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1032

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Secretion of HBV from the late endosome is regulated by the small GTPase Rab7

Jun Inoue1, Eugene W. Krueger1, Mark A. McNiven1,2;
1Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, MN; 2Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN

Background: The infectious life cycle of the hepatitis B virus (HBV) from endocytic internalization to infectious secretion is poorly defined. It has been shown that viral assembly appears to occur in the late endosomes/multivesicular bodies (MVBs) of the hepatocyte where mature virons are packaged and subsequently secreted into the blood space. Understanding the regulation of this trafficking step utilized by this virus is essential toward disrupting its growth and infection. The small GTPase Rab7 is known to act as a regulatory switch in mediating the transport of cargo from the MVB to the lysosome for degradation. The central GOAL of this study was to define the role of the MVB in HBV propagation, and, test if Rab7 regulates the life cycle of this virus by controlling its traffic between the MVB, the lysosome, and the cell surface during secretion/infection. Results: Confocal immunofluorescence microscopy (IF) of HBV genome-transfected HepG2.2.15 cells showed that two different HBV proteins (LHBs and HBc) localized with Rab7 and LAMP1 at the MVB and lysosome, respectively. Importantly, depletion of Rab7 by siRNA decreased the colocalization with LAMP1 and significantly increased both the retained levels of cytoplasmic LHBs protein and the HBV DNA secreted into the culture supernatant. This effect was rescued by overexpression of wild type Rab7. In support of these findings, electron microscopy (EM) of these cells showed that Rab7 depletion led to a striking increase in the number and size of virus-containing MVBs compared to the non-transfected parental HepG2 cells. Remarkably, many of the MVBs in the HBV-transfected cells exhibited a large number of tubules extending outward into the cytoplasm. Accordingly, Rab7 activity was markedly increased (7-fold) in the HBV-expressing HepG2.2.15 cells compared to parental HepG2 cells. To define which viral components are responsible for the increased Rab7 activity and the subsequent changes in MVB morphology, all five individual HBV proteins were expressed in HuH7, Hep3B, and Hela cells. Importantly, a 2-4 fold increase in Rab7 activation was observed in all 3 cell types expressing the exogenous HBeAg protein alone while the other proteins had no effect. Conclusion: These findings suggest that membrane traffic from the MVB plays an essential role in the infectious secretion cycle of HBV. Most surprising is that the virus itself appears to regulate Rab7 activity and MVB dynamics to aid in its own propagation.

Disclosures:

The following people have nothing to disclose: Jun Inoue, Eugene W. Krueger, Mark A. McNiven

1033

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HBV DNA Diagnostic Testing: Positivity is in the eye of the beholder

Carla Osiowy1,2, Anton Andonov1, Jamie Borlang1, Chris Huynh1, Julia Uhanova2, Gerald Y. Minuk2;
1Bloodborne Pathogens and Hepatitis, National Microbiology Laboratory, Winnipeg, MB, Canada; 2Section of Hepatology, Department of Medicine, University of Manitoba, Winnipeg, MB, Canada

Purpose: Qualitative molecular detection of hepatitis B virus (HBV) in serum or plasma is used as a marker of viral replication and infectivity in reference diagnostic laboratories. Qualitative testing is often relevant in cases of suspected occult HBV infection and when viral load quantities fall below the limit of quantification. However, the methodologies utilized are not standardized and are highly subjective, depending on primer sensitivity and the number of targets detected. This study investigated various parameters of qualitative HBV DNA detection to determine an optimal measure. Methods: The Canadian hepatitis B reference laboratory utilizes two nested PCR (nPCR) reactions (sensitivity 10 IU/ml) targeting the surface and core coding regions of the HBV genome for molecular detection of HBV. Detection of both targets is required for a positive result, while an indeterminate result is reported with positive/negative target discrepancies. Recently, a real time PCR (RT) protocol for qualitative detection was adopted involving 3 target regions: the surface/polymerase, Enhancer I, and X/Enhancer II regions of the genome. A positive result with two or more RT targets defined a positive diagnostic result. The results of 223 low or undetectable viral load diagnostic specimens (74/223 with quantifiable viral load; median 2.4 log 10 IU/ml, range 0.81 to 3.41 log10 IU/ml) assayed by both methods were compared. Additionally, the RT method criteria was investigated as a means of accurately detecting occult HBV with 1007 community-based HBsAg-negative specimens. Results: The RT method detected HBV DNA to a level of 3 IU/ml, with >80% sensitivity (95% CI = 0.57 to 0.99) between 6 and 12 IU/ml for all targets. High concordance between both methods was observed (53 positive and 126 negative by both methods). Twenty-five samples negative or indeterminate by nPCR were positive by RT. Similarly, 17 samples indeterminate by nPCR but RT negative were either truly negative by repeat nPCR testing (1 1/17) or had a viral load below the limit of quantification. OBI was detected in 8 (0.8%) of the HBsAg-negative specimens using the criteria of at least two RT targets positive; however, 55 (5.5%) samples were positive by at least one RT target, indicating a large discrepancy in reported OBI if the RT protocol were not followed. Conclusions: Results of this study suggest that the RT protocol had increased sensitivity, objectivity and reliability compared to the nPCR method. The results also support the present diagnostic criteria for OBI, of at least two target sites, in community-based populations.

Disclosures:

The following people have nothing to disclose: Carla Osiowy, Anton Andonov, Jamie Borlang, Chris Huynh, Julia Uhanova, Gerald Y. Minuk

1034

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Liver Stiffness Measurement by Transient Elastography to Assess Liver Fibrosis in a Multicenter Chronic Hepatitis B Study

Heng Chi1, Bettina E. Hansen1, Jordan J. Feld2, David K. Wong2, Erik H. Buster1, Robert J. de Knegt1, Harry L. Janssen1,2;
1Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands; 2Liver Clinic, Toronto Western and General Hospitall, University Health Network, Toronto, ON, Canada

Introduction: Liver stiffness measurement (LSM) using transient elastography (TE) can grade liver fibrosis non-invasively in chronic hepatitis B (CHB). However, diagnostic cut-offs differ and even ALT-stratified cut-offs have been proposed due to the confounding effect of ALT on LSM. Only a few small studies examined ALT-stratified cut-offs. Therefore, we sought to 1) develop optimal cut-offs to grade liver fibrosis, and 2) evaluate the diagnostic performance of ALT-stratified cut-offs in CHB patients. Methods: In this multicenter retrospective study, we enrolled CHB patients with paired liver biopsy and LSM between 2005 and 201 3. Liver biopsies had to be ≥15 mm in length and corresponding LSMs had to be valid (IQR/M ratio ≤0.30, ≥10 valid measurements, and success rate ≥60%). The LSMs were performed within 3 months of the liver biopsy. We excluded patients with HCC, hepatic decompensation, concomitant liver diseases, liver transplant, and HCV, HDV, HIV co infections. We calculated the AUROCs and net reclassification index (NRI) of non-stratified cut-offs (≥F2 - 7.2 kPa; ≥F3 - 8.1; F4 - 1 1.0) compared to ALT-stratified cut-offs (ALT ≤1 upper limit of normal [ULN]: ≥F2 - 6.0 kPa; ≥F3 - 9.0; F4 - 12.0. ALT <1 ULN: ≥F2 - 7.5 kPa; ≥F3 - 12.0; F4 - 13.4). Results: We analyzed 301 paired liver biopsies and LSMs. The fibrosis stage was F0 in 11.3% (34), F1 in 41.5% (125), F2 in 28.2% (85), F3 in 1 1.6% (35) and F4 in 7.3% (22). We found 219 (73.2%) patients with ALT >1 ULN, 138 (46.2%) with ALT >1.5 ULN, and 95 (31.8%) with ALT >2 ULN. The AUROCs to diagnose ≥F2, ≥F3, F4 were, 0.794, 0.830, 0.879, respectively. We used the maximum sum of sensitivity and specificity to calculate the cut-offs (in kPa): 7.1 for ≥F2, 8.8 for ≥F3, and 11.9 for F4. We observed a significantly higher LSMs in patients with ALT <1 ULN compared to ALT ≤1 ULN within the METAVIR group F1 (p=0.009), F2 (p=0.005), and F3 (p=0.009). However, there were no significant differences between AUROCs of non-stratified vs. ALT-stratified cut-offs for any of the METAVIR scores (all p>0.20). Furthermore, the AUROCs for ALT ≤1.5 ULN vs. >1.5 ULN and ALT ≤2 ULN vs. ALT >2 ULN demonstrated similar diagnostic performances (all p>0.09). The NRIs for ALT-strati-fied cut-offs were -0.03, -0.06, and -0.1 8 for ≥F2, ≥F3, and F4, respectively, suggesting no improvement in fibrosis stage reclas-sification with ALT stratification. Subsequently, all NRIs were negative (≤-0.03) for ALT ≤1.5 ULN vs. >1.5 ULN and ALT ≤2 ULN vs. ALT >2 ULN. Conclusions: In this study we propose new cut-offs to grade fibrosis in CHB patients. ALT-stratified cut-offs did not improve the diagnostic performance of TE in CHB.

Disclosures:

Jordan J. Feld - Advisory Committees or Review Panels: Roche, Merck, Vertex, Gilead, Abbott, Tibotec, Theravance, Achillion; Speaking and Teaching: Merck, Roche, Abbott

David K. Wong - Grant/Research Support: Gilead, BMS, Vertex, BI

Robert J. de Knegt - Advisory Committees or Review Panels: MSD, Roche, Norgine, Janssen Cilag; Grant/Research Support: Gilead, MSD, Roche, Janssen Cilag, BMS; Speaking and Teaching: Gilead, MSD, Roche, Janssen Cilag

Harry L. Janssen - Consulting: Abbott, Bristol Myers Squibb, Debio, Gilead Sciences, Merck, Medtronic, Novartis, Roche, Santaris; Grant/Research Support: Anadys, Bristol Myers Squibb, Gilead Sciences, Innogenetics, Kirin, Merck, Medtronic, Novartis, Roche, Santaris

The following people have nothing to disclose: Heng Chi, Bettina E. Hansen, Erik H. Buster

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ARC-520 RNAi therapeutic reduces hepatitis B virus DNA, S antigen and e antigen in a chimpanzee with a very high viral titer

Robert E. Lanford1, Christine I. Wooddell2, Deborah Chavez1, Claudia Oropeza3, Qili Chu2, Holly L. Hamilton2, Alan McLach-lan3, Bruce Given4, Christopher R. Anzalone4, David L. Lewis2;
1 Texas Biomedical Research Institute, San Antonio, TX; 2Arrowhead Madison, Arrowhead Research Corporation, Madison, WI; 3Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL; 4Corporate Office, Arrowhead Research Corporation, Pasadena, CA

Chronic hepatitis B virus (HBV) infection is a major disease world-wide for which there remains an unmet medical need. Current therapies have little effect on the viral proteins that allow sustained infection and progression of disease, and prevent the patient from mounting an effective immune response. We are developing a small interfering RNA (siRNA)-based therapeutic named ARC-520 that is designed to decrease viral protein load by the mechanism of RNA interference (RNAi). We have shown that a single intravenous injection of ARC-520 results in multi-log repression of viral RNA, proteins and DNA with long duration of effect (more than one month) in transient and transgenic mouse models of chronic HBV infection, without toxicity. Here, we present studies that demonstrate dose dependent reduction of HBV DNA in serum and liver, HBV transcripts in liver, HBsAg, HBeAg and core antigen from single or multiple doses of ARC-520 in mice. Multiple doses of ARC-520 enabled an extended duration of effect. Recently we extended our investigations of ARC-520 to treatment of a chimpanzee chronically infected with HBV since 1 979. This animal was 36 years old, weighed 51 kg and had a very high viral titer of HBV genotype B (1E+10 GE/ml serum). A single intravenous injection of 2 mg/kg of ARC-520 was well-tolerated and resulted in decreases in serum levels of HBsAg, HBeAg and HBV DNA. A subsequent injection of 3 mg/kg two weeks after the first injection correlated with increased pharmacological effect, giving 81-96% reductions in these HBV parameters at nadir. These reductions were similar in magnitude and duration of effect to those observed in the mouse HBV models receiving similar doses. The efficacy and safety of ARC-520 in a large primate demonstrate its promise as a new class of therapeutic for patients chronically infected with HBV.

HBsAg in chimpanzee

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Disclosures:

Robert E. Lanford - Grant/Research Support: Arrowhead Research

Christine I. Wooddell - Employment: Arrowhead Research Corporation

Qili Chu - Employment: Arrowhead Madison

Bruce Given - Board Membership: Icon plc, Calando Pharmaceuticals; Consulting: Leonardo Biosystems, Inc; Employment: Arrowhead Research Corp

David L. Lewis - Employment: Arrowhead Research Corporation

The following people have nothing to disclose: Deborah Chavez, Claudia Oropeza, Holly L. Hamilton, Alan McLachlan, Christopher R. Anzalone

1036

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HBsAg Loss Is Correlated With Distinct Patterns Of Baseline Viral Diversity Within Core and HBx Genes in Genotypes (GT) A and D, HBeAg+ Chronic Hepatitis B (CHB) Subjects Treated With Tenofovir DF (TDF) Longterm

Kathryn M. Kitrinos1, Paul N. Hengen1, Raul E. Aguilar Schall1, Phillip Dinh1, Hendrik W. Reesink2, Fabien Zoulim3, Prista Charu-worn1;
1Gilead Sciences, Foster City, CA; 2Department of Gas-troenterology and Hepatology, Academic Medical Center, Amsterdam, Netherlands; 3INSERM U1052, Hospices Civils de Lyon, Lyon, France

Background/Aims: Previous analyses demonstrated lower genetic distance within HBV polymerase/reverse transcriptase (pol/RT) and HBsAg genes in HBeAg+ GT A and D CHB subjects who lost HBsAg compared to control subjects who maintained high HBsAg levels through 192 weeks of TDF treatment. This study evaluated the differences in mean pairwise genetic distance across the core and HBx genes in this subject cohort. Methods: Study GS-US-174-0103 HBeAg+ subjects were randomized 2:1 to receive TDF or ADV for 48 weeks followed by open-label TDF. After 4 years, 23/266 (8.6%) experienced HBsAg loss, including 14 GT A and 7 GT D subjects. 17 GT A and 10 GT D subjects who maintained high HBsAg levels with similar baseline HBV DNA and ALT were selected as case controls. Population sequencing was performed on baseline samples and pair-wise genetic distance matrices for segments across HBx and core genes were used to calculate viral diversity. Non-parametric Levene test for homogeneity of variances in control and HBsAg loss groups was performed for each region, and equality of mean genetic distances within regions was evaluated using the Mann-Whitney-Wilcoxon test. The Hochberg procedure was used to control for multiple testing. Results: For GT A and GT D, in general, segments corresponding to non structural regulatory elements (URR, NRE, CURS, and EnhII within HBx gene and precore) showed higher viral diversity within HBsAg loss patients compared to controls. In contrast, the core gene, which encodes a structural element, the opposite pattern was observed with lower viral diversity in HBsAg loss patients. Similar to previous observations across the pol/RT and HBsAg genes, genotype-specific differences were observed across the core and HBx genes. For GT A, 6/9 segments had significant genetic diversity differences between HBsAg loss and control subjects, while only 4/9 segments had significant differences for GT D. In addition, GT A subjects had lower mean pairwise genetic distance in the majority of HBx and core gene segments evaluated compared to GT D subjects. Conclusions: In this cohort, lower viral diversity was observed in HBsAg loss subjects compared to controls within the core gene, which encodes a structural element. In contrast, specific regions across the HBx gene and precore that encode non structural regulatory or signaling elements, generally displayed higher viral diversity within HBsAg loss subjects compared to controls. These distinct patterns may reflect different mechanisms by which coding regions for structural or nonstructural elements respond to adaptive pressure resulting in the common endpoint of HBsAg loss.

Disclosures:

Kathryn M. Kitrinos - Employment: Gilead Sciences, Gilead Sciences; Stock Shareholder: Gilead Sciences, Gilead Sciences

Paul N. Hengen - Employment: Gilead Sciences

Raul E. Aguilar Schall - Employment: Gilead Sciences, Inc.

Phillip Dinh - Employment: Gilead Sciences

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

Fabien Zoulim - Advisory Committees or Review Panels: Gilead; Consulting: Roche; Grant/Research Support: Gilead, Scynexis, Roche; Speaking and Teaching: Novartis, Roche, Janssen, Bristol Myers Squibb, Gilead

Prista Charuworn - Employment: Gilead Sciences; Stock Shareholder: Gilead Sciences

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Sequence analysis of serum hepatitis B virus (HBV) RNA represents a novel method for HBV genome analysis of HBV variants in patients achieving undetectable HBV DNA during antiviral treatment

Florian van Boemmel1, Laura Schmalbrock1, Danilo Deichsel1, Eckart Schott2, Thomas Berg1, Stephan Böhm1;
1Hepatology Section, University Hospital Leipzig, Leipzig, Germany; 2Hepatology and Gastroenterology, Charité university Hospital, Berlin, Germany

Background: During treatment with highly potent nucleo(s)tide analogues serum HBV DNA levels become suppressed to undetectable levels during the first treatment year in most patients and HBV genome analysis becomes infeasible. However, HBV RNA remains detectable in serum in many patients with undetectable HBV DNA. We have investigated the evolution of HBV variants by sequence analysis of serum HBV RNA in patients with HBV resistance mutations who were achieving undetectable HBV DNA levels during consecutive treatment with tenofovir (TDF). Methods: Nineteen patients who received monotherapy with TDF 245 mg/day for a mean of 40±14 (range, 21-76) months after prior treatment failure to lamivu-dine (n=4), adefovir (n=1) or both (n=14) were retrospectively analyzed. Sixteen patients were male, 16 HBeAg positive, the mean HBV DNA was 6.3±1.5 (3.8-9.6) log 10 copies/mL and HBV genotypes A, B, D and E were present in 3,2, 13 and 1 patient, respectively. From serum samples stored at -20°C representing the start of TDF treatment and consecutive time points HBV DNA was amplified by a real time PCR targeting the HBV core region and HBV RNA after reverse transcription by a real time PCR targeting the x gene with HBV RNA specific RACE primers (minimal detectable quantity was 100 and 500 copies/mL, respectively). The rt region and the overlapping s gene of HBV were sequenced in all samples with HBV DNA or HBV RNA levels > 1000 copies/mL (n=103). Results: During TDF treatment HBV DNA decreased to levels < 1000 and < 100 copies/mL after a mean duration of 13±8 (3-32) and 34±14 (3-76) months, respectively. After this HBV RNA levels remained detectable at levels > 1000 and > 100 copies/mL during mean periods of 22±12 (0-44) and 18±11 (0-41) months, respectively. On HBV DNA basis the HBV resistance associated mutations rtL80V, rtV173L, rtL180M, rtM204V/I, rtS202T/C and rtN236T were detectable in 5, 4, 5, 7,1 and 2 patients. All these variants remained consistently detectable on HBV RNA basis. Additionally, the variants rtL180M+rtM204V, rt204V and rtA181T became detectable at months 25, 12, and 5 on HBV DNA or RNA basis in one patient each. Within the s gene the stop codons sC69*, sL216*, sW172*, sW182*, sW196* and sW199* were found in 2, 1, 1, 2, 1 and 1 patients on HBV DNA basis, and they remained detectable on HBV RNA basis. Conclusion: Sequencing of HBV RNA represents a novel and reproducible method for the detection of HBV variants in patients with undetectable HBV DNA during antiviral treatment with nucleos(t)ideanalogues. The value of this method should be investigated for variants conferring to resistance or to possible cytopathological effects on hepatocytes.

