoding HBV core antigen by deletion of amino acids at N-terminal. Methods: We constructed the DNA vaccines encoding HBV core antigen with truncation amino acid from N- amino terminal by site-directed mutagenesis. 5 DNA vaccines with deletion of the first and second amino acid ( named M12), the third and forth amino acid ( named M34), the fifth and sixth amino acid ( named M56), the third ( named M3) and forth ( named M4) were constructed successfully. 293T cells were transiently transfected with M12, M34, M56, M3, M4, wild-type HBc DNA vaccine (pJW4303/HBc) and pJW4303 vector. The expression of HBcAg was measured by western blot. Results: It was revealed DNA vaccine encoding HBV core antigen with mutation was successfully constructed and proved by digesting with PstI and BglII and sequencing. There are HBcAg in lysate and supernatant of 293T cells transfected with pJW4303/HBC, M12 and M56. Only a weak band of HBcAg was observed in the lysate of 293T cells transfected with M34 and M3, while none in the supernatant. However, HBcAg can be detected in both supernatant and lysate of 293T cells transfected with M4. Conclusion: Deletion of the third amino acid in the N-terminal may decrease the expression of HBcAg. The third amino acid may play an important role in the expression of HBcAg protein.

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Disclosures:

The following people have nothing to disclose: Yiping Xing, Jun Li, Zuhu Huang