Disclosures:

Florian van Boemmel - Advisory Committees or Review Panels: Roche Pharma; Board Membership: Gilead Sciences; Grant/Research Support: Gilead Sciences, Roche Pharma, BMS; Speaking and Teaching: Gilead Sciences, Roche Pharma, BMS, MSD, Janssen-Cilag, Siemens

Eckart Schott - Advisory Committees or Review Panels: Gilead, Roche, Bayer, BMS; Speaking and Teaching: Gilead, Novartis, Roche, MSD, Bayer, Falk, BMS

Thomas Berg - Advisory Committees or Review Panels: Gilead, BMS, Roche, Tibotec, Vertex, Jannsen, Novartis, Abbott, Merck; Consulting: Gilead, BMS, Roche, Tibotec; Vertex, Janssen; Grant/Research Support: Gilead, BMS, Roche, Tibotec; Vertex, Jannssen, Schering Plough, Boehringer Ingelheim, Novartis; Speaking and Teaching: Gilead, BMS, Roche, Tibotec; Vertex, Janssen, Schering Plough, Novartis, Merck, Bayer

The following people have nothing to disclose: Laura Schmalbrock, Danilo Deichsel, Stephan Böhm

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Antiviral effect of tenofovir disoproxil fumarate on drug-resistant HBV clones and different susceptibility between HBV genotype A and C

Eisuke Murakami, Masataka Tsuge, Nobuhiko Hiraga, Tomokazu Kawaoka, Atsushi Ono, Daiki Miki, Hiromi Abe, Michio Imamura, Hiroshi Aikata, Hidenori Ochi, C. Nelson Hayes, Kazuaki Chayama;
Department of Gastroenterology and Metabolism, Hiroshima university, Hiroshima, Japan

Background & Aims: Tenofovir disoproxil fumarate (TDF) has been approved for chronic hepatitis B treatment in several countries and is reported to have strong anti-viral effect and lower incidence of drug resistance. To date, differences in TDF susceptibilities among hepatitis B virus (HBV) genotypes and drug-resistant strains have been unclear. In this study, TDF susceptibilities between genotypes A and C were evaluated using several drug-resistant HBV clones in vitro and in vivo. Methods: HBV expression plasmids were constructed from sera of HBV carriers, and drug-resistant substitutions in HBV reverse transcriptase (RT) region were introduced by site-directed mutagenesis. HepG2 cells transiently transfected with HBV expression plasmids were treated with different concentrations of TDF for 72 hours. Core-associated HBV replication intermediates were quantified by real time PCR. HBV particles generated from transfected HepG2 cells were inoculated into human hepatocyte chimeric mice (PXB mouse). After infection, the mice were treated with 90mg/kg/day of TDF for 2 weeks. Results: Core-associated HBV replication intermediates of wild type clones, lamivudine-resistant clones (rtL180M/M204V) and lamivudine plus entecavir-resistant clones (rtL180M/S202G/M204V) in transiently HBV transfected HepG2 cells were suppressed by TDF in a dose-dependent manner. Clones with lamivudine plus adefovir-resistant mutations (rtA181T/N236T) showed resistance against TDF. Comparing the changes of serum HBV DNA levels in the mice by TDF treatment, the reduction of HBV DNA with rtA181T/N236T clone was less than with wild type (-2.0Log, -2.8Log, respectively). In the in vitro and in vivo analyses using a lamivudine-resistant clone (rtL180M/M204V) and a lamivudine plus adefovir-resistant clone (rtL180M/M204V/N236T), the latter developed a strong resistance to TDF. Therefore, rtN236T substitution could be a key mutation for TDF susceptibility. IC50 (inhibitory concentration 50) of genotype A clones was approximately 20% higher than that of genotype C clones in vitro. The reduction of serum HBV DNA in the mouse with wild type genotype A was also less than with wild type genotype C (-1.8Log, -2.8Log, respectively). These genotypic differences were determined by amino acid sequences at amino acids 223 and 224 in HBV RT region. Conclusions: The present study indicates that TDF susceptibilities vary among HBV genotypes and drug-resistant HBV clones. Examination of amino acid sequences in the HBV RT region will give us useful information to select nucleot(s)ide analogues to treat patients with chronic hepatitis B virus infection.

Disclosures:

Kazuaki Chayama - Consulting: Abbvie; Grant/Research Support: Dainippon Sumitomo, Chugai, Mitsubishi Tanabe, DAIICHI SANKYO, Toray, BMS, MSD; Speaking and Teaching: Chugai, Mitsubishi Tanabe, DAIICHI SANKYO, KYORIN, Nihon Medi-Physics, BMS, Dainippon Sumitomo, MSD, ASKA, Astellas, AstraZeneca, Eisai, Olympus, GlaxoSmithKline, ZERIA, Bayer, Minophagen, JANSSEN, JIMRO, TSUMURA, Otsuka, Taiho, Nippon Kayaku, Nippon Shinyaku, Takeda, AJINOMOTO, Meiji Seika, Toray

The following people have nothing to disclose: Eisuke Murakami, Masataka Tsuge, Nobuhiko Hiraga, Tomokazu Kawaoka, Atsushi Ono, Daiki Miki, Hiromi Abe, Michio Imamura, Hiroshi Aikata, Hidenori Ochi, C. Nelson Hayes

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Risk factors for significant liver fibrosis in genotype D HBeAg-negative chronic hepatitis B infection with persistently normal ALT and high serum HBVDNA levels

Asli Cifcibasi Ormeci1, Filiz Akyuz1, Bulent Baran1, Ozlem Mutluay Soyer1, Suut Gokturk1, Cetin Karaca1, Mine Gulluoglu3, Derya Onel2, Selim Badur2, Kadir Demir1, Fatih Besisik1, Sabahattin Kay-makoglu1;
1Department of Gastroenterohepatology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey; 2Microbiology, Istanbul University Istanbul Faculty of medicine, Istanbul, Turkey; 3Pathology, Istanbul University Istanbul Faculty of medicine, Istanbul, Turkey

Aim: It is controversial to monitor disease activity using ALT level in HBeAg (-) chronic hepatitis B (CHB) infection. Current guidelines favor liver biopsy in HBeAg (-) CHB with persistently normal ALT (PNALT) and high serum HBVDNA levels. In this study, we aimed to determine fibrosis stage and histological activity index (HAI) in HBeAg (-) CHB patients with PNALT and high HBVDNA. We evaluated the possible risk factors associated with significant histological abnormalities. Materials and Methods:Patients with genotype D HBeAg (-) CHB who have PNALT (≤40IU/ mL;4 values in 3 month intervals) and high HBVDNA (≥2000 IU/mL) during a 12 months screening period were included in the study.HBVDNA was measured at the beginning and at the end of the screening period .A liver biopsy was performed in patients who conform to the selection criteria.All of the patients had a normal physical examination, blood counts and ultrasonography.Fibrosis stage and HAI were scored according to Ishak scoring system.Multivariate logistic regression analyses were performed to find out independent factors associated with fibrosis stage≥2 and HAI≥6. Results:1 17 patients (56 male,mean age 43±11years) were included.The known duration of HBsAg positivity was 8.3 ± 5.4 (range,1-22) years.Median HBVDNA level was 111.641 IU/ml and 42.7% patients had a HBVDNA level >20.000 IU/ml.Median HAI was 3 (0-8) and 14.5% patients had a moderate-to-severe (HAI>6) histological activity.Distribution of patients according to fibrosis stage was as follows:38 (32.5%) patients with stage 0, 41 (35%) patients with stage 1 and 34 (29.1%) patients with stage 2 fibrosis and 4 (3.4%) patients with advanced fibrosis (stage 3-4).The number of patients with fibrosis stage≥2 and/or HAI≥6 was 43 (36.7%). In multivariate logistic regression analysis, only independent variable associated with fibrosis stage ≥2 was age (OR=1.04, 95% CI 1.004-1.079, p=0.031). Independent risk factors for HAI≥6 were; age (OR= 1.067, 95% CI 1.010-1.126, p=0.02) and male gender (OR=4.73, 95% GA 1.19-1 8.7, p=0.027). According to ROC analyses, an optimal cut-off for age to detect fibrosis stage≥2 was 46 years (sensitivity=0.58 /specificity=0.67) and age cut-off to determine HAI≥6 was 44 years (sensitivity=0.82/specificity=0.56). Discussion:A significant liver damage indicating treatment was detected in one third of CHB patients with PNALT and high serum HBVDNA level who underwent liver biopsy.Age is an independent predictor for moderate-to-severe liver fibrosis and histological activity regardless of the level of HBVDNA. It seems reasonable to perform liver biopsy in CHB patients older than 40 years to determine the degree of liver damage.

Disclosures:

The following people have nothing to disclose: Asli Cifcibasi Ormeci, Filiz Akyuz, Bulent Baran, Ozlem Mutluay Soyer, Suut Gokturk, Cetin Karaca, Mine Gulluoglu, Derya Onel, Selim Badur, Kadir Demir, Fatih Besisik, Sabahattin Kay-makoglu

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Caspase-cleaved fragments of cytokeratin-18 as a marker of inflammatory activity in chronic hepatitis B virus infection

Ho Joong Kim, Chang bum Bae, Joo An Hwang, Sung Won Cho, Jae Youn Cheong;
Ajou univ. medical center, Suwon city, Republic of Korea

Background: The differential diagnosis between inactive carrier and active hepatitis is important in patients with chronic hepatitis B (CHB) virus infection. Serum cytokeratin (CK)-18 fragments (M30-antigen) are proposed as biomarkers of apoptosis. Objectives: We investigated whether serum M30-antigen levels might help to characterize the various phases of CHB and predict the state of significant inflammation in patients with CHB. Study design: A total of 339 CHB patients who underwent liver biopsy, were included. Serum M30-antigen levels were compared with inactive carriers (n=21), patients with HBeAg- negative hepatitis (n=95), HBeAg-positive hepatitis (n=141) and liver cirrhosis (n=82). Results: Serum M30-antigen levels were correlated significantly not only with AST (r=0.544, p<0.001) and ALT (r=0.315, p<0.001) and but also inflammatory grading score on liver biopsy (r=0.240, p<0.001). Serum M30-antigen level in HBeAg-negative CHB was significantly higher than that of inactive HBV carrier (399.78 U/L vs 148.90 U/L, p<0.001). Multivariate analysis showed that AST (p<0.001), albumin (p=0.009) and M30-antigen (p=0.020) were the independent predictors of significant inflammation. Combined serum M30-antigen level (>344 U/L) and AST (>78 IU/L) measurement provided the most accurate identification of significant inflammation, showing 38.2% sensitivity, 96.1% specificity, 91.0% positive predictive value and 56.1% negative predictive value. Conclusions: Serum M30-antigen can be a predictive marker for distinguishing between inactive carrier and HBeAg-negative CHB. Serum M30 levels are associated with the presence of significant inflammation, especially in patients with normal or minimally elevated ALT in CHB patients.

Disclosures:

The following people have nothing to disclose: Ho Joong Kim, Chang bum Bae, Joo An Hwang, Sung Won Cho, Jae Youn Cheong

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Prediction of liver-related event development using transient elastography in patients with chronic hepatitis B showing complete virological responses to antiviral therapy

Hye Won Lee1, Seung Up Kim1,2, Beom Kyung Kim1,2, Jun Yong Park1,2, Do Young Kim1,2, Sang HoonAhn1,2, Kwang-Hyub Han1,2;
1Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea; 2Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Republic of Korea

Background: The hepatitis B virus (HBV) DNA level is a strong predictor of disease progression in chronic hepatitis B (CHB). However, few studies have investigated other prognostic factors when a complete virological response (CVR) is achieved with antiviral therapy. Liver stiffness (LS) transient elastography (TE) is useful for assessing not only liver fibrosis, but also the risk of forthcoming liver-related events (LREs). Here, we investigated the prognostic role of TE in predicting LREs development in CHB patients with a CVR. Methods: The study analyzed 192 consecutive patients with CHB who achieved a CVR (defined as HBV DNA < 20 IU/mL) with entecavir therapy. LS values were measured at the time of CVR. All subjects were followed regularly to detect LREs, defined as any cirrhotic complication (variceal bleeding, ascites, hepatic encephalopathy, hepatorenal syndrome, and spontaneous bacterial peritonitis), hepato-cellular carcinoma (HCC), and liver-related mortality. Results: The median patient age was 49 years, and 134 (69.8%) were male. Liver cirrhosis was identified in 90 (46.9%) patients and all had well-preserved liver function. The median LS value at CVR was 8.8 kPa and the median time to CVR was 8.3 months. During follow-up (median 34 months), LREs occurred in 21 (11.0%) patients. Age, alanine aminotransferase, and LS values were significantly higher in patients with LRE than those without (all P<0.05). When the study population was stratified into three groups (≤8 kPa, 8.1-13 kPa, >13 kPa), the proportion of patients with LRE increased significantly with the LS value: 1.3% for LS≤8 kPa, 14.8% for LS 8.1-13 kPa, and 21.2% for LS>13 kPa (P<0.001). In addition, the cumulative incidence of LREs increased significantly in association with LS values among the three groups (P=0.001). Patients with a LS value >13 kPa (hazard ratio [HR] 10.268, 95% confidence interval [CI] 1.253-84-129, P=0.046) and 8.1-13 kPa (HR=7.877, 95% CI, 1.150-65.312, P=0.046) were at significantly greater risk of developing LREs than those with a LS value ≤8 kPa, as a reference. On multivariate analysis using the Cox regression method, age and LS values were identified as independent predictors of LRE development (both P<0.05). Conclusion: The LS value at the time of CVR is a useful predictor of future LREs. Despite complete viral suppression through antiviral therapy, more careful surveillance is necessary, particularly in patients with a LS >8 kPa.

Disclosures:

The following people have nothing to disclose: Hye Won Lee, Seung Up Kim, Beom Kyung Kim, Jun Yong Park, Do Young Kim, Sang Hoon Ahn, Kwang-Hyub Han

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Clinical and Immunological Characteristics of Acute Hepatitis B with Viral Clearance or Persistence

Ying Sun, Baosen Li, Zhengsheng Zou;
302 Military Hospital, Beijing, China

Objective: this study was to investigate the clinical,virological, and immunological course of acute HBV infection in order to gain more information of HBV infection characteristics responsible for viral clearance or persistence.Methods: A total of 348 patients with acute hepatitis B were prospectively followed up for more than 24 weeks(acute Hepatitis B with viral persistence for at least 48 weeks), biochemical, virological, and immunological parameters of these patients detected at different time-points of the follow-up were analyzed. Results: Among the 348 acute hepatitis B patients, 286 subjects were male, and the mean age was 35.38±11.46 years. 328 patients resolved HBV spontaneously, while the remaining 20 patients developed chronic HBV infection. There were statistically significant differences in age(35.77±11.41 vs29.00±10.62years,P<0.01), peak ALT(1358.62±645.17vs893.64±385.13U/L, P<0.05), serum HBV DNA(2.79±0.05vs8.82±0.40 log10 IU/ml, P<0.01),and the ratio of acute icteric hepatitis (78.45%vs40.00%)between patients who developed spontaneous viral clearance and those who did not. HBV DNA in patients with viral clearance became undetectable accompanied by normalization of alanine aminotransferase levels in 24w; but HBV DNA in the subjects with viral persistence demonstrated persistent viremia and abnormal alanine aminotransferase levels in 80% patients. All these individuals with viral persistence were treated with Peg-interferon antiviral therapy, the serum HBV DNA disappearance was observed in 65 percents at the 24W of follow up, and 50 percents of these patients showed HBsAg serologic loss and HBsAg seroconversion. Intrahepatic total HBV DNA and cccDNA levels at baseline were detectable in 24 patients,who had been identified with spontaneous HBV clearance at 24 weeks follow-up, the results showed there was significant correlation between serum HBV DNA levels with Intrahepatic cccDNA levels(r= 0.608, P<0.05), intrahepatic total HBV DNA also correlated with intrahepatic cccDNA(r=0.739, P< 0.01),but there was no significant correlations with Intrahepatic total HBV DNA levels(r= 0.108, P>0.05).Serum HBsAg titers was not correlated with intrahepatic total HBV DNA and intrahepatic cccDNA levels(r=0.041, P>0.05;r=-0.244, P>0.05).Patients with spontaneous viral clearance displayed a higher IL-17A and TNF-α levels,but lower IL-10 compared with persistently infected subjects.Conclusions: patients with acute icteric hepatitis B, high ALT,IL-17A and TNF-α level have a high rate of spontaneous viral clearance antiviral therapy with pegylated interferon seem to be effective to some patients with Hepatitis B virus persistence.

Disclosures:

The following people have nothing to disclose: Ying Sun, Baosen Li, Zhengsheng Zou

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Over-expression of microRNA-30a inhibits HBx-induced autophagosome formation in hepatic cells

Satendra Kumar1, Parul Gupta1, Sweta Khanal1, Aashirwad Shahi1, Preeti Damania2, Senthil K. Venugopal1;
1Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India; 2Research, Institute of Liver and Biliary Sciences, New Delhi, India

Background: Hepatitis B Virus (HBV) enters the host and survives itself by adopting several mechanisms. One of the ways that HBV survives and replicates in the host cells is by inducing autophagy. miRNAs are small, non-coding RNA molecules, which regulate gene expression at post-transcriptional level. Several reports have shown that microRNAs modulate the HBV infection and proliferation. Previous reports have shown that miRNA-30a inhibits autophagosome formation in cancer cells. Hence, we hypothesized that over-expression of miRNA-30a could inhibit HBV-induced autohphagosome formation in hepatic cells. Methods: Both Hep G2 cells and Hep G2.2.1.5 (HBV stably expressing cells) were used in all the experiments. microRNA-30a was over-expressed in these cells using siPORT NeoFX reagent. After 72 hours, the cells were collected either for RNA or protein isolation. Total RNA enriched with miRNAs was isolated, cDNA was synthesized and real time PCR for miRNA-30a was performed. The cellular protein was isolated and Western blots were performed for beclin-1 and β-actin. Effect of miRNA-30a over-expression on apoptosis was studied by conducing Western blots for cleaved caspase-3 in the cell lysates. To identify the role of HBx on the autophagosome formation, Hep G2 cells were transfected with pSG5-HBx plasmid and the effect on miRNA-30a and beclin-1 was determined. Results: Over-expression of miRNA-30a resulted in a significant 20-fold increase (n=3; p<0.001) in the intracellular levels of miRNA-30a. The expression of beclin-1 was at least 4-fold higher in Hep G2.2.1.5 cells compared to Hep G2 cells. miRNA-30a over-expression in Hep G2 and Hep G2.2.1.5 cells resulted in a significant decrease in the expression of beclin-1 protein levels in both these cells (8-fold and 4-fold respectively; n=3; p<0.05). To determine the role of HBx on beclin-1 expression, Hep G2 cells were transfected with pSG5-HBx plasmid or empty vector. After 48 hours, the cells were isolated and the expression of HBx protein was determined by Western blots and found to be significantly increased. There was a significant increase in beclin-1 expression (6-fold increase compared to the empty vector transfected cells). There was no effect of HBx on miRNA-30a was found. Over-expression of miRNA-30a significantly increased cleaved caspase-3 protein levels, suggesting that over-expression of miRNA-30a induces apoptosis. Conclusion: These data demonstrate that HBx induces beclin-1 expression and over-expression of miRNA-30a could successfully inhibit the autophagy induced by HBx and possibly increasing apoptosis in hepatic cells.

Disclosures:

The following people have nothing to disclose: Satendra Kumar, Parul Gupta, Sweta Khanal, Aashirwad Shahi, Preeti Damania, Senthil K. Venugopal

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The Safety and Efficacy of Entecavir and Tenofovir Combination Therapy for Chronic Hepatitis B in Patients with Previous Nucleos(t)ide Treatment Failure

Maciej S. Jablkowski1, Mircea. Diculescu2, Harry L. Janssen3,4, Fabien Zoulim5, Joerg Petersen6, Patrick Marcellin7,8, Soumaya Bendahmane9, Isabelle Klauck10, Krzysztof. Simon11;
1Department of Infectious and Liver Diseases, Medical University of Lodz, Lodz, Poland; 2Centre of Gastroenterology and Hepatology, Fundeni Clinical Institute, Bucharest, Romania; 3Department of Gastroenterology & Hepatology, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, Netherlands; 4Liver Clinic, Toronto Western & General Hospital, University Health Network, Toronto, ON, Canada; 5Hepatology Department, Hospices Civils de Lyon, INSERM U1052, Lyon University, Lyon, France; 6IFI Institute for Interdisciplinary Medicine, Asklepios Klinik St Georg, Hamburg, Germany; 7Hôpital Beaujon, Assistance Publique Hôpitaux de Paris, University of Paris 7, Paris, France; 8INSERM Unité 773, Centre de Recherches Claude Bernard sur les Hepatites Virales, Clichy, France; 9Bristol-Myers Squibb, Braine-I'Alleud, Belgium; 10Bristol-Myers Squibb, Paris, France; 11Division of Infectious Diseases and Hepatology, Wroclaw University of Medicine, Wroclaw, Poland

Introduction: Combination of potent antivirals with non-overlapping resistance profiles, such as entecavir (ETV) and tenofovir disoproxil fumarate (TDF), may provide superior antiviral efficacy compared with single agents for chronic hepatitis B (CHB) treatment. This study evaluated the efficacy and safety of ETV+TDF in patients with CHB who had failed previous nucleos(t)ide therapy. Methods: Single-arm, open-label, multi-center study assessing once-daily ETV 1 mg plus TDF 300 mg for up to 96 weeks, with 24-week follow-up. The primary end point was the proportion of patients with a virologic response (HBV DNA <50 IU/mL, Roche COBAS TaqMan-HPS Assay) at Week 48 (non-completer = failure). Secondary end points included HBeAg and HBsAg loss and seroconversion and emergence of resistance mutations on treatment. Treatment-emergent adverse events (TEAEs) and serious adverse events (SAEs) were assessed cumulatively. Results: Baseline characteristics and efficacy data for the 92 patients who received at least one dose of ETV+TDF are summarized in table 1. At Week 48, 17/20 (85%) patients previously failing lamivudine (LVD), and 37/48 (77%) and 6/11 (55%) patients previously failing ETV or TDF, respectively, achieved HBV DNA <50 IU/mL. There was no genotypic evidence of treatment-emergent resistance. Twenty seven patients (29%) experienced at least one TEAE suspected to be related to study treatment; fatigue (10%) and nausea (9%) were most frequently reported. All treatment-related TEAEs were Grade 1/2. Three patients (3%) experienced five SAEs; none were considered related to study treatment. Conclusions: The combination of ETV+TDF therapy for 48 weeks resulted in virologic response in around three-quarters of patients who had failed prior nucleos(t)ide therapy for CHB, and no treatment-emergent resistance was observed. The combination of ETV and TDF was well tolerated. This study is ongoing, with additional efficacy and safety analyses to be completed.

Table 1. Baseline characteristics and efficacy data (N = 92)
  1. All data are n, (%) unless otherwise stated; *data missing for one patient; ADV, adefovir.

Patient characteristics at baseline 
Median age, years (range)43(19-85)
Male69 (75)
HBV DNA, median (range)3.7 log10 lU/mL (1.5-9.3)
HBeAg positive56(62)
Treatment monotherapy regimens prior to study entry* 
ETV48 (53)
LVD20 (22)
TDFH(12)
Resistance mutations 
LVDr48 (52)
ETVr24 (26)
ADVr6(7)
Week 48 efficacy results 
HBVDNA<50IU/mL70/92 (76)
HBVDNA<6IU/ml17/92 (19)
HBeAg loss3/56 (5)
HBe seroconversion2/56 (4)
HBsAg loss ± seroconversion0

Disclosures:

Maciej S. Jablkowski - Advisory Committees or Review Panels: Gilead; Consulting: BMS, Gilead, Roche, MSD; Speaking and Teaching: BMS, Roche, MSD, Janssen-Cilag

Harry L. Janssen - Consulting: Abbott, Bristol Myers Squibb, Debio, Gilead Sciences, Merck, Medtronic, Novartis, Roche, Santaris; Grant/Research Support: Anadys, Bristol Myers Squibb, Gilead Sciences, Innogenetics, Kirin, Merck, Medtronic, Novartis, Roche, Santaris

Fabien Zoulim - Advisory Committees or Review Panels: Gilead; Consulting: Roche; Grant/Research Support: Gilead, Scynexis, Roche; Speaking and Teaching: Novartis, Roche, Janssen, Bristol Myers Squibb, Gilead

Joerg Petersen - Advisory Committees or Review Panels: Bristol-Myers Squibb, Gilead, Novartis, Merck, Bristol-Myers Squibb, Gilead, Novartis, Merck; Grant/Research Support: Roche, GlaxoSmithKline, Roche, GlaxoSmithKline; Speaking and Teaching: Abbott, Tibotec, Merck, Abbott, Tibotec, Merck

Patrick Marcellin - Consulting: Roche, Gilead, BMS, Vertex, Novartis, Janssen-Tibotec, MSD, Boehringer, Pfizer, Abbott, Alios BioPharma; Grant/Research Support: Roche, Gilead, BMS, Novartis, Janssen-Tibotec, MSD, Alios BioPharma; Speaking and Teaching: Roche, Gilead, BMS, Vertex, Novartis, Janssen-Tibotec, MSD, Abbott

Isabelle Klauck - Employment: Bristol-Myers Squibb

Krzysztof. Simon - Advisory Committees or Review Panels: BMS, MSD, Roche, GIlead

The following people have nothing to disclose: Mircea. Diculescu, Soumaya Ben-dahmane

1045

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Impact of Genetic Variability in the PreS1/S2 and Hepatitis B Surface Antigen (HBsAg) Regions of the Hepatitis B Virus (HBV) on Quantitative HBsAg Levels

Kathryn M. Kitrinos1, Henry Lik-Yuen Chan2, Edward J. Gane3, Scott Fung4, Phillip Dinh1, Lanjia Lin1, Amoreena C. Corsa1, Michael D. Miller1, Mani Subramanian1, Alexander J. Thompson5, Maria Buti6;
1Gilead Sciences, Foster City, CA; 2Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong S.A.R., Hong Kong; 3New Zealand Liver Transplant Unit, Auckland, New Zealand; 4Toronto General Hospital, Toronto, ON, Canada; 5Gastroenterology, St Vincent's Hospital, Melbourne, VIC, Australia; 6Liver Center, Hospital Vall d'Hebron, Barcelona, Spain

Objective: It has been reported (Pollicino et al. Hepatol. 2012; 56:434-443) that predominantly genotype D, chronic hepatitis B (CHB) patients with preS1/S2 and HBsAg genetic variability have lower plasma HBsAg levels with no impact on HBV DNA levels. PreS1/S2 and HBsAg genetic variability may confound analyses identifying clinically relevant HBsAg levels for treatment response. We evaluated preS1/S2 and HBsAg genetic variability in CHB patients across multiple genotypes. Methods: Sixty-one patients from Studies GS-US-174-0102, 174-0103, and 203-0101 with HBV DNA >1×108 cp/mL (1.7×107 IU/mL) and HBsAg <10,000 IU/mL were selected (low HBsAg group) for comparison with a matched set of 61 patients with HBsAg >10,000 IU/mL (high HBsAg group). Patients were matched by HBV DNA, genotype, HBeAg status, and ALT level. PreS1/S2 and HBsAg population sequencing was conducted and sequences were evaluated for insertions, deletions, and start codon mutations in preS1/S2, premature stop codons in preS1/S2 and HBsAg, and a-determinant mutation(s) in HBsAg. Comparisons were conducted using Fisher's exact test with the Hochberg's procedure to control for multiple testing. Results: Groups were well matched; median HBV DNA 8.53 log10 cp/mL, 75% ALT ≥2x ULN, 58% HBeAg-, and genotype C (33%) and D (43%) were most common. Median HBsAg was significantly different between low and high groups (4465 vs. 19,965 IU/mL, p= <0.0001). There was no significant difference between groups in regard to the percentage of patients with preS1/S2 start codon mutations, HBsAg premature stop codons, or HBsAg a-determinant mutations. No patient had a preS1/S2 premature stop codon. There was a trend for a higher percentage of patients with preS1/S2 insertions or deletions in the low HBsAg group compared to the high HBsAg group. A sub-analysis evaluating genotype D patients (n=52) found no difference between the low and high HBsAg groups for any variable assessed, with no trend for the percentage of patients with pre S1/S2 insertions and deletions (p=0.722). Conclusions: HBsAg genetic variability does not appear to correlate with quantitative HBsAg levels in patients with HBV DNA > 1 × 108 cp/mL. There is a trend for a higher percentage of pre S1/S2 insertions or deletions among patients with low HBsAg and high HBV DNA across genotypes.

  Low HBsAg (N=61)High HBsAg (N=61)Adjusted p-value a
  1. aAdjusted by Hochberg's procedure

PreSl/S2Insertion or Deletion31.1%11.5%0.070
 Premature Stop Codon0%0%NA
 Start Codon Mutation14-8%11.5%1.000
HBsAg

a-determinant

Mutation(s)
41%39.3%1.000
 Premature Stop Codon16.4%11.5%1.000

Disclosures:

Kathryn M. Kitrinos - Employment: Gilead Sciences, Gilead Sciences; Stock Shareholder: Gilead Sciences, Gilead Sciences

Henry Lik-Yuen Chan - Advisory Committees or Review Panels: Gilead, Vertex, Bristol-Myers Squibb, Abbott, Novartis Pharmaceutical, Roche, MSD

Edward J. Gane - Advisory Committees or Review Panels: Roche, AbbVie, Novartis, Tibotec, Gilead Sciences, Janssen Cilag, Vertex, Achillion; Speaking and Teaching: Novartis, Gilead Sciences, Roche

Scott Fung - Advisory Committees or Review Panels: Merck, Vertex; Grant/Research Support: Gilead Sciences, Roche; Speaking and Teaching: Gilead Sciences, BMS

Phillip Dinh - Employment: Gilead Sciences

Lanjia Lin - Employment: Gilead; Stock Shareholder: Gilead

Amoreena C. Corsa - Employment: Gilead Sciences Inc.; Stock Shareholder: Gilead Sciences Inc.

Michael D. Miller - Employment: Gilead Sciences, Inc.; Stock Shareholder: Gilead Sciences, Inc.

Mani Subramanian - Employment: Gilead Sciences

Alexander J. Thompson - Advisory Committees or Review Panels: Merck, Inc, Roche, Janssen (Johnson & Johnson), BMS, GSK Australia, Novartis, GILEAD Sciences, Inc; Consulting: GILEAD Sciences, Inc; Grant/Research Support: Merck, Inc, Roche, GILEAD Sciences, Inc; Speaking and Teaching: Merck, Inc, Roche, BMS

Maria Buti - Advisory Committees or Review Panels: Boerhinger Inghelm, Boer-hinger Inghelm; Speaking and Teaching: MSD, Bristol-Myers Squibb, Novartis, Gilead, Janssen, MSD, Bristol-Myers Squibb, Novartis, Gilead, Janssen

1046

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Dynamics of Quasispecies Complexity and Main Regulatory Motifs of Hepatitis B Virus (HBV) X Gene/Basal Core Promoter/preCore Region Analyzed by Ultradeep Pyrosequencing (UDPS) Technology

Andrea Caballero1, Josep Gregori2,3, Maria Homs1,2, David Tabernero1,2, Maria Blasi1,2, Rosario Casillas1,3, Leonardo Nieto4, Irene Belmonte Mula1, Xose Costa5, Carolina Gonzalez1, Rafael Esteban2,6, Maria Buti2,6, Francisco Rodriguez-Frias1,2;
1Biochemistry, Vall d'Hebron Hospital, Barcelona, Spain; 2CIBERehd, Barcelona, Spain; 3Liver Diseases, Vall d'Hebron Research Institute, Barcelona, Spain; 4Microbiology, Vall d'Hebron Hospital, Barcelona, Spain; 5Microbiology, Hospital Santiago de Com-postela, Santiago de Compostela, Spain; 6Hepatology, Vall d'Hebron Hospital, Barcelona, Spain

Background The HBV X region (HBX) overlapped with preCore, includes essential BCP motifs: TATA boxes TA1-TA4 and the conserved DR1 motif with the target sequence (AS) for the 4-nucleotide primer (4nt) that starts HBV replication Aim To characterize HBV quasispecies complexity in HBX, TA1-TA4, and DR1 by UDPS Patients and Methods UDPS (GS Junior Roche) analysis of HBX from 10 chronic HBV patients, all LMV nonresponders, in 30 serum samples: baseline (BA), untreated (UT), and after LMV. nt variations were studied. Quasispecies complexity was estimated by Shannon entropy (SE), mutation frequency, and nucleotide diversity (ND) Results UDPS yielded 415,726 sequences. TA1, TA2 and DR1 were more variable than TA3 or TA4 (Table). TA1 and TA2 variability was mainly due to T1753C and T1762A, respectively. In 6 patients, there was no identity between 4nt and AS (Table). Without treatment (BA/UT), quasispecies complexity was higher in HBeAg(-) than HBeAg(+) cases (SE 0.55 vs 0.35, p=0.029); after LMV it was greater in HBeAg(+) than HBeAg(-) (SE 0.38 vs 0.21, p=0.007), and near significantly greater in genotype A than D (ND 0.016 vs 0.01, p=0.070) Conclusions Better conservation of TA3 and TA4 suggests a more essential role for these motifs than for TA1 and TA2 The remarkable variability in DR1 suggests existence of alternative mechanisms for “4nt primer jumping”, starting HBV replication Highest complexity in seroconverted cases suggests enhancement of evolution rate by immune pressure once HBeAg disappears. The increase in quasispecies complexity after LMV in genotype A and HBeAg(+) cases suggests lower sensitivity to this treatment. Funding Instituto CarlosIII (PI 12/1893) cofinanced by ERDF

(<)Less than 0.25%; (*)No viral breakthrough

(Λ)No identity between 4nt and ASDR1 sequences

The variability in TA1 and TA2 does not include variability of positions 1753 and 1762

 %TATA boxes(TA1-TA4)%DR1
CaseSampleGenotypeHBe1 (1753)2 (1762)34Total(Λ)
1BasalA/DN11.00<27.9<<2.4<
 UntreatedAP<<<19.1<<<<
 After LMVAP<<<16.6<<<<
2BasalAP1.9<0.387.50.4<<0.38
 UntreatedAP<<<92.9<<<1.74
 After LMVAP<<<13.9<14<5.84
3BasalA/DN<<<27.6<<<<
 UntreatedA/DN2.1<<23.2<<<<
 After LMV *A/DN<88.00<84.3<<1.51.51
4BasalDP<<<<<<0.40.60
 UntreatedDP<<<1.2<<2.92.30
 After LMV*DP0.3<<18.5<<<<
5BasalDP0.8<<<0.30.32.91.05
 UntreatedDP0.3<0.3<0.3<0.60.29
 After LMVDP<<<<<<1.20.88
6BasalAP<<<0.9<0.30.50.77
 UntreatedAP0.50.50<<<0.4150.42
 After LMVAP<<<<0.3<0.42.64
7BasalA/DN6.66.60<18<<<<
 UntreatedDN<<<<<<<<
 After LMV *DN<<<<<<2.5<
8BasalAP<<<98<<<<
 untreatedDN<<<<<<<<
 After LMVAP<<<4<<<<
9BasalDP0.4<6.30.650.40.60.60.53
 UntreatedAP<<<99.4<<<<
 After LMVAP<7.50<<<<<<
10BasalDP/N<<<63.5<<<<
 UntreatedDN1.9<<100<<<<
 After LMVAN/P<<<<<<3<

Disclosures:

Rafael Esteban - Speaking and Teaching: MSD, BMS, Novartis, Gilead, Glaxo, MSD, BMS, Novartis, Gilead, Glaxo, Janssen

Maria Buti - Advisory Committees or Review Panels: Boerhinger Inghelm, Boer-hinger Inghelm; Speaking and Teaching: MSD, Bristol-Myers Squibb, Novartis, Gilead, Janssen, MSD, Bristol-Myers Squibb, Novartis, Gilead, Janssen

The following people have nothing to disclose: Andrea Caballero, Josep Gregori, Maria Homs, David Tabernero, Maria Blasi, Rosario Casillas, Leonardo Nieto, Irene Belmonte Mula, Xose Costa, Carolina Gonzalez, Francisco Rodriguez-Frias

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A serum microRNA signature associated with both natural and therapy induced immune controls of chronic genotype D hepatitis B virus infection

Maurizia R. Brunetto1, Daniela Cavallone1, Francesco Moriconi1, Piero Colombatto1, Filippo Oliveri1, Pietro Ciccorossi1, Barbara Coco1, Veronica Romagnoli1, Carlotta Rastelli1, Maria W. Teilum2, Thorarinn Blondal2, Ferruccio Bonino3;
1Hepatology Unit, University Hospital of Pisa, Pisa, Italy; 2Division of Dx and Services,, Exiqon A/S, Vedbaek, Denmark; 3General Medicine 2 Unit, University Hospital of Pisa, Pisa, Italy

Background & aims. MicroRNAs (miRs) are implicated in viral immune control: we studied their serum dynamics in chronic inactive HBsAg carriers (IC) and chronic hepatitis B (CHB) patients with different responses to antiviral therapy. Methods. Sera (143) were obtained from 75 (male/female 48/27, median age 43, 18-67 y.) HBeAg negative chronic genotype-D-HBV carriers followed for 8-13 y. IC (15) had persistently serum HBV-DNA levels ≤2000 IU/ml and normal ALT. CHB patients (60) were treated with peg-IFN or nucleos(t)ide-analogs. Off therapy response was defined by HBsAg loss and/or persistence of HBV-DNA ≤2000 IU/ml for at least 12 months after the end of treatment (EOT). Total RNA was extracted from serum and immunoprecipitated HBsAg-particles (that carry liver miRs) using miRNeasyTM Mini kit (Qiagen Inc.) and reverse transcribed using miRCURY LNA™ Universal RT cDNA synthesis kit (Exiqon A/S). RT-q-PCR profiling: cDNA was assayed using miRCURY LNA™ Universal PCR System (Exiqon A/S) and each microRNA by qPCR on version 2 ready-to-use human panels I and II containing 739 microRNA assays and 3 spike-ins and inter-plate calibrators. The amplification was performed in a LightCycler 480 RT- PCR System (Roche A/S) in 384 well plates. Data were filtered on amplification efficiency, melting curves and peaks and non-specific background using non-template controls with a cutoff of Cq <37. Serum samples were normalized using global mean. TIGR's Multiple Experiment Viewer (MEV) version 4.8 was used for statistical analysis. Results. IC miRs signatures were compared to those of CHB patients at baseline and different time-points during/after treatment according to treatment response: miRs were differentially expressed in IC and CHB patients and the IC-associated miRs signature held true in sustained responders. A mirB-index was defined using 5 miRs (hsa-miR-122-5p, hsa-miR-99a-5p, hsa-miR-126-3p, hsa-miR-192-5p, hsa-miR335-5p) identifying HBV carriers with spontaneous or treatment achieved control of HBV infection. Three miRs of the mirB-index (hsa-miR-122 -5p, hsa-miR-99a-5p and hsa-miR-192-5p) showed significantly different expression patterns in IC and baseline and EOF patients without response to therapy and were upregulated in liver derived HBsAg particles. The remaining 2 miRs in the classifier were up-regulated in responders as compared to non-responders. Conclusions. A serum miRNA signature of miRNAs associated with both natural and therapy induced immune control of HBV infection was identified in chronic HBsAg carriers; the relative mirB-index classifier is worth of being validated in an independent set of samples and tested in clinical practice.

Disclosures:

Maurizia R. Brunetto - Speaking and Teaching: Roche, Gilead, Schering-Plough, Bristol-Myers Squibb, Abbott, Roche, Gilead, MSD, Novartis

Filippo Oliveri - Consulting: Bristol-Myers Squibb EMEA sarl Maria W. Teilum - Employment: Exiqon Thorarinn Blondal - Employment: Exiqon AS

Ferruccio Bonino - Advisory Committees or Review Panels: Roche, MSD; Speaking and Teaching: Gilead, Novartis, BMS

The following people have nothing to disclose: Daniela Cavallone, Francesco Moriconi, Piero Colombatto, Pietro Ciccorossi, Barbara Coco, Veronica Romag-noli, Carlotta Rastelli

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Serum cytokeratin 18 M30 as a potential marker of both biochemical and histological activity of chronic hepatitis B

Magdalena widerska, Jerzy Jaroszewicz, Anna Parfieniuk-Kow-erda, Magdalena Rogalska-Plonska, Anatol Panasiuk, Robert Flisiak;
Department of Infectious Diseases and Hepatology, Medical University of Bialystok, Bialystok, Poland

Background. Assessment of both biochemical and histopathological activity is important in selection of chronic hepatitis B (CHB) candidates for therapy. Currently used methods for histological advancement are either invasive (liver biopsy) or cost consuming (Fibroscan). Serum levels of liver specific cytokeratin 18 epiotope M30 (CK-18) have been associated with progression of chronic hepatitis C, while data on CK-18 in persistent HBV is limited. Therefore we aimed to analyze a potential usefulness of serum CK-18 measurement in a large cohort of patients with chronic HBV-infection not receiving anti-HBV therapy. Patients and methods. Studied cohort consisted of 195 HBeAg(-) patients (116 male, median age 33) with persistent HBV-infection, including 122 with normal and 73 with elevated ALT activity, among them 8 with HBV-related HCC. Liver biopsy results were available in 71 patients. Serum CK-18 levels were measured by ELISA (Peviva, Sweden). Results. Serum CK-18 levels were significantly higher in CHB patients with elevated ALT activity (413±50 vs 253±24 U/L, P=0.002) as well as in those with liver cirrhosis (679±222 vs 297±23, P=0.005). CK-18 showed a correlation with liver injury ALT (r=0.33, P<0.001), but also with platelets count (r=-0.26, P=0.002) and with APRI score (r=0.35, P<0.001), reflecting liver fibrosis. On the other hand, no associations with liver function or HBV viral load were noted. Most importantly, serum CK-18 was highly associated with histological advancement of liver fibrosis (ANOVA P=0.0003) and degree of inflammation (ANOVA P=0.01). Interestingly serum CK-18 was more than trifold lower in patients with mild (S1) vs moderate/severe (S2-S4) fibrosis (Scheuer S1: 177±34, S2: 613±161, S3: 956±360, S4: 676±222 IU/mL, P<0.001). ROC showed good discrimination ratio for patients with mild vs moderate/severe fibrosis (AUC 0.84, P<0.001), with 81% sensitivity and 80% specificity for CK-18 M30 value of 200 IU/mL. Conclusion. Based on a large cohort of CHB patients CK-1 8 serum levels reflect both biochemical and histological activity of disease, suggesting its potential usefulness as simple biomarker predicting the need for anti HBV-therapy in patients with replication of HBV. Serum CK-18 >200 IU/mL has good sensitivity and specificity in discriminating mild vs moderate/severe fibrosis in CHB, stressing its value in its non-invasive assessment.

Disclosures:

Jerzy Jaroszewicz - Speaking and Teaching: Roche, Gilead, Abbott, MSD, BMS

Robert Flisiak - Advisory Committees or Review Panels: Gilead, Merck, Roche, Bristol Myers Squibb, Janssen, Novartis, Achillion, Abbvie; Grant/Research Support: Roche, Bristol Myers Squibb, Janssen, Novartis, Gilead, Vertex, Merck; Speaking and Teaching: Janssen, Merck, Roche, Bristol Myers Squibb, Gilead

The following people have nothing to disclose: Magdalena widerska, Anna Parfieniuk-Kowerda, Magdalena Rogalska-Plonska, Anatol Panasiuk

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High expression of beta2-glycoprotein I is significantly associated with the earliest steps in hepatitis B virus infection

Yaming Liu, Zhongfeng Wang, Pujun Gao;
Department of Hepatology, First Hospital ofJilin University, Changchun, China

BACKGROUND&AIM: Human beta2-glycoprotein I(beta2-GPI) binds to recombinant hepatitis B surface antigen(rHBsAg).The affinity of beta2-GPI and HBsAg is strong in plasma and gly-cosylation of beta2-GPI has no effect on this conjugation.The aim of this study was to investigate that binding of beta2-GPI to HBsAg played a role in the earliest steps of hepatitis B virus(HBV) infection. METHODS: Expression of beta2-GPI was investigated by western-blot and real time qPCR in six different cell types: L02,SMMC-7721 ,HepG2,HepG2.2.15,HEK293T and CHO-K1 cell lines.Then,the cells were incubated with beta2-GPI and/or rHBsAg proteins,and tested the ability of HBsAg to bind to the cell surfaces by cell ELISA. Subse-quently,beta2-GPI and HBsAg were observed via confocal microscopy in HepG2.2.15 cells.Furtherly, to evaluate whether beta2-GPI expression was mediated by HBV and even HBV envelope proteins,we co-transfected HEK293T cells with beta2-GPI plasmid(VR-beta2-GPI-myc) and HBV or HBsAg expression vector(i.e.VR-LHBsAg-flag,VR-MHBsAg-flag and VR-SHBsAg-flag).Western-blot was carried out to examine the expression of beta2-GPI and Abbott chemiluminescence immunoassay was used to detect HBsAg level. RESULTS: Beta2-GPI up-regulation at mRNA and protein level was confirmed by real time qPCR and western-blot on HepG2.2.15 cells.We also discovered that beta2-GPI increased the ability of HBsAg to attach to human hepatocyte L02 and HepG2 cells and also non-hepatocyte HEK293T cells. Especially,beta2-GPI enhanced the binding of HBsAg to HEK293T cells.Further study revealed that beta2-GPI and HBsAg co-localized to the cytosol in HepG2.2.15 cells.Moreover,co-transfection of HBV or LHBsAg expression vector with beta2-GPI plasmid increased the expression of beta2-GPI in HEK293T cells. And the middle and the small surface protein had no effect on enhanced expression. CONCLUSIONS: HBV and also LHBsAg upregulate beta2-GPI expression,and binding to beta2-GPI is critical for HBsAg to attach to cell surfaces.These data demonstrate that beta2-GPI is significantly associated with the early steps in HBV entry and provide a new insight on the mechanisms of human hepadnavirus route of cell entry.

Disclosures:

The following people have nothing to disclose: Yaming Liu, Zhongfeng Wang, Pujun Gao

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Analysis of Novel Complex Structural Variants in Hepatitis B Virus

Kei Fujiwara, Noboru Shinkai, Shunsuke Nojiri, Mio Endo, Etsuko Iio, Takashi Joh;
Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

Background and Aims: Basic core promoter (BCP) mutations (nt.1762/1764) and pre-core mutation (nt.1896) are clinically and virologically very important mutations in hepatitis B virus (HBV), and they are associated with hepato-carcinogenesis, progression to liver cirrhosis, or fulminant hepatitis. These mutations are canonical point mutations. Recently, the notion of non-canonical complex structural variants (SV), including complex of canonical mutations has been reported. Complex SVs are defined by clustered breakpoints that arose through a single mutation but cannot be explained by one simple end-joining or recombination event. We previously reported a novel complex mutation that included an HNF1 binding site insertion/core promoter deletion and an internal tandem duplication, and provisionally named “replacement mutation” (RM) (1). We found that the RM is included in the complex SVs. Therefore, we investigated the prevalence of complex SVs in HBV, and furthermore, analyzed patterns, characteristics, and clinical significance of complex SVs in HBV. Methods: Complex SVs in HBV were investigated from international database and published articles. We analyzed the prevalence, positions, and various characteristics of complex SVs in HBV. We further investigated clinical significance of complex SVs in HBV. Results: From the international database and published articles, we found six strains of HBV with complex SVs. HBV genotype distribution was genotype A in two, genotype B in one, genotype D in one, and genotype E in two. All the complex SVs in HBV were observed in the region containing X open reading frame (ORF) and BCP. Patterns of complex SVs were deletion and duplication in two, deletion, insertion, and duplication in three, and deletion and insertion in one. Median deletion nucleotide length was 21 bases (range 8 -847 bases). In four strains with insertion, the median insertion nucleotide length was 23 bases (range 12-36 bases). In five strains with duplication, the median duplication nucleotide length was 31 bases (range 20-67 bases). Two were found in patients with hepato-cellular carcinoma, and other two were found in severe liver disease patients with post-renal transplantation. Conclusion: Novel genetic variants, complex SVs, were observed in six HBV strains. Complex SVs were observed in the region between X ORF and BCP. Complex SV in HBV was combination of canonical mutations. Though the cause and detailed mechanism still are not clear, it seems that this genetic variation is associated with severe liver disease, such as hepatocellular carcinoma or hepatic failure. (1) Fujiwara K, J Virology, 2005, 79(22), 14404.

Disclosures:

The following people have nothing to disclose: Kei Fujiwara, Noboru Shinkai, Shunsuke Nojiri, Mio Endo, Etsuko Iio, Takashi Joh

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Hepatitis delta virus RNA can be quantified in paraffin-embedded liver tissue and has a strong correlation with serum HDV-RNA

Maria Homs1,2, Maria Blasi1,2, Maria Teresa Salcedo4, Francisco Rodriguez-Frias1,2, David Tabernero1,2, Marc Luetgehetmann5, Rafael Esteban2,3, Maura Dandri5, Maria Buti2,3;
1Biochemistry, Vall d'Hebron Hospital, Barcelona, Spain; 2CIBERehd, Barcelona, Spain; 3Hepatology, Vall d'Hebron Hospital, Barcelona, Spain; 4Pathology, Vall d'Hebron Hospital, Barcelona, Spain; 5Internal Medicine, University Medical Center Hamburg, Hamburg, Germany

Background and aim In clinical practice, serum HDV RNA level is used as a marker of viral replication. However, knowledge about its relationship to intrahepatic HDV markers is scant and there is no available data on the stability of HDV RNA in formalin-fixed paraffin-embedded liver samples (FFPE-LS). The aim of this study was to determine HDV RNA in FFPE-LS using a new technique and to compare the findings with HDV RNA levels in serum. Material and Methods Among 40 untreated CHD patients, 13 had FFPE-LS and a simultaneous serum sample testing positive for HDV RNA by qualitative assays. A patient with anti-HDV who tested negative for serum HDV RNA was also included. FFPE-LS were obtained between 1999 and 2012. Serum and liver HDV RNA were analyzed by quantitative realtime PCR. A new HDV RNA standard was used, and the sensitivity of the method was 10E3 to 10E6 copies HDV/uL. Results Liver HDV RNA was detected in 13/13 CHD patients (Table). The median liver HDV RNA level was 1.1×10E7 copies/mg (range 3.85×10E4-9.2×10E8). Retested serum HDV RNA yielded a median of 3.5×10E6 copies/uL (range 3.85×1 0E4-9.2×10E8). Serum and liver HDV RNA presented a good correlation (R2=0.89). HDV RNA was not detected in the liver of the anti-HDV-positive patient testing negative for HDV RNA. No statistically significant differences were found between histology findings and quantification of HBV and HDV in serum and liver. Conclusions HDV RNA is stable in FFPE-LS for more than 10 years and can be quantified by real-time PCR. A good correlation was found between intrahepatic and serum HDV RNA, suggesting that serum HDV RNA may be an excellent marker for viral replication in untreated patients. Further studies looking at the effect of therapy on intrahepatic HDV RNA loads are needed to better evaluate this correlation.

CHD PtSERUMLIVER
HBV DNA (IU/mL)HBeAgALTHDV RNA (copies/uL)IshakHDV RNA (copies/mg)
11,20E+03N2044,50E+0511,99E+08
21,70E+03N732,28E+1019,20E+08
31,50E+05N946,00E+0631,12E+07
4<20N1303,15E+0731,65E+08
55,60E+03N2235,33E+0738,18E+06
61,30E+05N2031,70E+0648,02E+07
71,70E+03N1551,70E+0757,93E+05
8<20N446,34E+0564,08E+05
91,30E+06N474,05E+0562,90E+06
101,50E+04N707,46E+0862,32E+07
11l,60E+07p571,02E+0462,00E+05
121,10E+05N1251,20E+0463.85E+04
13<20P493.49E+0662.21E+08

Disclosures:

Rafael Esteban - Speaking and Teaching: MSD, BMS, Novartis, Gilead, Glaxo, MSD, BMS, Novartis, Gilead, Glaxo, Janssen

Maria Buti - Advisory Committees or Review Panels: Gilead, Janssen, Vertex; Grant/Research Support: Gilead, Janssen; Speaking and Teaching: Gilead, Janssen, Vertex, Novartis

The following people have nothing to disclose: Maria Homs, Maria Blasi, Maria Teresa Salcedo, Francisco Rodriguez-Frias, David Tabernero, Marc Luetgehetmann, Maura Dandri

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The expression of miR-125b-5p is increased in serum of patients with chronic hepatitis B infection, and miR- 125b-5p interferes with the detection of hepatitis B virus surface antigen

Masashi Ninomiya1, Yasuteru Kondo1, Takayuki Kogure1, Eiji Kakazu2, Osamu Kimura2, Tatsuki Morosawa1, Tomoaki Iwata1, Yasuyuki Fujisaka1, Tooru Shimosegawa1;
1Tohoku University Hospital, Division of Gastroenterology, Sendai, Japan; 2Tohoku Medical Megabank Organization, Department of Community Medical Supports, Sendai, Japan

Background: MicroRNAs are small endogenous RNA molecules with specific expression patterns for some diseases. Some miR-NAs were reported to be differentially expressed in hepatitis B virus (HBV) serum. This study examines whether the serum expression levels of miRNAs by deep sequencing can serve as biomarkers and clarify the mechanism of miRNA with chronic hepatitis B (CH-B) infection. Methods: We detected circulating miRNAs using an Illumina deep sequencer. 20 cases of CH-B were enrolled, and 30 cases of CH-C and healthy subjects as a control. 1) Short read sequences of 32-mer were generated. The sequence reads were mapped with miRBase. ANOVA was applied to extract differentially expressed miRNAs among the three groups. Adjustment of the p-value by multiple comparisons was performed by calculating FDR. 2) The validation study of differentially expressed miRNA was conducted by qRT-PCR with TaqMan MicroRNA assay. 3) Computer software RNAhybrid 2.2 was used to scan the genome of HBV for the presence of target sites for the differentially expressed miRNA. 4) To investigate interfering activity of miRNA in cultured hepatic cells, HepG2 and Huh-7 cells were transfected with the luciferase-based reporter plasmid psiCheck-2 containing the HBV genomic segment. Then, to analyze the scilencing effect of miRNA, HepG2 cells were transfected with the psiCheck-2 containing the wild-type HBV clone along with miRNA mimics or miRNA inhibitors. Result: We obtained about 9.0 million 32-mer short reads on average per sample, and mapping rates to miRBase were 15.5%. In the statistical analysis, the p-value and the expression levels of 110 miRNAs were found to be differentially expressed in the 3 groups. 16 miRNAs were up-regulated in CH-B patients with miR-3591-5p being the most enriched. To set up the condition of miRNA-mRNA pairings with perfect matching of the seed region, RNAhybrid 2.2 analysis predicted that human hepatic cells might use 8 out of 16 miRNAs to down regulate the expression of HBV P and S genes. Then, eight HBV genomic segments containing putative target sites for human miRNAs were separately cloned in the psiCheck-2 vector. The HBV genomic segment predicted to be targeted by miR-125b-5p inhibited remarkably the expression of the reporter in HepG2 and Huh-7 cells. Transfection of miR-125b-5p mimic in HepG2 cells increased the reporter silencing effect. Moreover, transfection of the inhibitor in the cells reduced the reporter silencing activity. Conclusion: We demonstrated that 16 miRNAs were up-regulated in patients with HBV chronic infection. miR-125b-5p, one of the 16 miRNAs, may be responsible for the silencing effect on the HBV genome segment.

Disclosures:

The following people have nothing to disclose: Masashi Ninomiya, Yasuteru Kondo, Takayuki Kogure, Eiji Kakazu, Osamu Kimura, Tatsuki Morosawa, Tomoaki Iwata, Yasuyuki Fujisaka, Tooru Shimosegawa

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Reapparence of HBV DNA with changes in HBV genome after stopping tenofovir in HBeAg-negative patients with complete suppression of HBV DNA for more than 7 years

Maria Buti1,2, David Tabernero1,3, Francisco Rodriguez-Frias1,3, Rosario Casillas2, Josep Gregori2,4, Carolina Gonzalez3, Irene Bel-monte Mula3, Maria Homs1,3, Maria Blasi1,3, Josep Quer1,2, Leonardo Nieto5, Silvia Camos3, Andrea Caballero3, Rafael Esteban1,2;
1Centro de Investigación Biomédica en Red de Enfer-medades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; 2Hepatology Department, University Hospital Vall d'Hebron, Uni-versitat Autònoma de Barcelona, Barcelona, Spain; 3Biochemistry Department, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain; 4Statistics Department, Universitat de Barcelona, Barcelona, Spain; 5Microbiology Department, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain

Background and Aims: Long-term treatment with tenofovir (TDF) is effective in suppressing viral replication in HBeAg-ve and +ve chronic hepatitis B (CHB) patients, but loss of HBsAg is rare. The effect of stopping TDF in a CHB patient with long-term HBV-DNA suppression or HBsAg loss is uncertain. Methods: Among 25 TDF-treated CHB patients with persistently undetectable HBV-DNA for a median time of 7.46 years, therapy was stopped in 7 prospectively followed-up patients. Hepatitis B virus (HBV) quasispecies between codons rt163-rt278 was studied by ultra-deep pyrosequencing (UDPS, GS-FLX/Junior, Roche) in patients in whom amplification of HBV-DNA at baseline (BA) and after TDF discontinuation (Post) was possible. Results: At BA, median HBV-DNA and HBsAg levels (qHBsAg) were 5.61 (4.82-8.59) and 3.33 (1.95-5.31) logIU/mL respectively, median age 54.81 (43.07-62.86) years, HBV genotype A 1, D 4 and F 1. At end of treatment, all patients were HBeAg-ve, HBV-DNA undetectable, and median qHBsAg was 2.99 (2.02-3.70) logIU/mL. After stopping TDF fora median of 7.29 weeks, no patient cleared HBsAg and HBV-DNA was detectable in all cases (median, 4.52 [1.75-5.34] logIU/mL) with no changes in ALT or qHBsAg. UDPS analysis of HBV quasispecies in 3 patients showed no changes in the master sequence between BA and Post despite 7 years' complete suppression of HBV replication, but low-frequency variants between positions rtG21 0 and rtS238 were detected at Post: 1 patient showed variants rtV214A (0.26%), rtQ215S (0.99%), rtL217R (0.99) and rtN238T (0.71%) related with low sensitivity to adefovir (ADV), and the other two displayed variants, such as rtA211T (0.57%), rtQ215H (3.91%), rtA219S (13.32%), rtA223T (0.71) and rtA223S (0.46%), likely related to ADV. Conclusions: After more than 7 years' complete viral suppression and despite relative reductions in HBsAg level, none of the 7 HBeAg-ve patients cleared HBsAg after discontinuing TDF. After TDF, there is an increase in HBV reverse transcriptase variability mainly due to variants with low sensitivity to ADV, which seems to reflect a cross-resistance mechanism between ADV and TDF. This phenomenon could reflect evolutive pressure of TDF over the cccDNA reservoir during suppression of detectable viral replication. Study funded by Instituto de Salud Carlos III, grant PI 1 1/01973, co-financed by the European Regional Development Fund (ERDF).

Disclosures:

Maria Buti - Advisory Committees or Review Panels: Boerhinger Inghelm, Boer-hinger Inghelm; Speaking and Teaching: MSD, Bristol-Myers Squibb, Novartis, Gilead, Janssen, MSD, Bristol-Myers Squibb, Novartis, Gilead, Janssen

Rafael Esteban - Speaking and Teaching: MSD, BMS, Novartis, Gilead, Glaxo, MSD, BMS, Novartis, Gilead, Glaxo, Janssen

The following people have nothing to disclose: David Tabernero, Francisco Rodriguez-Frias, Rosario Casillas, Josep Gregori, Carolina Gonzalez, Irene Bel-monte Mula, Maria Homs, Maria Blasi, Josep Quer, Leonardo Nieto, Silvia Camos, Andrea Caballero

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Hepatocellular carcinomas replicating hepatitis B DNA have a low risk of vascular invasion and specific tran-scriptional phenotype

Christophe Desterke2, Guillaume Fallot2, Boris Halgand4, Lise Riviere3, Christine Neuveut3, Marie-Annick Buendia2, Didier Samuel2, Cyrille Feray1;
1INSERM 955, Hopital Henri Mondor, Creteil, France; 2INSERM 785, Hopital Paul Brousse, Villejuif, France; 3CNRS URA 3015, Institut Pasteur, Paris, France; 4EA 4271, Hotel Dieu, Nantes, France

We recently reported that despite antiviral treatment, HBV ccc DNA persists in hepatocellular carcinoma (HCC) and is correlated to the absence of vascular invasion. The tumoral (T) and non-tumoral(NT) liver transcriptomes are now available. Patients and methods. 63 HBsAg-positive patients ( anti-HCV, anti-HDV and anti-HIV negative), resected for HCC. Most were cirrhotic (82% of cases) with low viremia under antiviral therapy (79%). Replication in T and NT was assessed by detection of full length HBV DNA, quantification of HBV ccc DNA or of total HBV DNA. Agilent micro arrays were used for transcriptomic experiments. Results. HBV ccc DNA was strongly correlated with transcriptome analysis. Principal component analysis (PCA) comparing differential gene expression between T and NT demonstrated in the first dimension, a clustering according to the tumoral vascular invasion and in the second dimension, a clustering according to the detection of HBV ccc DNA in T. Fatty acid metabolism, complement and coagulation, retinol metabolism and PPAR pathways were upregulated in absence of vascular invasion. Vascular smooth muscle contraction, ECM-receptor interaction and Gap junction pathways were upregulated in case of tumoral replication of HBV. Comparisons with other data on HCC showed in HCC not replicating HBV, an enrichment of genes related to vascular invasion. Conclusion. HBV replication in the HCC correlated to unfrequent vascular invasion, better prognosis and specific pathways.

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Disclosures:

Didier Samuel - Consulting: Astellas, MSD, BMS, Roche, Novartis, Gilead, LFB, Janssen-Cilag

The following people have nothing to disclose: Christophe Desterke, Guillaume Fallot, Boris Halgand, Lise Riviere, Christine Neuveut, Marie-Annick Buendia, Cyrille Feray

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Antiviral Activity of Flavocoxid against Hepatitis B Virus

Teresa Pollicino1, Cristina Musolino2, Alessandra Bitto2, Giovanni Raimondo2, Francesco Squadrito2, Domenica Altavilla1;
1Pediatric, Gynecologic, Microbiological and Biomedical Sciences, University Hospital of Messina, Messina, Italy; 2Clinical and Experimental Medicine, University Hospital of Messina, Messina, Italy

Recent evidence has shown that flavonoids may exert antiviral activities (i.e., against HIV, HSV, influenza virus and HCV) by targeting virus entry, reverse transcription or gene expression. Aims. To investigate the potential antiviral effect of Flavocoxid (containing the natural flavonoids, baicalin and catechin) against HBV and to verify whether the antiviral control exerted by Entecavir (ETV) in HBV-replicating cells may be enhanced by its use in combination with FLAV. Methods. HepG2 cells were transfected with linear wild-type HBV genomes. HBV replicating cells were treated with different dosages of Flavocoxid to determine the drug inhibitory concentrations (IC50). Treatment with Flavocoxid or ETV or with drugs combination started 3 hours after transfection and was renewed every other day for 7 days. Total HBV replicative intermediates, viral transcripts and cccDNA levels were evaluated in untreated and treated HepG2 cells by quantitative real-time PCR, Southern and Northern blots experiments. To analyse the epigenetic modulation of HBV cccDNA the cccDNA-ChIP assay was applied to untreated and treated cells Results. The analysis of HBV transcription/replication in the presence or absence of Flavocoxid enabled to determine that IC50 for the drug was 75 μg/mL. HBV replicative intermediates in cell treated with ETV, FLAV, or FLAV + ETV were decreased by 47%, 68%, and 83%, respectively, compared with untreated HBV-replicating HepG2 cells. After exposure to ETV, FLAV or FLAV + ETV, Northern blot analysis showed that HBV pregenomic RNA levels were decreased by 31%, 87%, and 85% respectively, compared with untreated HBV-replicating cells. Levels of HBV cccDNA in the nuclei of cells treated with FLAV or FLAV + ETV were reduced by 34% and 23%, respectively, whereas treatment with ETV, failed to decrease cccDNA. HBsAg amount was reduced by 20%, 75%, and 60% in the supernatant of cells treated with ETV, FLAV, and FLAV + ETV, respectively, compared with untreated cells. Epigenetic analysis showed that cccDNA-bound H4 histones were hypoacetylated in cells treated with ETV, FLAV, or FLAV + ETV and that the recruitment of HDAC1 histone deacetylase was increased at higher levels both in FLAV and FLAV + ETV treated cells. The binding to the cccDNA of NFkB transcriptional regulator was strongly reduced in all treated cells Conclusions. The results of our study demonstrate that Flavocoxid: (a) is capable to inhibit HBV replication, (b) exerts its antiviral activity against HBV at multiple levels and (c) acts synergistically with ETV in a cell-based HBV replication system.

Disclosures:

Teresa Pollicino - Speaking and Teaching: Gilead

Giovanni Raimondo - Speaking and Teaching: BMS, Gilead, Roche, Merck, Janssen

The following people have nothing to disclose: Cristina Musolino, Alessandra Bitto, Francesco Squadrito, Domenica Altavilla

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Longitudinal Assessment of Liver Fibrosis By Liver Stiffness Measurements Using Transient Elastography in Chronic Hepatitis B Patients

Heng Chi1, Bettina E. Hansen1, Erik H. Buster1, RobertJ. de Knegt1, Harry L. Janssen1,2;
1Department of Gastroenterology and Hepa-tology, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands; 2Liver Clinic, Toronto Western and General Hospital, University Health Network, Toronto, ON, Canada

Introduction: Limited evidence supports the use of liver stiffness measurement (LSM) to monitor liver fibrosis in chronic hepatitis B (CHB) longitudinally. We aimed to investigate the changes of LSM values over time and the applicability of LSM to monitor liver fibrosis in patients with longitudinal LSMs and liver biopsies. Methods: We retrospectively studied CHB patients with a paired liver biopsy and LSM, and at least one follow-up LSM between 2005 and 2013. Liver biopsies had to be ≥15 mm in length and LSMs had to be valid (IQR/M ratio ≤0.30, ≥10 valid measurements and success rate ≥60%). The LSMs were performed within 3 months of the liver biopsy. We excluded patients with HCC, hepatic decompensation, concomitant liver diseases, liver transplant and HCV, HDV, HIV co-infections. We defined histologic progression as any increase in the METAVIR score in the follow-up biopsy. Results: We analyzed 124 patients with a mean follow-up of 3.6 years. 85 (68.5%) patients were treated during follow-up, mostly (79/85) with oral antivirals. Treated patients showed significantly decreasing LSMs per year (p<0.001), while non-treated had no change in LSMs (p=0.841).The LSM decrease was greater in treated than non-treated patients: -1.4 vs. -0.1 kPa/year, p=0.017. Among patients with normal ALT (25/124) at both baseline and follow-up, LSM decreased over time from 7.0 to 5.6 kPa (p=0.012). Among these patients, a significant LSM decline was observed in those with antiviral therapy (7.3 vs. 5.6 kPa, p=0.042), but not in those without (6.4 vs. 5.5 kPa, p=0.15). 28 patients had at least two paired liver biopsies. 18 (64%) patients were treated during follow-up. Five (18%) patients had histologic progression. Patients without histologic progression had decreasing LSMs (p<0.001), whereas the LSM remained stable in patients with histologic progression (p=0.367). The LSM change was different between patients with and without histologic progression (0.5 vs. -1.4 kPa/year, p=0.019). Patients with decreasing LSMs had decreasing METAVIR scores, while those with increasing LSMs had increasing METAVIR scores (-0.35 vs. 0.25, p=0.045). None of the treated patients had histologic progression, while five non-treated patients (50%) had progression (p=0.003). Conclusions: In this longitudinal study, CHB patients with repeatedly normal ALT levels had decreasing LSMs over time, suggesting disease remission and fibrosis regression. Interestingly, this beneficial effect was observed in treated patients only. Fibrosis progression assessed by longitudinal liver biopsies was associated with stable LSMs at follow-up. LSM may be a useful instrument to monitor liver fibrosis during follow-up.

Disclosures:

Robert J. de Knegt - Advisory Committees or Review Panels: MSD, Roche, Norgine, Janssen Cilag; Grant/Research Support: Gilead, MSD, Roche, Janssen Cilag, BMS; Speaking and Teaching: Gilead, MSD, Roche, Janssen Cilag

Harry L. Janssen - Consulting: Abbott, Bristol Myers Squibb, Debio, Gilead Sciences, Merck, Medtronic, Novartis, Roche, Santaris; Grant/Research Support: Anadys, Bristol Myers Squibb, Gilead Sciences, Innogenetics, Kirin, Merck, Medtronic, Novartis, Roche, Santaris

The following people have nothing to disclose: Heng Chi, Bettina E. Hansen, Erik H. Buster

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Inactive hepatitis B carriers are prone to HBsAg loss rather than reactivation: a 5-year prospective follow-up study

Onur Keskin, Mustafa Yakut, Cagdas Kalkan, Senem C. Karatayli, Ersin Karatayli, Ramazan Idilman, A Mithat Bozdayi, Cihan Yurdaydin;
Gastroenterology, Ankara University School of Medicine, Ankara, Turkey

The HBsAg inactive carrier (IC) stage is considered to have a good prognosis. However, this knowledge is based mostly on retrospective data and insensitive HBV DNA assays. The aim of the current study is to better characterize the IC stage through a well characterized prospectively followed single center cohort. Of the initial cohort of 129 patients diagnosed as ICs, at year 5, 22 had been excluded (19 lost to follow-up (FU) and 3 anti HDV positive). The rest of 107 (64 f,43 m, median age 48 [19-74]) were prospectively followed with monthly serum ALT, HBVDNA determinations for the first year and 3 monthly thereafter. Quantitative serum HBsAg were determined q6 months. HBVDNA was determined with TaqMan PCR and HBsAg with Architect assay (Abbott). HBV DNA, ALT and HBsAg levels were lower in ICs compared to control HBe Ag negative CHB patients (p<0.0001 and p<0.001 and <0.001 respectively). AUROC for HBsAg was 0,86 (95%CI: 0.80-0.92). A cut-off of 3705 IU/ml revealed sensitivity of 73% and specificity of 84% for diagnosing the IC. In 92 patients with liver biopsy, fibrosis score was 0 in 77 and necroinflammatory score was <6 in 82. Patients were divided into two groups: stable group (HBVDNA continiously <2000 IU/ml, n=64) and unstable groups (n:43) with HBV DNA fluctuations between < and > 2000 IU/mL based on monthly HBVDNA determinations in the first year of FU. Gender, HAI, fibrosis score, BMI and ALT levels were similar in the stable and unstable groups. Stable group patients had lower baseline HBsAg levels compared to those with unstable HBVDNA (967±1862 IU/mLvs3803±4481 p<0.001). 4 patients developed reactivation. All of them were in unstable group. Majority of unstable patients (65%) continued to have fluctuating HBV DNA levels whereas 35% became stable carriers. HBsAg clearance occurred in 15 patients (14 stable and 1 in the unstable group) during 60 months of FU. Cumulative probability of HBsAg seroconversion was 1.8%, 6.1%, 10.8% and 14.5% at the end of 2, 3,4 and 5 year FU respectively. Baseline HBsAg levels in patients who developed seroconversion were lower compared to the rest of patients (20[0.1-280] vs 884 [1.6-17360], p<0.0001). 13 of 15 patients who developed HBsAg clearance had baseline HBsAg < 60IU/mL. Conclusion:1 .Quantitative HBsAg should be considered as an adjunct for the diagnosis of the IC state. 2. Distinction is needed between a “stable inactive carrier” and an unstable carrier. The former group may be the true IC whereas the latter group may evolve to the true IC state. 3. The IC is unlikely to reactivate but may be more likely to clear HBsAg. HBsAg levels <100 IU/mL may herald HBsAg clearance.

Disclosures:

Cihan Yurdaydin - Advisory Committees or Review Panels: Janssen, Roche, Merck, Gilead

The following people have nothing to disclose: Onur Keskin, Mustafa Yakut, Cagdas Kalkan, Senem C. Karatayli, Ersin Karatayli, Ramazan Idilman, A Mithat Bozdayi

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Genotypic and phenotypic characteristics of HBV entecavir resistance in Chinese patients

Yan Liu, Zhihui Xu, Liming Liu, Xiaodong Li, Jiuzeng Dai, Zengtao Yao, Li Chen, Siyu Bai, Shaojie Xin, Dongping Xu;
Institute of Infectious Diseases and Liver Failure Research Center, Beijing 302 Hospital, Beijing, China

Background/Aim: The study aimed to investigate HBV ETV resistance profile of Chinese patients in clinical practice. Methods: Serum samples from 18,419 patients collected from July 2007 to June 2012 in Beijing 302 Hospital were screened. Genotypic resistance was detected by direct PCR sequencing and confirmed by clonal sequencing if necessary. Phenotypic resistance was analyzed by measuring HBV replication capacity under drug pressure in HepG2 cells. Results: ETV-resistant mutations were detected from 646 samples and the incidence had been increased in the past five years (1.91%, 2.23%, 3.54%, 3.96%, and 4.77%). Mutational pattern analysis showed that concomitant with rtM204V/rtM204I, mutations at rt1 84, rt202, rt250, and two of rt1 84/rt202/rt250 sites were 57.4%, 22.4% and 14.1%, and 6.1%, respectively (Figure 1). Nineteen percent (123/646) of ETV-resistant samples harbored rtM204I±L80I/L180M-based pattern rather than rtL180M+M204V-based pattern. Among them 70 samples only harbored two resistant mutations (rtM204I+T184I × 39, rtM204I+M250L × 26, rtM204I+M250I × 5). All ETV-resistant strains exhibited varied lower natural replication capacity compared to wild-type strains in vitro. ETV susceptibility of rtL180M+M204V-based ETV-resistant mutants was usually lower than rtM204I-based ETV-resistant mutants. Replication of all tested ETV-resistant strains could be effectively suppressed by tenofovir and adefovir in vitro. In clinical practice, adding-on adefovir was more efficacious than switching-to adefovir as a rescue therapy for ETV-resistant patients. Conclusions: The occurrence of ETV-resistant HBV infection kept growing in the past five years in Chinese patients. ETV-resistant mutational pattern diversified and rtM204I-containing ETV-resistant strains occupied near 1/5 of the patients. ETV-resistant strains could be suppressed by tenofovir or adefovir in vitro; and currently, adding-on adefovir was a practical rescue therapy in clinical practice in China.

image

Figure 1. Incidence of entecavir-resistant mutations in recent five years

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Disclosures:

The following people have nothing to disclose: Yan Liu, Zhihui Xu, Liming Liu, Xiaodong Li, Jiuzeng Dai, Zengtao Yao, Li Chen, Siyu Bai, Shaojie Xin, Dongping Xu

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ENPP2 is an Intracellular Antiviral Factor Against Hepatitis B Virus

Min Yang1,2, Hong Li1,2, Xiwei Wang1,2, Hongmin Zhang1,2, Yix-uan Yang1,2, Huaidong Hu1,2, Peng Hu1,2, Dazhi Zhang1,2, Hong Ren1,2;
1Department of Infectious Diseases, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China; 2Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China

Background: Hepatitis B virus (HBV) infection plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Therefore, it is of prime importance to understand the mechanisms of HBV-host interactions during malignant transformation in chronic hepatitis B (CHB) infection to identify novel therapeutic anti-HBV targets. Methods: We utilized Isobaric Tags for Relative and Absolute Quantitation(iTRAQ) to identify the secretory proteins that are differentially expressed in the HBV DNA-transfected HepG2.2.15 cell line and its parental HepG2 cell line. IHC was employed to assess the clinical relevance of the observations. Small interfering (si)RNA-based silencing transfection methods were carried out to study the function of ENPP2. Results: Totally, 827 unique proteins were detected and 145 of them were identified as differentially expressed in HepG2.2.15 cell line compared with that of its parental HepG2 cell line. Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 precursor (ENPP2) is one of the most significantly up-regulated secretory proteins associated with HBV replication. This differential expression of ENPP2 was further validated by real-time quantitative RT-PCR, Western Blot and immunohistochemical analysis. To study the function of ENPP2, we knockdown ENPP2 expression in HepG2.2.15 cell line by RNA interference. SiRNA-mediated ENPP2 silencing resulted in a significant increase of HBV titer by nearly 3-fold, which is concomitant with elevated levels of hepatitis B surface antigen and e antigen in the culture medium. The affect of ENPP2 on HBV titer is associated with IFN signaling pathway, which is determined by real-time quantitative RT-PCR. Conclusion: In conclusion, the present study demonstrates for the first time that ENPP2 functions as an endogenous anti-HBV factor during HBV infection via the IFN signaling pathway. It may provide valuable novel insights into the underlying mechanisms of HBV infection. Acknowledgements: This research was supported by the National Natural Science Foundation of China (81171560, 30930082, 81171561, 30972584), the National Science and Technology Major Project of China (2008ZX10002-006, 2012ZX1002007001, 2011ZX09302005, 2012ZX09303001-001, 2012ZX1 0002003), The National High Technology Research and Development Program of China(2011AA020111), the Key Project of Chongqing Science and Technology Commission (cstc2012gg-yyjsB10007), the Chongqing Natural Science Foundation(cstc2011jjA1 0025), the Medical Research Fund by Chongqing Municipal Health Bureau (2009-1-71).

Disclosures:

The following people have nothing to disclose: Min Yang, Hong Li, Xiwei Wang, Hongmin Zhang, Yixuan Yang, Huaidong Hu, Peng Hu, Dazhi Zhang, Hong Ren

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Hepatitis D virus infection in Victoria, Australia: changing epidemiology and evidence of gaps in clinical practice

Jennifer H. MacLachlan1,2, Benjamin C. Cowie1,2;
1WHO Regional Reference Laboratory for Hepatitis B, Victorian Infectious Diseases Reference Laboratory, Melbourne, VIC, Australia; 2Department of Medicine, University of Melbourne, Melbourne, VIC, Australia

Purpose: This study investigated whether the evolving global epidemiology of hepatitis D virus (HDV) is reflected in Australia, and analysed diagnostic testing and monitoring for HDV in people living with chronic hepatitis B. Methods: Data regarding HDV diagnoses in Victoria during 2000-2009 were obtained from health department notifiable diseases surveillance and public health laboratory testing records. Notifications data were analysed to determine risk factors and demographics of HDV diagnoses, while laboratory records for serological and nucleic acid testing were used to determine practices of screening and follow-up of patients. During the period examined, 2,604 HDV serological tests were conducted on 2,327 patients residing in Victoria; of these, 110 patients (4.7%) tested positive for HDV antibody and/or antigen. The number of patients positive and the number of tests conducted increased over time, more than doubling between 2004 and 2009. Of those patients who tested antibody positive, less than half (44 patients, 40.0%) were subsequently evaluated by qualitative HDV PCR, and the majority of those who were (29 patients, 70.5%) tested HDV RNA positive. Surveillance data showed reasonable concordance with laboratory diagnoses, with 88 notifications for new diagnoses of HDV reported to the Victorian Department of Health as required by public health regulations over this period (representing 80% of patients with positive test results). An increasing proportion of notifications during the time period were from Australians born overseas, particularly in Asia and Africa, while there was a concurrent decrease in the number reporting injecting drug use as a risk factor, from 47.5% of notifications in 2000-2004 to 23.4% during 2005-2009 (p=0.02). Conclusion: The proportion positive observed (4.7%) corresponds with global estimates of 5% HDV prevalence in people living with chronic hepatitis B, while the trend of risk factors shifting from injecting drug use towards migrants born in endemic regions reflects the pattern seen recently in a number of other countries. Increased testing for HDV in Victoria over the last decade has resulted in an escalating number of HDV diagnoses and highlights the potential for undiagnosed HDV infection in those living with chronic hepatitis B; however gaps still remain in the appropriate follow-up of patients known to be infected, in order to inform effective clinical management.

Disclosures:

The following people have nothing to disclose: Jennifer H. MacLachlan, Benjamin C. Cowie

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Hepatitis B Virus Surface Immune Escape Variants by Ultra-deep Pyrosequencing and Variations during Treatment with Nucleoside Analogs

David Tabernero1,2, Francisco Rodriguez-Frias1,2, Rosario Casil-las3, Josep Gregori3,4, Irene Belmonte Mula2, Maria Homs1,2, Maria Blasi1,2, Josep Quer1,3, Leonardo Nieto5, Silvia Camos2, Andrea Caballero2, Carolina Gonzalez2, Rafael Esteban1,3, Maria Buti1,3;
1Centro de Investigación Biomédica en Red de Enfer-medades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; 2Biochemistry Department, University Hospital Vall d'Hebron, Uni-versitat Autònoma de Barcelona, Barcelona, Spain; 3Hepatology Department, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain; 4Statistics Department, Universitat de Barcelona, Barcelona, Spain; 5Microbiology Department, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain

Background and Aims: Selection of amino acid (AA) changes in hepatitis B virus (HBV) surface (S) proteins may be associated with immune response or antiviral treatment. Frequencies of AA changes (FreqAA) in S proteins during treatment with nucleoside analogs (Nucs) were assessed by ultra-deep pyrosequencing (UDPS). Methods: FreqAA in S gene codons, s92-s200, in 2 serum samples from 20 patients with chronic HBV and lamivu-dine resistance (LMVr) were analyzed by UDPS (GS-FLX/Junior, Roche). Frequency of non-polymorphic AA changes and variations in mean FreqAA>1% between baseline (BA) and LMVr were considered. Results: 595 371 sequences analyzed. Overall, a FreqAA increase was observed in 11 codons, five overlapped with LMV resistance positions and related to immune escape (figure). Decreased FreqAA was observed in sQ101 and sW1 82, mainly due to decreased sW1 82stop (3.12% to 0.38%), which overlaps with rtV1 91I associated with LMVr (figure). In “a” determinant, 45% of patients showed changes at BA and 40% at LMVr, mainly between sG1 30 and sP1 35 (no cases of sG145R). Immune escape mutations were detected in 5 cases at BA, but could have been detected by direct sequencing (sM133T, 41.58%) in only 1 case. Conclusions: The increased frequency of AA changes in S gene unrelated to LMV resistance suggests enhanced immune escape; further studies are needed to clarify whether this is related to Nuc pressure or natural history of the disease. In “a” determinant, immune escape variants other than the common sG1 45R were detected, suggesting a different pattern in our area. Study funded by Instituto de Salud Carlos III, grant PI 11/01973, cofinanced by the European Regional Development Fund (ERDF).

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Disclosures:

Rafael Esteban - Speaking and Teaching: MSD, BMS, Novartis, Gilead, Glaxo, MSD, BMS, Novartis, Gilead, Glaxo, Janssen

Maria Buti - Advisory Committees or Review Panels: Boerhinger Inghelm, Boer-hinger Inghelm; Speaking and Teaching: MSD, Bristol-Myers Squibb, Novartis, Gilead, Janssen, MSD, Bristol-Myers Squibb, Novartis, Gilead, Janssen

The following people have nothing to disclose: David Tabernero, Francisco Rodriguez-Frias, Rosario Casillas, Josep Gregori, Irene Belmonte Mula, Maria Homs, Maria Blasi, Josep Quer, Leonardo Nieto, Silvia Camos, Andrea Caballero, Carolina Gonzalez

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Hepatitis B surface antigen levels after hepatitis B e-antigen seroclearance: A longitudinal follow-up study

James Fung, Wai-Kay Seto, Danny Wong, Ching-Lung Lai, Man-Fung Yuen;
Medicine, University of Hong Kong, Hong Kong, China

Background: The role of quantitative hepatitis B surface antigen (HBsAg) after hepatitis B e-antigen (HBeAg) seroclearance is not well defined Aim: To determine the role of HBsAg levels in predicting significant viremia and hepatitis flares after HBeAg seroclearance, and to describe the trend of HBsAg levels and its correlation with HBV DNA in a longitudinal study. Methods: 228 chronic hepatitis B patients with spontaneous HBeAg seroclearance were included, with a minimum of 5 years follow-up after seroclearance. Patients were followed up regularly at 3-6 monthly intervals with routine liver biochemistry and hepatitis B serology. Levels of HBV DNA and HBsAg were measured at yearly intervals for up to 5 years after HBeAg seroclearance. Results: The median age at the time of seroclearance was 38 years (range, 14-77), with a similar male:female ratio (51%:49%). Of the 228 patients, 218 (95.6%) had evidence of seroconversion with detectable anti-HBe, with the remaining 1 0 (4.4%) patients remaining anti-HBe negative. The median log HBsAg and HBV DNA level after HBeAg seroclearance was 3.52 IU/mL and 4.13 IU/mL respectively, with no significant correlation (p=0.572). There was a gradual but significant decline in HBV DNA with increasing time from HBeAg seroclearance. The HBV DNA at HBeAg seroclearance was 4.13 log IU/mL, compared with 3.12 log IU/mL after 5 years (p<0.001). In contrast, the level of HBsAg remained consistent throughout each time-point after HBeAg seroclearance in non-treated patients. At the time of HBeAg seroconversion, the median HBsAg level was 3.52 log IU/mL, and this was comparable to the level of 3.50 log IU/mL (p=0.991) at 5 years. Hepatitis B flares occurred in 103 (45.2%) patients. Patients who developed hepatitic flares compared with those without hepatitic flares were older (39 vs 35 years, p=0.002), had a higher HBV DNA at the time of HBeAg seroclearance (4.63 vs 3.75 log IU/mL, p=<0.001), and more likely to be males (59.8% vs 29.7%, p<0.001) respectively. There was no difference in HBsAg levels between those with and without hepatitis flare (3.53 vs 3.52 log IU/mL respectively, p=0.465). Conclusion: HBV DNA levels, but not HBsAg levels, after HBeAg seroclearance were associated with subsequent significant viremia and hepatitic flares. Male gender and older age was associated with significant viremia.

Disclosures:

James Fung - Speaking and Teaching: Bristol Myers Squibb

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

Ching-Lung Lai - Advisory Committees or Review Panels: Bristol-Myers Squibb, Gilead Sciences Inc; Consulting: Bristol-Myers Squibb, Gilead Sciences, Inc; Speaking and Teaching: Bristol-Myers Squibb, Gilead Sciences, Inc

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

The following people have nothing to disclose: Danny Wong

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Association between spontaneous hepatitis B e antigen mutations and severity of liver disease

Martine Lapalus1, Michelle Martinot-Peignoux1, Ana Carolina Cardoso1, Roberto J. Carvalho-Filho1, Cédric Laouénan2, Simon Gos-set2, Zhang Qian1, Emilie Estrabaud1, Olivier Lada1, Nathalie Boyer1, FeryelMouri1, Tarik Asselah1, Patrick Marcellin1;
1INSERM U773/CRB3 et Service d'Hépatologie, Université Paris-Diderot -Hôpital Beaujon, Clichy, France; 2Service de Biostatistiques, APHP - Hôpital Bichat, Paris, France

Background and aims: Chronic hepatitis B (CHB) patients with moderate to severe liver disease need to be treated. Currently, little is known about the association between the severity of the liver disease and both host factors (including IL28B rs12979860) and viral factors such as mutations in Basal Core Promoter (BCP) and Precore (PC) regions, HBsAg and HBV-DNA levels and HBV genotypes. Therefore, we investigated these relationships in a large cohort of unselected, well-characterized, treatment-naïve CHB patients. Methods: 406 HBsAg positive patients were included. Liver biopsy was available for all patients. HBsAg and HBV-DNA levels, HBV genotypes, BCP and PC variants were determined the day of the liver biopsy. BCP and PC variants were detected by reverse hybridization using the Inno-LIPA HBV PreCore assay (Innogenetics). Histo-logical lesions were assessed using the METAVIR scoring system. Results: Out of the 406 CHB patients: 101 were HBeAg(+), 305 HBeAg(-). Fibrosis stage F0-1, F2, F3 and F4 was observed in 61% 23%, 8% and 8% of the patients, respectively. PC, BCP and BCP+PC variants were found in 25%, 29% and 28% of the patients, respectively. The HBV genotype distribution was: A, 26%; B, 11%; C, 9%; D, 24% and E, 30%. The IL28B genotype distribution was: CC, 43%; CT, 31% and TT, 26%. Patients with fibrosis stage ≥F2 had higher ALT level (3.28±4.93 times of the normal) as compared to patients with F0-1 fibrosis (1.70±2.07) (p<0.0001) and higher activity (≥A2) (12% vs 54% in F0-1 and F2-4 patients, respectively, p<0.0001). A higher proportion of patients with HBsAg level >4.3 logIU/mL (p=0.009) and HBV DNA level ≤4.3 log IU/mL (p=0.0001) was observed in F0-1 patients. Presence of BCP variant was significantly associated with a more severe liver disease (p<0.0001). Conversely, PC variant proportion was higher in F0-1 patients. IL28B CC genotype was more frequent in patients with HBsAg level <3.3 log IU/mL (p=0.004). In mul-tivariate analysis, fibrosis stage was independently associated with age (p=0.0002), HBeAg status (p=0.01), activity (p<0.0001) and BCP variant presence (p=0.008). The ROC analysis showed an AUC of 0.82 for the predictive model (age + HBeAg status + activity + HBeAg mutations) to identify F2-4 patients. Conclusion: Our study shows that patients with BCP variants were more at risk of cirrhosis. A strong correlation between age, activity grade, HBeAg status, HBV variants and fibrosis stage allows an accurate identification of subjects with moderate to severe liver disease who need to be treated. Our results suggest that detection of HBV variants is clinically relevant for the assessment of the severity of HBV related liver disease.

Disclosures:

Olivier Lada - Grant/Research Support: Gilead

Nathalie Boyer - Board Membership: MSD, JANSSEN; Speaking and Teaching: BMS

Tarik Asselah - Consulting: BMS, Boehringer-Ingelheim, Roche, Merck-Schering Plough, Gilead, Janssen

Patrick Marcellin - Consulting: Roche, Gilead, BMS, Vertex, Novartis, Janssen-Tibotec, MSD, Boehringer, Pfizer, Abbott, Alios BioPharma; Grant/Research Support: Roche, Gilead, BMS, Novartis, Janssen-Tibotec, MSD, Alios BioPharma; Speaking and Teaching: Roche, Gilead, BMS, Vertex, Novartis, Janssen-Tibotec, MSD, Abbott

The following people have nothing to disclose: Martine Lapalus, Michelle Mar-tinot-Peignoux, Ana Carolina Cardoso, Roberto J. Carvalho-Filho, Cédric Laoué-nan, Simon Gosset, Zhang Qian, Emilie Estrabaud, Feryel Mouri

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Investigation of HBV rtA181T mutation in a large number of Chinese patients with chronic HBV infection

Xiaodong Li, Yan Liu, Liming Liu, Pan Zhao, Jiuzeng Dai, Zengtao Yao, Shaojie Xin, Dongping Xu;
Institute of Infectious Diseases and Liver Failure Research Center, Beijing 302 Hospital, Beijing, China

Aim: The study aimed to investigate HBV rtA181T mutation profile in clinical practice and its clinical implications. Methods: Serum samples from 1 8,41 9 patients collected from July 2007 to June 2012 in Beijing 302 Hospital were investigated. Around 92% patients experienced nucleos(t)ide analogs. The rtA181T mutation and HBV genotype were determined by direct sequence analysis. Viral replication capacity, drug susceptibility and HBsAg secretion were determined in HepG2 cells that had been transfected with replication-competent HBV vectors containing reverse-transcriptase/S genes. Results: rtA181T was detected from 750 patients. The incidence escalated in the past five years (1.97%, 2.47%, 3.84%, 5.21%, and 6.35%); rtA181T emerged either alone or with other drug-resistant mutations (37.3% alone, 48.6% with adefovir-resistant mutation rtA181V/N236T, 12.1% with lamivudine-resistant mutation rtM204V/rtM204I, and 2.0% with entecavir- or multidrug-resistant mutations, respectively). In patients harboring rtA181T, 96.7% experienced adefovir and/or lamivudine, including 19.5% experienced single adefovir, 12.0% experienced single lamivudine, 49.4% experienced both adefovir and lamivudine, and 15.8% experienced adefovir/lamivudine plus telbivudine/entecavir). Only four patients with rtA181T were antivirals-naive. HBV genotype C/B were 93.0%/7.0% for rtA181T positive patients, and 84.6%/15.4% for rtA181T negative patients (P <0.01), suggesting there was a positive link between rtA181T with genotype C HBV. Most but not all rtA181T led to sW172* (stop codon) mutation on overlapping S-gene and coexistence of rtA181T/sW172* with wild-type sequence was frequently detected (22.0% presented as sW172* alone, 69.9% presented as sW172* concomitant with the wild-type, 5.7% presented as sW1 72* with sW1 72/L or sW172S, etc). Phenotypic analysis of representative rtA181T/sW172* strains showed that the 50% effective concentration (EC50) values of adefovir for the mutants were 1.8-fold to 2.9-fold higher than that of wild-type strain. By contrast, the EC50 of adefovir for rtA1 81V or rtN236T mutants was at least 3.5-fold higher than that of wild-type strain. A defect in HBsAg secretion and a decreased replication capacity of rtA181T (sW172*) strain were observed in comparison with wild-type strain in vitro; while no significant difference in average serum HBsAg and HBV DNA levels was observed between patients with and without rtA181T/sW172*. Conclusions: The emergence of HBV rtA181T mutation is closely associated with adefovir and lamivudine exposure, but it may not directly cause adefovir resistance. The clinical significance of rtA181T should be properly interpreted.

Disclosures:

The following people have nothing to disclose: Xiaodong Li, Yan Liu, Liming Liu, Pan Zhao, Jiuzeng Dai, Zengtao Yao, Shaojie Xin, Dongping Xu

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Quasispecies analysis of HBV strains isolated from chronic hepatitis B patients treated with Peg-inter-feron+Tenofovir therapy

Olivier Lada1, Qian Zhang1, Cédric Laouénan2, Michelle Martinot-Peignoux1, Martine Lapalus1, Emilie Estrabaud1, Nathalie Boyer1, Tarik Asselah1, Patrick Marcellin1;
1Hepatologie, Hopital Beaujon and INSERM U773 CRB3,, Clichy, France; 2Modèles et Méthodes de l’évaluation thérapeutique des maladies chroniques, Inserm UMR 738, UFR de Médecine– Université de Paris Diderot – Paris 7 – Site Bichat, Paris, France

Introduction: in chronic Hepatitis B (CHB), loss of hepatitis B surface antigen (HBsAg) and seroconversion to anti-HBs is generally considered as the ultimate goal of antiviral therapy. New combination therapy of Pegylated interferon (PegIFN) with potent HBV Inhibitors such as tenofovir (TDF) is assessed in order to improve the rate of HBsAg loss. The HBsAg gene contains the “a determinant” epitope located within the major hydrophilic region (MHR). In this study we investigate the S-gene variability of patients at baseline of PegIFN plus TDF combination therapy in order to determine the role of HBsAg variants on response to treatment. Methods: CHB patients received 180 μg of Peg-IFN/week plus 300 mg of TDF /day during 48 week. Patients were seen every 3 months. Sustained virologic response (SVR) was defined as HBV DNA< 2000UI/mL 48 weeks after end of therapy. Non-sustained virologic response (N-SVR) was defined as HBV DNA > 2000 UI/mL 48 weeks after end of therapy. HBs Ag-encoding gene from each patient's serum at baseline was PCR-amplified, cloned and sequenced. At mean of 17 clones per patient were analysed. Results: Forty CHB patients were included in this study. The median age of patients was 45 years (range=27-67 years). Thirty-two patients (80%) were male. Mean value of serum HBV DNA was 5.1 logUI/ml (range 1.8-8.8 logUI/ml) and mean value of serum ALT was 116 IU/L (range 24-437 IU/L). Twelve patients (30%) were HBeAg-positive. After 48 weeks of follow-up, 7/40 patients (17.5%) achieved a SVR. Among them, 3/7 SVR patients (43%) had a loss of HBsAg, 5/7 (71%) have a HBsAg level below 100 IU/mL and 7/7 (100%) have a HBsAg below 1000 UI/mL. Comparison of variability along the S protein by clonal analysis showed a higher percentage MHR variants in N-SVR compared to SVR patients (p=0.048). Furthermore, a higher frequency of mutated clones was observed in the “a determinant” region of N-SVR vs SVR (p=0.049). This is known to be the main anti-HBs targets. The most frequent changes observed in N-SVR patients were located at position S126 and S133, which are known as immune escape variants. Conclusion: In patients receiving PEG-IFN plus TDF combination therapy, a SVR was observed in 17.5% of patients. N-SVR patients showed more variability along the S protein. The Accumulation of residue substitutions in and around the “a” determinant at baseline should be a sensitive predictor of response to combination of PegIFN and TDF therapy in CHB patients.

Disclosures:

Olivier Lada - Grant/Research Support: Gilead

Nathalie Boyer - Board Membership: MSD, JANSSEN; Speaking and Teaching: BMS

Tarik Asselah - Consulting: BMS, Boehringer-Ingelheim, Roche, Merck-Schering Plough, Gilead, Janssen

Patrick Marcellin - Consulting: Roche, Gilead, BMS, Vertex, Novartis, Janssen-Tibotec, MSD, Boehringer, Pfizer, Abbott, Alios BioPharma; Grant/Research Support: Roche, Gilead, BMS, Novartis, Janssen-Tibotec, MSD, Alios BioPharma; Speaking and Teaching: Roche, Gilead, BMS, Vertex, Novartis, Janssen-Tibotec, MSD, Abbott

The following people have nothing to disclose: Qian Zhang, Cédric Laouénan, Michelle Martinot-Peignoux, Martine Lapalus, Emilie Estrabaud

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Hepatitis B Virus (HBV) Genotype (GT) and Drug Resistance (DR) Distribution among Clinical Specimens Submitted to a U.S. National Reference Testing Laboratory

Jeffrey J. Germer, Yuna Choi, Jayawant N. Mandrekar, Joseph D. Yao;
Mayo Clinic and Mayo Medical Laboratories, Rochester, MN

Background & Aim: Nucleos(t)ide analogues currently approved by the U.S. FDA for treatment of chronic HBV infection (CHB) include lamivudine (3TC), adefovir (ADV), entecavir (ETV), telbivudine (LdT), and tenofovir (TDF). Current U.S. and European treatment guidelines for CHB recommend ETV or TDF as first-line therapy due to their increased potency and higher genetic barrier to DR than first-generation nucleos(t)ide analogues. We assessed frequency and distribution of HBV DR mutations in a large cohort of clinical specimens of U.S. origin submitted to a national reference testing laboratory (Mayo Medical Laboratories) from April 2007 to November 2012 for routine HBV GT and DR testing (performed at Quest Diagnostics, San Juan Capistrano, CA). Methods: Analyses were limited to HBV GT and DR distribution relative to gender, and geographic origins of specimens. Geographic locations of submitting laboratories were grouped according to the U.S. National Notifiable Diseases Surveillance System (Centers for Disease Control and Prevention, Atlanta, GA). Regions included New England (NE), Mid-Atlantic (MA), East North Central (ENC), West North Central (WNC), South Atlantic (SA), East South Central (ESC), West South Central (WSC), Mountain (M), and Pacific (P). Results: A total of 2,974 specimens were submitted from 44 states representing all 9 U.S. geographic regions. Median age was 44 years (range, 0.02 to 91) with 59.3% of specimens from males. Despite a significant decline in assay success between 2007 and 2012 (81.0% vs. 59.7%, P <0.0001), 1,933 of 2,974 (65.0%) specimens yielded HBV sequences: GT A (37.0%), B (19.2%), C (21.2%), D (12.5%), E (5.1%), F (1.1%), G (2.9%), and H (0.6%), with 7 unresolved sequences (0.4%). GT G (n=56) occurred in males with greater frequency than GT A, B, C, D, E, F, or H (P <0.01). GT A occurred in males with greater frequency than GT B or GT C (P <0.0001) and occurred more frequently in NE than in ENC, WNC, SA, ESC, or WSC (P <0.015). GT C occurred more frequently in P than in NE, MA, ENC, WNC, SA or WSC (P <0.05). While no DR was identified in 90.1% of HBV sequences, rates of DR to 1, 2, and 3 drugs were 2.2%, 6.9%, and 0.8%, respectively. Resistance to 3TC and LdT was more frequent in WSC than in NE, ENC, WNC, or SA (P <0.003), while resistance to ETV, 3TC, and LdT was more frequent in P than ENC (P <0.03). Conclusion: Significant differences in HBV GT and DR exist within a large cohort of clinical specimens of U.S. origin and submitted to a national reference testing laboratory for HBV GT and DR testing. Regional differences may reflect differences in population demographics not considered in these analyses.

Disclosures:

Joseph D. Yao - Consulting: Abbott Molecular, Inc., Roche Diagnostics Corp.; Grant/Research Support: Abbott Molecular, Inc., Roche Diagnostics Corp.

The following people have nothing to disclose: Jeffrey J. Germer, Yuna Choi, Jayawant N. Mandrekar

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Evaluation of HBV DNA Decay Kinetics in Patients Containing both rtM204V/I Mutant and Wild-type HBV Subpopulations During Tenofovir DF (TDF) Monotherapy or Combination Therapy with Emtricitabine (FTC/TDF)

Yang Liu1, Kathryn M. Kitrinos1, Phillip Dinh1, John F. Flaherty1, Evguenia S. Svarovskaia1, Michael D. Miller1, Edward J. Gane2, Scott Fung3;
1Gilead Sciences, Foster City, CA; 2New Zealand Liver Transplant Unit, Auckland, New Zealand; 3Toronto General Hospital, Toronto, ON, Canada

Objective: To compare the viral load decay kinetics of mutant vs. wild-type (WT) virus in chronic hepatitis B infected patients harboring rtM204V and/or rtM204I prior to treatment with TDF or FTC/TDF therapy. Methods: Baseline samples were quantified for rtM204V/I subpopulations from all patients (n=280) in Study GS-US-174-0121, a study that evaluated TDF vs. FTC/TDF in patients harboring LAM-R at screening. Allele-specific PCR assays (AS-PCR) were designed to detect rtM204V or rtM204I mutant and rtM204M WT populations in serum samples with HBV DNA ≥1000 copies/mL, with an assay sensitivity of 0.5% of the total population. Seventeen patients (TDF, n=8; FTC/TDF, n=9) with a mixture of WT and rtM204V/I populations at baseline were evaluated for the percentage of rtM204V/I subpopulations at all study visits until HBV DNA was <1,000 copies/mL. Differences in HBV DNA decline rates and the percentage of rtM204V/I subpopulations on treatment were evaluated using the Wilcoxon Rank Sum and signed rank tests. Results: The rtM204V or rtM204I mutation was detected in 272/280 (97.1%) baseline samples with a range of 0.7% to >95%. Overall, 205/272 patients (75.4%) had >95% rtM204V or rtM204I at baseline. The majority of patients (227/ 272, 83.5%) had either rtM204V or rtM204I at baseline, while a minority of patients (45/272, 16.5%) had a mixture of rtM204V/I. For the 17 patients evaluated on treatment, the median change in HBV DNA through week 12 for the WT and rtM204V/I mutant population was similar, -2.65 and -3.34 log10 copies/mL respectively, as determined by AS-PCR quantification of each population (p=0.161). Additionally, there was no significant difference in HBV DNA decline rates for either the WT or rtM204V/I mutant viruses through week 12 (p=1.000 and 0.401 respectively) when comparing TDF to FTC/TDF treatment. Overall, there was a significant decrease in the relative amount of rtM204V/I mutant virus at the last on treatment visit compared to baseline (p=0.002); the decrease in the relative proportion of rtM204V/I during treatment was not significantly different when comparing TDF and FTC/TDF treatment (p=0.885). Conclusions: Among patients with mixtures of WT and LAM-R HBV at baseline, the rtM204V/I mutant showed similar HBV DNA decline kinetics to WT virus during treatment with either TDF or FTC/TDF. A significant decline in rtM204V/I populations was observed in patients on TDF monotherapy or FTC/TDF combination therapy. These results demonstrate that TDF is equally active against both WT and LAM-R HBV.

Disclosures:

Yang Liu - Employment: Gilead Sciences

Kathryn M. Kitrinos - Employment: Gilead Sciences, Gilead Sciences; Stock Shareholder: Gilead Sciences, Gilead Sciences

Phillip Dinh - Employment: Gilead Sciences

John F. Flaherty - Employment: Gilead Sciences Inc.; Stock Shareholder: Gilead Sciences Inc.

Evguenia S. Svarovskaia - Employment: Gilead Sciences Inc; Stock Shareholder: Gilead Sciences Inc

Michael D. Miller- Employment: Gilead Sciences, Inc.; Stock Shareholder: Gilead Sciences, Inc.

Edward J. Gane -Advisory Committees or Review Panels: Roche, AbbVie, Novar-tis, Tibotec, Gilead Sciences, Janssen Cilag, Vertex, Achillion; Speaking and Teaching: Novartis, Gilead Sciences, Roche

Scott Fung - Advisory Committees or Review Panels: Merck, Vertex; Grant/Research Support: Gilead Sciences, Roche; Speaking and Teaching: Gilead Sciences, BMS

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Application of a newly-developed high sensitivity HBsAg chemiluminescent enzyme immunoassay “Lumipulse HBsAg-HQ” for hepatitis B patients with HBs Ag seroclearance

Noboru Shinkai1,2, Etsuko Iio2, Tsunamasa Watanabe1, Kentaro Matsuura2, Mio Endo2, Kei Fujiwara2, Shunsuke Nojiri2, Joh Takashi2, Yasuhito Tanaka1;
1Departments of Virology & Liver unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 2Departments of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

Background/Aim: A highly sensitive chemiluminescent enzyme immunoassay (CLEIA) was developed and automated for quantitative hepatitis B surface antigen (HBsAg) detection by a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving the conjugation technique (Lumipulse HBsAg-HQ). Object: Of 471 HBV carriers seen 2009-2012 in our hospital, 26 were HBsAg-seronegative by quantitative HBsAg detection system (Abbott ARCHITECT). Methods: Lumipulse HBsAg-HQ performance, that is full-automatic and convenient (30 min), was compared with a quantitative HBsAg detection system (Abbott ARCHITECT) and the Roche COBAS TaqMan HBV-DNA assay (CTM, lower limit of detection 2.1 copies/ml) using serum samples from patients determined to be HBsAg-seronegative by Abbott ARCHITECT. Results: Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NA), two were HBsAg-seronegative after stopping lamivudine therapy, and 6 during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg-seronegative. Of all 26 patients, 16 were HBsAg-positive by Lumipulse HBsAg-HQ but negative by Abbott ARCHITECT. Differences between the two assays in detectable HBsAg persisted for a long time in the spontaneous loss group (median 10 months, figure), followed by the NA-treated group (3 months) and the AH group (0.5 months). In 9 patients, Lumipulse HBsAg-HQ detected HBsAg when HBV DNA was negative by CTM. HBsAg was also detected by Lumipulse HBsAg-HQ in 4 patients with anti-HBs above 10 mIU/ml, 3 of whom had no HBsAg escape mutations. Conclusions: The automatic highly sensitive HBsAg CLEIA “Lumipulse HBsAg-HQ” is a convenient and precise assay for HBV monitoring.

HBsAg duration of Abbott ARCHITECT (-) and Lumipulse HBsAg-HQ (+) in spontaneous HBsAg loss group

Patient No.Duration of Abbott ARCHITECT(-) / Lumipulse HBsAg-HQ (+) [month]Re-Appearance of HBsAg
N17 
N210 
N3>26 
N4>4(+)
N5>35 
N6>11(+)
N710 
N84 
N913 
N1010 

Disclosures:

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

The following people have nothing to disclose: Noboru Shinkai, Etsuko Iio, Tsunamasa Watanabe, Kentaro Matsuura, Mio Endo, Kei Fujiwara, Shunsuke Nojiri, Joh Takashi

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A comparison of Ultra-Deep Pyrosequencing and Clone-Based Sequencing in Analysis of HBV RT Quasispecies Heterogeneity

Ling Gong, Yue Han, Li Chen, Feng Liu, Xin-xin Zhang;
Department of Infectious Diseases, Institute of Infectious & Respiratory Dis-eases,Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

OBJECTIVE: We previously reported that Hepatitis B virus (HBV) heterogeneity within reverse transcriptase (RT) was a predictor of antiviral efficacy based on clone-based sequencing (CBS). Here, by comparing ultra-deep pyrosequencing (UDPS) with CBS in characterizing the genetic heterogeneity of HBV quasispecies within the RT region, we evaluated the performance of UDPS in the analysis of HBV biodiversity. METHODS: HBV genomic DNA was extracted from serum samples of thirty one antiviral treatment naïve chronic hepatitis B (CHB) patients. The RT region's quasispecies were parallel analyzed using CBS and UDPS (three sequential overlapping segments covering RT coding region in UDPS). Quasispecies heterogeneity characterization was conducted using bioinformatics analysis. Quasispecies complexity was calculated based on Shannon entropy formula (Sn=-2i(pilnpi)/lnN) RESULTS: UDPS determined much more qualified viral quasispecies than CBS did (P<0.001). Genotyping results using the data from both methods matched. Pearson analysis showed that there was positive correlation of quasispecies complexity at nucleotide level between the two methods (P<0.05), while complexity derived from UDPS was higher than that from CBS (P<0.001). Prevalence study of variations within RT region showed that CBS detected an average of 9.7±1.1 amino acid substitutions/sample, and UDPS detected an average of 16.2±1.4 amino acid substitutions/sample. The phylogenetic tree constructed from UDPS data was more delicate than that from CBS data. CONCLUSIONS: Viral heterogeneity determination by UDPS technique was more sensitive and efficient in terms of low abundant variations detection and quasispecies simulation than that by CBS method, thus sheds light on the future clinical application of UDPS in HBV quasispecies studies.

Disclosures:

The following people have nothing to disclose: Ling Gong, Yue Han, Li Chen, Feng Liu, Xin-xin Zhang

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Quantification of Large, Middle and Small Hepatitis B virus (HBV) surface protein (HBsAg) fractions in patients with Hepatitis Delta

Steffen B. Wiegand1, Birgit Bremer1, Andreas Geipel2, Corinna M. Bremer2, Anika Wranke1, Michael P. Manns1, Heiner Wedemeyer1 , Dieter Glebe2, Markus Cornberg1;
1Department of Gas-troenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; 2National Research Center for Hepatitis B and D viruses, Institute of Medical Virology, Giessen, Germany

Background and aims: HBsAg itself is regarded as the sum of three HBV-surface-proteins present on virions and subviral particles. They are co-carboxyterminal proteins called large (L-), middle (M-) and small (S-)HBs that differ in aminoterminal sequences and glycosylation status (preS1/preS2 in LHBs; N-glycosylated preS2 in MHBs, SHBs in all proteins). Commercial HBsAg-tests can only determine the total amount of HBsAg but variations in their protein composition and posttranslational modifications are not covered that could reflect specific host responses, since preS-domains cover B-and T-cell epitopes. LHBs contains preS1 and is necessary for receptor-binding and thus entry of HDV into hepatocytes. So far no study explored HBsAg fractions in Hepatitis Delta patients. This may be relevant for the development of biomarkers, i.e. to predict treatment response to IFN. Patients and methods: We used well-defined monoclonal Abs (mAbs) against the preS1-domain (LHBs), the N-glycosylated preS2-domain (only in MHBs) and the S-domain (L-, M-, SHBs) covering HBV genotypes A-H to detect and quantify differences in the composition of serum HBsAg concerning the three surface proteins. We analyzed HBsAg fractions in twenty-five well-defined patients with HDV infection and compared our findings with results of HBsAg fractions in fifteen acute hepatitis B (AHB) patients and twenty-one patients with chronic hepatitis B virus monoinfection. Results: Hepatitis delta infection resulted in highest ratios in LHBs compared to AHB and CHB with 14,10± 7,70%, 4,62± 3,23% and 10,03± 5,29% respectively (p<0,001; p<0,05), lower MHBs compared to CHB with 3,07± 3,31% to 13,21± 9,95% (p<0,001) and lower SHBs compared to AHB 82,84± 9,80% to 90,91 ±7,01% (p<0,01). Conclusion This is the first study investigating the ratio of L-, M-, SHBs in patients with Hepatitis Delta, demonstrating differences in HBsAg fractions between Hepatitis Delta, acute and chronic HBV monoinfection. Higher LHBs-ratios in Hepatitis Delta might be one reason for a strong infectivity of Hepatitis Delta. Future studies have to elaborate if LHBs levels may be a better marker compared to total HBsAg to predict response to IFN during HDV-therapy.

Disclosures:

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

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

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

The following people have nothing to disclose: Steffen B. Wiegand, Birgit Bremer, Andreas Geipel, Corinna M. Bremer, Anika Wranke, Dieter Glebe

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A novel non-invasive fibrosis score based on cytokines and clinical parameters for the use in chronic hepatitis delta

Benjamin Heidrich1,2, Anika Wranke1, Cihan Yurdaydin3, Judith Stift4, Florin A. Caruntu5, Manuela G. Curescu6, Kendal Yalcin7, Selim Gurel8, Stefan Zeuzem9, Andreas Erhardt10, Stefan Lüth11, George V. Papatheodoridis12, Birgit Bremer1, Jan Grabowski1, Jan-ina Kirschner1, Kerstin Port1, Markus Cornberg1,13, Falk Christine2, Hans Peter Dienes4, Svenja Hardtke1,13, Michael P. Manns1,2, Heiner Wedemeyer1,2;
1Dept. of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; 2Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Hannover, Germany; 3Med-ical Faculty Ankara University, Ankara, Turkey;4Medical University of Vienna, Vienna, Austria; 5Institut de Boli Infectioase, Bucharest, Romania; 6Spitalul Clinic de Boli Infectioase, Timisoara, Romania; 7Dicle University Medical Faculty, Diyarbakir, Turkey; 8Uludag University Medical Faculty, Bursa, Turkey; 9Johann Wolfgang Goethe University Medical Center, Frankfurt/Main, Germany; 10Heinrich Heine University, Düsseldorf, Germany; 11University Medical Centre Hamburg-Eppendorf, Hamburg, Germany; 12Athens University School of Medicine, Athen, Greece; 13HepNet Study-House, Hannover, Germany

Introduction: Hepatitis delta is the most severe form of viral hepatitis with a fast progression of fibrosis to cirrhosis. Treatment options are still very limited as PEG-interferon alfa is effective only in a minority of patients. Therefore, appropriate determination of stage of liver disease is desired. Non-invasive fibrosis scores used for other liver diseases including APRI-score, AST/ALT ratios or FIB-4 index do not perform well in hepatitis delta. We here aimed to develop novel non-invasive fibrosis scores optimized for patients with hepatitis delta. Methods: In the ongoing HIDIT-2 treatment trial 120 patients with chronic hepatitis delta were recruited. Liver biopsies were evaluated centrally by two independent pathologists. Additionally, 50 cytokines, chemokines, growth factors and angiogenic factors were measured in sera of 100 of the 120 patients using multiplex technology (Bio-Plex System). Anti-HDV-IgM-testing was performed in all patients by the ETI-DELTA-IGMK-2 assay (Diasorin). T-test was used to identify factors associated with cirrhosis or fibrosis. With ROC curve analysis and calculation of the Youden index cut offs were determined differentiating cirrhosis and non-cirrhosis as well as fibrosis and non-fibrosis for each factor. In a last step logistic regression was used to identify the most important factors differentiating fibrosis and cirrhosis in order to create the new score. Results: Four factors were identified differentiating between cirrhosis and Ishak scores <5 (MIF, AST/ALT ratio, age, HGF). Defined cut-offs were determined for each factor (MIF >3400 ng/ml, AST/ALT >0.8, age >35 and HGF >370 ng/ml) which were then included in the following equitation (1 point × the indicated factor for each variable if above the cut-off): 5*MIF+2*(AST/ALT)+AGE+HGF. The AUC of the new score was 0.84; >2 points predicted cirrhosis with a sensitivity of 85%, a specificity of 69%, a PPV of 72% and a NPV of 83%. In order to differentiate between fibrosis (Ishak-score >2) and non-fibrosis, another score was similarly determined based on 6 variables: 0.5*MIP-1b+2*IP-10+(AST/ALT)+3*age+RANTES+0.2*HDV-IgM (cutoffs MIP-1b >40 ng/ml, IP-10 >1000 ng/ml, AST/ALT >0.8, age >35 years, RANTES >1200 ng/ml and HDV-IgM >0.2 OD) resulting in an AUC of 0.83. A value of >3.2 points predicted liver fibrosis with a sensitivity of 71% and a specificity 79% (PPV 86%, NPV 59%). Discussion: We here suggest novel non-invasive fibrosis scores to distinguish hepatitis delta patients with and without cirrhosis and with and without significant fibrosis. These scores need to be validated in independent cohorts.

Disclosures:

Cihan Yurdaydin - Advisory Committees or Review Panels: Janssen, Roche, Merck, Gilead

Selim Gurel - Speaking and Teaching: Glead, BMS, Roche, MSD, Glead, BMS, Roche, MSD

Stefan Zeuzem - Consulting: Abbvie, Achillion Pharmaceuticals, Boehringer Ingel-heim GmbH, Bristol-Myers Squibb Co., Gilead, Novartis Pharmaceuticals, Merck & Co., Idenix, Janssen, Roche Pharma AG, Vertex Pharmaceuticals, Presidio, Santaris, Inc

George V. Papatheodoridis - Advisory Committees or Review Panels: Janssen, Abbott, Boehringer, Novartis, BMS, Gilead, Roche; Consulting: Roche; Grant/Research Support: BMS, Gilead, Roche; Speaking and Teaching: Janssen, Novartis, BMS, Gilead, Roche, MSD

Kerstin Port - Speaking and Teaching: Roche

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

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

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: Benjamin Heidrich, Anika Wranke, Judith Stift, Florin A. Caruntu, Manuela G. Curescu, Kendal Yalcin, Andreas Erhardt, Stefan Lüth, Birgit Bremer, Jan Grabowski, Janina Kirschner, Falk Christine, Hans Peter Dienes, Svenja Hardtke

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The aa 15-17 Amino Acid Sequence in the Terminal Protein Domain of HBV Polymerase as a Viral Factor Affecting in Vivo as Well as in Vitro Replication Activity of the Virus

Yoshihito Uchida, Kiyoko Yoshino, Kayoko Sugawara, Jun-ichi Kouyama, Kayoko Naiki, Mie Inao, Nobuaki Nakayama, Satoshi Mochida;
Department of Gastroenterology and Hepatology, Saitama Medical University, Iruma-gun, Saitama, Japan

[Background and Aim] The viral factors affecting sustained response after discontinu-ation of treatment with nucleoside analogs in patients with viral hepatitis B are uncertain. Thus, the amino acid sequences responsible for the replication activity of HBV were evaluated both in vivo and in vitro. [Methods] (1) In Vivo Analysis: The subjects were 203 patients with HBV infection who had been treated with nucleoside analogs. Therapy was discontinued when the fol-lowing criteria were fulfilled in the patients; HBe antigen- negative and serum HBV-DNA level <2.1 Log copies/mL for at least 1 year, with core-related antigen titers <3.0 Log IU/mL. Serum HBV-DNA levels were measured every 2 or 4 weeks for 48 weeks following the treatment discontinuation, and the full amino acid sequences of the HBV detected in the patients' sera were compared in relation to the rate of increase of the serum HBV-DNA levels following the treatment discontinuation. (2) In Vitro Anal-ysis: Full-sequence genome of HBV strains isolated from patients following nucleotide analog discontinuation were transfected to Huh7 cells, and HBV-DNA levels were measured in culture medium by the TaqMan PCR method. [Results] (1) A total of 34 patients (16.7%) fulfilled the criteria for treatment discontin-uation, and the serum HBV-DNA titers increased to more than 4.0 Log copies/mL in 26 of these patients (76.5%); within 4 weeks in 5 patients (group-A), between 4 and 12 weeks in 13 patients (group-B), and later than 12 weeks in 8 patients (group-C). The amino acid sequences of HBV were evaluated in 22 of these patients, and were found to differ among the 3 groups, especially in the terminal protein domain of polymerase; mutations from acidic to neutral amino acids between aa15 and aa17 showing DDE mo-tifs were absent in group A, while these were present in 1 of 12 patients in group B, and 5 of 6 patients in group C. (2) HBV-DNA levels in the medium 6 hours following the culture was about 10 times and 5 times higher in HBV strains isolated from patients in group-A and group-B, respectively than in HBV strains from those in group-C. [Conclusion] Amino acid sequences between aa15 and aa17 in the terminal protein domain of polymerase may affect the in vivo as well as in vitro replication activity of HBV.

Disclosures:

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: Yoshihito Uchida, Kiyoko Yoshino, Kayoko Sugawara, Jun-ichi Kouyama, Kayoko Naiki, Mie Inao, Nobuaki Nakayama

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Serum ceruloplasmin levels correlate negatively with stages of liver pathology and can be used in a new noninvasive model for predicting liver fibrosis in chronic hepatitis B-virus related liver disease

Da-wu Zeng, Jing Dong, Yu-rui Liu, Yue-Yong Zhu, Su LIN, Jing Chen, Jia-ji Jiang;
the first affiliated hospital of Fujian Medical University, Fuzhou, China

Background to investigate the relationship between CP levels and liver pathological stages in patients with chronic hepatitis B (CHB) and to establish a noninvasive model to predict cirrhosis. Methods Liver biopsy samples and sera were collected from 198 CHB patients who were randomized into a training group and a validation group. CP levels were determined using nephelometric immunoassays. Spearman rank correlation analysis was used to analyze the relationship between CP and liver pathological stages and ROC curves were used to evaluate the diagnostic value of CP for liver pathological stages. The liver pathology-predicting model was built using multivariate analysis by forward logistic regression to identify relevant indicators. Results CP levels were lower in patients with inflammation stage G4 compared to other stages and lower in cirrhotic compared to non-cirrhotic patients. Using AUC values, we showed that CP levels could distinguish different stages of inflammation and fibrosis. Multivariate analysis showed that CP levels, along with PT, PLT, AFP, were all significantly associated with cirrhosis and these markers were used to develop a model to predict fibrosis in CHB patients. Our APPCI model had a significantly better predictive performance compared to the FI, APRI, GPI, and APGPI models, respectively. Conclusions CP levels were negatively and indirectly correlated with inflammation and fibrosis stages in CHB patients. Our model used routine laboratory variables along with CP to accurately predict liver fibrosis in CHB.

Disclosures:

The following people have nothing to disclose: Da-wu Zeng, Jing Dong, Yu-rui Liu, Yue-Yong Zhu, Su LIN, Jing Chen, Jia-ji Jiang

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Performance of HBV core-related antigen testing in hepatitis B patients infected with HBV genotypes A and D

Benjamin Maasoumy1, Steffen B. Wiegand1, Jerzy Jaroszewicz2, Birgit Bremer1, Patrick Lehmann1, Katja Deterding1, Michael P. Manns1, Markus Cornberg1, Heiner Wedemeyer1;
1Gastroenterol-ogy, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; 2Department of Infectious Diseases and Hepatology, Medical University in Bialystok, Bialystok, Poland

Background: HBeAg and HBcAg share an identical sequence of 149 amino-acids. Hence, they are collectively called Hepatitis B virus core-related antigens (HBcrAg). HBcrAg levels have been shown to correlate with both HBsAg and HBV DNA in Asian patients infected with HBV genotypes B and C. Moreover HBcrAg but not HBsAg levels were independently associated with the development of HCC in Japanese patients. The aim of this study was to investigate the performance of an HBcrAg assay in European patients mainly infected with HBV genotypes A and D. Methods: Plasma samples of 302 HBsAg positive patients, including 124 (41%) HBeAg-positive samples, were tested for quantitative HBsAg levels (Abbott Architect) and HBV DNA (Cobas Taqman). In addition, HBcrAg was determined using the Lumipulse® G HBcrAg assay (Fujirebio), which measures HBeAg, HBcAg and the precore protein p22cr (aa28-150). Results: HBcrAg tested positive in 167 out of the 302 patients including all 124 HBeAg-positive and 43 out of 178 HBeAg-negative samples. A very robust linearity of HBcrAg levels for up to 4 log dilutions was observed across HBV genotypes A, B, C and D. There was no significant difference in detection limits between HBV genotypes. HBcrAg showed a very high correlation with HBV DNA (Spearman correlation: r=0.88; linear regression r2=0.84; p<0.0001) and a modest correlation with HBsAg levels (r=0.78; r2=0.47; p<0.0001). Correlation with HBV DNA was independent from HBV genotype A and D, and was also not influenced by HBeAg status. Conclusions: The HBcrAg assay shows a high accuracy across HBV genotypes A, B, C and D. Thus it can also be used in patients with HBV genotypes A and D. The clinical utility of HBcrAg testing to individualize management of patients with chronic HBV should be evaluated in further studies.

Disclosures:

Benjamin Maasoumy-Advisory Committees or Review Panels: Abbott Molecular; Speaking and Teaching: MSD, Roche Diagnostics, Roche Pharma

Jerzy Jaroszewicz - Speaking and Teaching: Roche, Gilead, Abbott, MSD, BMS

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 Germamny), Roche, Gilead, Novartis; Grant/Research Support: Merck (MSD Germamny), 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: Steffen B. Wiegand, Birgit Bremer, Patrick Lehmann, Katja Deterding

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Amino acid polymorphisms of S region of hepatitis B virus in acute hepatitis B patients in Japan

Norie Yamada1,2, Ryuichi Sugiyama1, Hiroshi Yotsuyanagi3, Chi-aki Okuse4, Michihiro Suzuki4, Fumio Itoh4, Kiyomi Yasuda2, Kyoji Moriya3, Kazuhiko Koike3, Takaji Wakita1, Takanobu Kato1;
1Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; 2Department of Internal Medicine, Center for Liver Diseases, Kiyokawa Hospital, Tokyo, Japan; 3Department of Internal Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 4Department of Internal Medicine, Division of Gastroenterology and Hepatology, St. Marianna University School of Medicine, Kawasaki, Japan

Amino acid substitutions in the major antigenic a determinant region of hepatitis B virus (HBV) S region are often observed in patients of immunoprophylaxis failure, and are considered to be responsible for the vaccine-escape mutants. However, the rate of emergence of the amino acid substitutions in this region is still unknown among patients of acute hepatitis B. The aim of this study is to evaluate the rate of amino acid polymorphisms of S region including the a determinant region among the patients of acute hepatitis B in Japan. From 2002 to 2012, serum samples were collected from 91 patients (male: female 81:10, median age 32.6 ± 10.6 ) diagnosed with acute hepatitis B in our institutions. None had received any vaccination against HBV. Anti-HIV was tested in 48 patients under informed consent, and 3 (6.3%) of them were positive. A fragment of HBV S region (nt. 155-835) was obtained by nested PCR amplification and was subsequently analyzed by direct sequencing. The HBV genotypes of isolated strains were determined by phylogenetic analysis. Deduced amino acid sequences were compared with the consensus sequence of each genotype in the database. Of the 91 patients, 62 (68.1%) were infected with genotype (gt) A, 14 (15.4%) were with gt B, and 15 (16.5%) were with gt C. In these isolated strains, 19 (20.9%) had the amino acid polymorphisms in S region (aa 1- 227), 9 (9.9%) were in the major hydrophilic region (aa 110 - 160), and 6 (6.6%) were in the a determinant region (aa 124 - 147). Identified polymorphisms in the a determinant region were T126S (gt B), T131P/A (gt C), M133L (gt B), F134Y (gt A), and P135H (gtA, Anti-HIV positive). Clinical features (age, gender), laboratory data (maximum ALT, T-Bil, HBV DNA) were not different in the 19 patients with polymorphisms in S region as compared with patients without them. In conclusion, the emergence of HBV strains with amino acid polymorphisms in S region was observed in about 20% of the patients with among acute hepatitis B in Japan. The 6 of 19 identified polymorphisms were in the a determinant region. These strains may affect antigenicity and reduce binding capability to neutralizing antibodies. The efficacy of current HBV vaccine used all over the world widely and the necessity of booster vaccination should be evaluated particularly in these strains with polymorphisms.

Disclosures:

Kazuhiko Koike - Speaking and Teaching: Bristol-Myers Squibb

The following people have nothing to disclose: Norie Yamada, Ryuichi Sugiyama, Hiroshi Yotsuyanagi, Chiaki Okuse, Michihiro Suzuki, Fumio Itoh, Kiyomi Yasuda, Kyoji Moriya, Takaji Wakita, Takanobu Kato

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Dynamic Study of Hepatitis B Virus (HBV) Quasispecies in X Gene/Basal Core Promoter/preCore Region by Ultradeep Pyrosequencing (UDPS) Technology: High Rates of Deletions, De Novo Stop Codons, and Viral Genotype Mixtures

Josep Gregori1,2, Andrea Caballero3, David Tabernero3,4, Maria Homs3,4, Maria Blasi3,4, Rosario Casillas2, Josep Quer2,4, Leonardo Nieto5, Xose Costa-Alcaide6, Rafael Esteban2,4, Maria Buti2,4, Francisco Rodriguez-Frias3,4;
1Statistics Department, Uni-versitat de Barcelona, Barcelona, Spain; 2Hepatology Department, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain; 3Biochemistry Department, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain; 4Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; 5Microbiology Department, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain; 6Microbiology Department, Clinic University Hospital of Santiago de Compostela, Santiago de Compostela, Spain

Background: HBV X region (HBX) (HBx protein, basal core promoter [BCP], overlapped with preCore [PC]) variants (var) are linked to liver disease severity. Aim: To characterize HBV quasispecies in HBX by UDPS, including var with deletions. Patients and Methods: UDPS analysis (GS Junior Roche) of HBX nt1596-1912 in 30 serum samples from 10 chronic HBV patients failing lamivudine (LMV): basal, untreated, after LMV. nt substitutions/deletions, aa var in HBx (aa75-154), and PC were studied. Results: 415,726 sequences obtained. Main HBX var: xK130M, xV131I (19 samples), associated with BCP ntA1762T/G1764Avar. HBX deletions (1->50nt) in all patients (Table), causing early HBX stop in 90.7% of these var, 33% were 8nt deletion in region nt1 758-1776 (9 samples/6 patients). Two main single-nt deletions: 1825 (12%) in 7 samples (4 patients) yields a long HBx, and 1746 (4.9%) causes HBx early stop. Genotype D cases (classified by LiPA) clustered with genotype E in HBX, suggesting D-E recombination (recD-E). Moreover; A/recD-E mixtures were frequent, 14/30 samples (9/10 patients) with changes during follow-up. After LMV, genotype A increased when recD-E was dominant (4 cases). All patients showed PC nonexpression var (Table), deletions being most frequent (17/30 samples, 0.3%-12%). Conclusions: The systematic presence of deletions in X/BCP/PC, mainly causing an early HBX stop, suggests a new multicoding HBV mechanism. Deletions are most frequent cause of PC nonexpression. The recD-E genotype may be prevalent in our region; the dynamics of genotype mixtures seem to be associated with a different sensitivity to antiviral therapy. Funding by Instituto CarlosIII (PI 12/1983), cofinanced by ERDF

Table 2. ID, patient number; Gen. HBV genotype by LiPa; BA. basal sample; UT. untreated sample; LA, sample after lamivudine failure; (<). less than 0.25% frequency; (*), no VBK; (#), G1896A main PC variant; %,PC var, var associated with PC nonexpression (non-start, stops or deletions); (a) non-recombinant gonotype D
     % HBX-BCP DeletionsGenotypes
IDSampleGenHBe%PC VarTotalWithStop%A%D-E/D
1BAA/DN1.031.301.4271.428.6
 UTAP<0.270.25100<
 LAAP<0.290.27100<
2BAAP1.184.904.87100<
 UTAP1.184.904.87100<
 LAAP<0.650.67100<
3BA(#)A/DN2.400.25<61.8538.2
 UTA/DN0.342.101.3776.7623.25
 LA(*)(#)A/DN83.740.640.5215.7384.25
4BADP0.851.981.843.896.2
 UTDP0.501.161.004.395.6
 LA(*)DP0.740.890.9115.7384.25
5BADP1.962.982.25<100
 UTDP2.320.393.500.8199.19
 LADP1.551.921.663.896.2
6BAAP0.282.506.93100<
 UTAP1.668.221.4798.681.32
 LAAP2.031.50<97.772.23
7BA(#)A/DN8.43<<82.0116.2/1.88
 UTDN0.300.380.38<100
 LA(*)DN1.170.420.29<100
8BAAP0.436.505.91100<
 UTDN<0.620.52<100
 LAAP<1.211.211000
9BADP3.5115.1112.93<100
 UTAP8.000.260.2691.938.07
 LAAP11.9612.308.70100<
10BA(#)DP/N23.8010.2010.25<36.96/63.4*
 UT(#)DN97.233.403.38<100*
 LAAN/P1.323.002.8196.83.19

Disclosures:

Rafael Esteban - Speaking and Teaching: MSD, BMS, Novartis, Gilead, Glaxo, MSD, BMS, Novartis, Gilead, Glaxo, Janssen

Maria Buti - Advisory Committees or Review Panels: Boerhinger Inghelm, Boer-hinger Inghelm; Speaking and Teaching: MSD, Bristol-Myers Squibb, Novartis, Gilead, Janssen, MSD, Bristol-Myers Squibb, Novartis, Gilead, Janssen

The following people have nothing to disclose: Josep Gregori, Andrea Caballero, David Tabernero, Maria Homs, Maria Blasi, Rosario Casillas, Josep Quer, Leonardo Nieto, Xose Costa-Alcaide, Francisco Rodriguez-Frias

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Clinical Exploration of Acute Exacerbation in Asymptomatic Patients with Chronic Hepatitis B Virus Infection

Woo Hee Cho, Hyoung Joon Kim, Sun Young Cho, Young Kwang Choo, Sung Soo La, Suk Bae Kim, Il Han Song;
Division of Hepatology and Gastroenterology, Department of Internal Medicine, Dankook University College of Medicine, Cheonan, Republic of Korea

Background/Aim: Asymptomatic patients with chronic hepatitis B virus (HBV) infection often experience hepatitis flare so-called acute exacerbation depending on the host condition, virologic factors or environmental status. Clinical courses of acute exacerbation in patients with chronic HBV infection vary from asymptomatic to transient self-limited, typical hepatitis symptoms, rarely hepatic decompensation or liver failure. The aim of this study was to explore the causes, clinical features and outcomes of acute exacerbation in asymptomatic patients with chronic HBV infection. Methods: A total of 208 asymptomatic chronic HBV-infected patients with acute exacerbation were consecutively enrolled for this study from January 2003 to December 2012. Acute exacerbation of chronic HBV infection was defined as an increase of serum alanine aminotransferase (ALT) level more than five times the upper limit of normal. We analyzed the causes and the clinical course of these patients with acute exacerbation. Results: The most frequent cause of acute exacerbation arised from hepatitis B viral factors; spontaneous reactivation(48.1%) and HBeAg seroconver-sion(10.0%). The next arised from hepatotoxicity; alcohol(8.1%) and drugs including herbal medicines(7.6%). Accompanying other diseases(12.9%), coinfection by hepatitis A virus(7.2%) or hepatitis D virus(1.9%), the development of hepatocellular carcinoma (HCC)(1.9%), and liver injury(1%) were the rest. Spontaneous reactivation of HBV showed the longest period to ALT normalization among the causes of acute exacerbation of which the average duration was 134.5 ± 184.2 days. A total of four patients rapidly deteriorated to fulminant hepatic failure; three of them died, one transferred to receive liver transplantation. Herbal medicine, alcohol, HCC development and traumatic liver laceration were the causes of liver failure, respectively. Conclusions: The main causes of acute exacerbation in asymptomatic HBV infection were spontaneous reactivation of HBV and HBeAg seroconversion which tented to recover well through antiviral therapy or spontaneously. Otherwise, The greatest care should be taken in managing acute exacerbation of HBV-infected patients by hepatotoxicity or HCC development.

Disclosures:

The following people have nothing to disclose: Woo Hee Cho, Hyoung Joon Kim, Sun Young Cho, Young Kwang Choo, Sung Soo La, Suk Bae Kim, Il Han Song

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Clinical significance of core promoter and precore mutations in chronic hepatitis B

Michelle Martinot-Peignoux1, Martine Lapalus1, Cédric Laouénan2, Ana Carolina Cardoso1, Roberto J. Carvalho-Filho1, Ahmed El Ray3, Simon Gosset2, Nathalie Boyer1, Tarik Asselah1, Patrick Mar-cellin1;
1Université Paris Diderot, INSERM U773/CRB3 et Service d'hépatologie, Clichy, France; 2Service de Biostatistique, Hopital Beaujon, Paris, France; 3Theodor Bilhaz Institut, Giza, Egypt

Background/Aim. Most common occurring HBV variants include precore stop codon (PC) and the dual mutation in basal core promoter region (BCP). We aimed to determine prevalence of PC and BCP in a multi ethnic chronic hepatitis B population and establish association of these variants with demographical, clinical, virological and histological data. Methods. At inclusion a liver biopsy and a serum sample the same day. Demographical, clinical and biochemical data were collected. HBeAg status [(e+) or (e-)], HBV variants, HBV DNA and HBsAg titers, HBV and IL28B genotypes, histological lesions were determined the day of liver biopsy. Results: 406 consecutive CHB patients, 101 e(+) and 305 e(-). Wild type (WT), BCP, PC, and BCP+PC found in 18%, 29%, 25% and 28%, respectively. Mean age was 40±12 years, 76% were male, 42% Caucasian, 18% Asian, and 40% Black African. HBV genotype A, B, C, D, and D found in 26%, 11%, 9%; 24%, and 30%, respectively, IL28 genotype CC, TT and CT found in 43%, 26%, and 31%, respectively. Fibrosis stage >F1 found in 39%, Activity grade >1 found in 29%. HBV DNA titers <3.3, 3.3 to 4.3 and >4.3 log IU/ml found in 21%, 20% and 59%, respectively. HBsAg titers <3.3, 3.3 to 4.3 and >4.3 log IU/ml found in 26%, 48% and 26%, respectively. Prevalence of HBV variants was significantly related to: sex (p=0.02), male were more likely to have BCP and BCP+PC; age (p=0.01), WT associated with younger patients; ethnicity (p<.001) WT and BCP found in Caucasians (51% and 49%), PC and BCP+PC found more frequently in black African (55% and 43%); HBV genotypes (p<.0001) WT and BCP found in genotype A (43% and 52%) while PC and BCP+PC found in D (32% and 36%) and E (50% and 41%) ; fibrosis stage (p<.0001) BCP, BCP+PC were associated with F>1 (52% and 50%) and WT and PC associated with F<1 (81% and 76%), HBsAg titers (p<.0001) WT associated with >4.3 log IU/ml (63%) while BCP, PC and BCP+PC associated with a titer <4.3logIU/ml (82%, 77%, 83%), HBV DNA levels (p<.0001) WT associated with a level >4.3 log IU/ml (73%) while PC associated with a level <3 .3log IU/ml (37%), In multivariate analysis HBV variants were significantly and independently associated: WT with e(+) (p<.002) and high HBsAg titer (p<.01); BCP with more severe fibrosis (F >1) (p<.001); BCP+PC with more severe fibrosis (F >1)(p<.002), e(-) (p<.0001), genotypes D (p<.01) and E (p<.0001). Conclusions: PC and BCP+PC variants found more frequently in e(-) status. Patients with BCP and BCP+PC variants were more likely to have more severe fibrosis (F >1). We confirm a strong correlation between HBV variants and HBV genotypes independently from ethnicity and IL28B genotypes.

Disclosures:

Nathalie Boyer - Board Membership: MSD, JANSSEN; Speaking and Teaching: BMS

Tarik Asselah - Consulting: BMS, Boehringer-Ingelheim, Roche, Merck-Schering Plough, Gilead, Janssen

Patrick Marcellin - Consulting: Roche, Gilead, BMS, Vertex, Novartis, Janssen-Tibotec, MSD, Boehringer, Pfizer, Abbott, Alios BioPharma; Grant/Research Support: Roche, Gilead, BMS, Novartis, Janssen-Tibotec, MSD, Alios BioPharma; Speaking and Teaching: Roche, Gilead, BMS, Vertex, Novartis, Janssen-Tibotec, MSD, Abbott

The following people have nothing to disclose: Michelle Martinot-Peignoux, Mar-tine Lapalus, Cédric Laouénan, Ana Carolina Cardoso, Roberto J. Carvalho-Filho, Ahmed El Ray, Simon Gosset

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Plasma levels of HBV pregenomic RNA before and after nucleos(t)ide analogue treatment

Louis Jansen1,2, Karel A. van Dort1, Hans L. Zaaijer3, Neeltje A. Kootstra1, Hendrik W. Reesink2;
1Department of Experimental Immunology, Academic Medical Center (AMC), Amsterdam, Netherlands; 2Department of Gastroenterology and Hepatology, Academic Medical Center (AMC), Amsterdam, Netherlands; 3Department of Virology, Academic Medical Center (AMC), Amsterdam, Netherlands

Background and Aim: Replication of the hepatitis B virus (HBV) DNA genome proceeds via an RNA pregenome (HBV RNA), transcribed from cccDNA present in the nuclei of infected hepatocytes. Treatment of HBV infection with nucleos(t)ide analogues (NUC) suppresses HBV DNA synthesis by blocking reverse transcription, but does not affect HBV RNA synthesis. We hypothesized that during NUC therapy HBV pregenomes continue to be incorporated in viral particles and subsequently are secreted into the bloodstream. For this, we developed a sensitive assay to measure HBV RNA in plasma. Patients and Methods: HBV RNA (see below), HBV DNA (COBAS TaqMan - Roche), and HBsAg (Architect - Abbott) were measured in plasma of 10 chronic hepatitis B patients (5 HBeAg-positive and 5 negative) who received NUC therapy (7 entecavir and 3 tenofovir). Pre-treatment levels were compared to levels at end of follow-up and evaluated with a paired T test. Total RNA was isolated from 280 uL of plasma and treated with DNAse. HBV RNA was then concentrated, reverse transcribed, and quantified by qPCR using HBV specific primers, adapted from Laras et al. (Hepatology 2006; 44-3). Lower limit of quantification of RNA was 143 C/ml. To confirm that no HBV DNA was measured, qPCR was additionally performed before reverse transcription (‘no-RT controls’). Results: The mean duration of patient follow-up was 75 weeks (range 58 - 90). In all patients mean plasma HBV DNA had declined strongly at end of follow-up from 8.67 (SD 0.93) to 2.43 (SD 1.55) log C/ml (p<0.001) in HBeAg-positive patients, and from 6.33 (SD 0.60) to 1.44 (SD 0.80) log C/ml (p<0.001) in HBeAg-negative patients. The decline in HBsAg levels was limited. HBV RNA was detectable in all patients before treatment, with mean levels of 6.01 (SD 1.14), and 2.73 (SD 0.98) log C/ml in HBeAg-positive and negative patients. In HBeAg-positive patients mean level of HBV RNA decreased to 4.22 (SD 1.75) log C/ml at end of follow-up, but remained significantly higher than HBV DNA levels (p=0.01). The same pattern was seen in HBeAg-negative patients, although 3/5 had detectable RNA levels below the limit of quantification at end of follow-up. The ‘no-RT control’ procedure confirmed the absence of influence of DNA on RNA measurements. Conclusion: In chronic hepatitis B patients HBV RNA can be quantified in plasma. NUC treatment, causing a strong decline in HBV DNA, influences the level of HBV RNA to a much lesser extent. More research is needed to elucidate the virological characteristics of HBV RNA containing particles in plasma and its possible clinical application, e.g. as marker of therapy response.

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, San-taris, SGS, Idenix, BMS

The following people have nothing to disclose: Louis Jansen, Karel A. van Dort, Hans L. Zaaijer, Neeltje A. Kootstra

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Serum cytokines and chemokines in patients with different phase of HBeAg negative chronic hepatitis B virus infection

Steffen B. Wiegand1, Anika Wranke1, Falk Christine2, BehrendJ. Zacher1, Katja Deterding1, Michael P. Manns1, Heiner Wede-meyer1, Markus Cornberg1;
1Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; 2Institute of Transplant Immunolgy, IFB-Tx, Hannover Medical School, Hannover, Germany

Background:Patients with chronic hepatitis B (CHB) show dynamics in their natural course of infection. Discrimination of HBeAg negative hepatitis (ENH) and low replicative inactive carrier status (LRC) can be difficult as HBV DNA and ALT can fluctuate. Quantitative HBsAg <1000 IU/ml and HBV DNA <2000 IU/ml has been shown to correlate with LRC, but the predictive value is limited. A third group of patients with HBV DNA levels between 2000 and 20000 IU/ml and repeatedly normal ALT values may also belong to inactive carrier (high replicative carrier, HRC). Our aim was to evaluate serum cytokines and chemokines as additional predictive markers to discriminate different phases of HBeAg negative CHB patients. Methods: Cross sectional analysis of 205 HBeAg negative patients. Serum cytokines and chemokines including: IL-2, -4, -7, -10, -12, -16, -17, IP-10, IFN-γ, MIP-1β, TNF-α, TGF-β1, -2, -3 were quantified using multiplex technology. Principal component analysis and multivariate analysis was performed. Results: 72 patients with ENH (HBV-DNA >2000 IU/ml; elevated ALT), 88 LRC (HBV-DNA < 2000 IU/ml; normal ALT) and 45 HRC patients with HBV-DNA 2000-20000 IU/ml and normal ALT were studied. A One-way ANOVA including HBsAg and cytokines showed, that groups differ concerning IFN-γ, IL-12, TNF-α (p*), IL-2, -16, TGF-β1, -2, -3 (p**), HBsAg, IP-10 and MIP-1 β(p***). Multivariate analysis of the parameters separates all groups by TGF-β2 (p***) and TGF-β3 (p**). Comparing ENH with LR there are following differences: IL-12, IFN-γ and TGF-β2 (p*); IL-2, -16 and TGF-β1, -3 (p**); HBsAg, IL-17, IP-10, MIP-1 β (p***). The strongest factor was IP-10. ENH patients show 2,59± 0,04 log pg/ml IP-10, compared to 2,19± 0,04 log pg/ml in LR patients (p***). χ2 test for IP-1 0 revealed a cut-off of 2,3log pg/ml as discriminative factor (p***). The differences between ENH and HRC: MIP-1β and TGF-β3 (p*); TGF-β2 (p**); IL-16, -17 and IP-10 (p***). The means of choice was TGF-β2. ENH patients have significantly higher TGF-β2 levels of 3,76± 0,02 log pg/ml as compared to HRC with 3,65± 0,02 (p***). χ2 test for TGF-β2 with cut-off of 3,8log pg/ml yielded significant results (p***). TGF-β2 and HBsAg discriminated LRC and HRC (p***). However, none of the cytokines were significant using multivariate analysis. Discussion: ENH patients are characterized by higher IP-10 and TGF-β2 levels. PCA analysis showed that in addition to HBsAg these two cytokines were significantly associated with the different phases. These markers should be further investigated in CHB. Effector cytokines were not different between the two groups of inactive carrier and may explain normal ALT values.

Disclosures:

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

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

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

The following people have nothing to disclose: Steffen B. Wiegand, Anika Wranke, Falk Christine, Behrend J. Zacher, Katja Deterding