The Liver Meeting
55th Annual Meeting of the American Association for the Study of
Liver Diseases

October 29 - November 2
Boston, Massachusetts
John B. Hynes Convention Center




Poster 19

PRODUCTION OF IMMUNOGENIC HBSAG-PULSED HUMAN DENDRITIC CELLS (DCS): EVALUATION OF SAFETY AND EFFICACY OF HBSAG-PULSED DCS IN NORMAL VOLUNTEERS, HB VACCINE NONRESPONDERS AND PATIENTS WITH CHRONIC HEPATITIS B

Sk. Md. Fazle Akbar, Shinya Furukawa, Norio Horiike, Morikazu Onji, Ehime University School of Medicine, Shitukawa, Japan.

 

 

Background:

Antigen loaded on antigen-presenting dendritic cell (DC) [antigen-pulsed DC] has shown potent therapeutic efficacy in animal models of human diseases.

 

Aims:

Indeed, administration of hepatitis B surface antigen (HBsAg)-pulsed DCs to hepatitis B virus (HBV) transgenic mouse led to the production of antibody to HBsAg (anti-HBs) in the sera. However, the immune modulatory potential of HBsAg-pulsed DCs has not been evaluated in human due to two major factors: (1) the methodology of production of immunogenic HBsAg-pulsed human DCs has not been optimized and (2) there are major concerns about the safety of HBsAg-pulsed human DCs in human. The aim of this study was: (1) to prepare immunogenic HBsAg-pulsed human DCs, (2) to assess their safety in human and (3) to evaluate their immunogenicity in vivo.

 

Methods:

Human DCs were obtained by culturing an adherent population of peripheral blood mononuclear cells with granulocyte-macrophages colony stimulating factor and interleukin-4 for 7 days. DCs were incubated with a commercial vaccine containing 5-10 microgram of HBsAg (subtype, adw) for 8-24 hours. After washing for 5 times in media, the expressions of HLA DR and CD86 of HBsAg-pulsed DCs were assessed by flow cytometry. Five million HBsAg-pulsed DCs were administered, interdermally, once to two anti-HBs-positive normal subjects, five HB vaccine nonresponders and one patient with chronic hepatitis B (CHB).

 

Results:

HBsAg-pulsed DCs did not contain any free HBsAg, endotoxin, toxoplasma and mycoplasma. HBsAg-pulsed DCs expressed higher levels of HLA DR and CD86 compared to unpulsed DCs. No volunteers exhibited any physical or biochemical evidences of inflammation, autoimmunity, liver or kidney dysfunction (at day 1, 3, 7, 14, 28 and 56 after administrations of HBsAg-pulsed DCs). Patient with CHB only showed slight elevation of serum alanine aminotransferase. The levels of serum anti-HBs increased 5-20 times due to a single injection of HBsAg-pulsed DCs in two anti-HBs-positive normal volunteers. To our surprise, all HB vaccine nonresponders developed detectable levels of anti-HBs within 2 weeks of a single injection of HBsAg-pulsed DCs. Anti-HBs was detected in the sera within 2 weeks of administration of HBsAg-pulsed DCs in one patient with CHB in the sera.

 

Conclusions:

This is the first report about the preparation of antigen-pulsed DCs for human usage in which antigen pulsing resulted in upregulation of HLA DR and CD86 on DCs. HBsAg-pulsed DCs were completely safe and induced anti-HBs in vivo in all subjects. HBsAg-pulsed human DCs represent a new and noble prophylactic vaccine for HB vaccine nonresponders. HBsAg-pulsed DCs could also be used as a therapeutic tool for patients with CHB.

 

 


Poster 20

PEGINTERFERON ALFA-2A (40KD) (PEGASYS(r)) MONOTHERAPY AND IN COMBINATION WITH LAMIVUDINE IS MORE EFFECTIVE THAN LAMIVUDINE MONOTHERAPY IN HBEAG-POSITIVE CHRONIC HEPATITIS B: RESULTS FROM A LARGE, MULTINATIONAL STUDY

George Lau, Queen Mary Hospital, Hong Kong, Hong Kong Special Administrative Region of China; Teerha Piratvisuth, Songklanakarin Hospital, Songkla, Thailand; Kang Xian Luo, Nangfang Hospital, Guangzhou, China; Patrick Marcellin, Hôpital Beaujon, Clichy, France; Satawat Thongasawat, Chiang Mai University, Chiang Mai, Thailand; Graham Cooksley, Royal Brisbane Hospital, Herston, Australia; Edward Gane, Middlemore Hospital, Otahuhu, New Zealand; Michael Fried, University of North Carolina, Chapel Hill, NC; Wan Cheng Chow, Singapore General Hospital, Singapore, Singapore; Seung Woon Paik, Samsung Medical Centre, Seoul, Republic of Korea; Wen Yu Chang, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan Republic of China; Thomas Berg, Charité Humboldt Universität zu Berlin, Berlin, Germany; Robert Flisiak, University of Bialystok, Bialystok, Poland; Friederike Zahm, Roche, Basel, Switzerland; Nigel Pluck, Roche, Welwyn, United Kingdom.

 

Background:

Recent data show that peginterferon alfa-2a (40KD) (PEGASYS(r)) gives significantly higher post-therapy response rates than lamivudine in HBeAg-negative chronic hepatitis B (CHB). Combining peginterferon alfa-2a and lamivudine did not improve response rates over peginterferon alfa-2a alone [Marcellin et al, J Hepatol 2004].

 

Aims:

In this study, the efficacy and safety of peginterferon alfa-2a with and without lamivudine vs lamivudine alone has been evaluated in HBeAg-positive CHB.

 

Methods:

Randomized, partially double-blind multinational study. Patients with HBeAg-positive CHB (n=814) received (1:1:1):

1) Peginterferon alfa-2a (40KD) (PEGASYS(r)) 180 µg once weekly (qw) + placebo once daily (qd)

2) Peginterferon alfa-2a (40KD) (PEGASYS(r)) 180 µg qw + lamivudine 100 mg qd

3) Lamivudine 100 mg qd.

Patients were treated for 48 weeks and assessed after 24 weeks of treatment-free follow-up.

 

Results:

 Baseline characteristics were comparable in all treatment groups. The overall patient population was predominantly Asian (85-87%). After 24 weeks follow-up (week 72), the proportion of patients achieving predefined co-primary endpoints (HBeAg seroconversion or HBV DNA <100,000 copies/ml), and secondary endpoints (HBeAg loss and ALT normalization), was significantly higher with peginterferon alfa-2a monotherapy or combination therapy than with lamivudine monotherapy (see table).

HBsAg seroconversion at week 72 was reported in 16 patients receiving peginterferon alfa-2a (± lamivudine) compared with none receiving lamivudine monotherapy.

Withdrawals from treatment for safety reasons were low across all groups (<=3%). The majority of adverse events were mild in nature and incidence of serious adverse events was low in all treatment groups (2-6%). Adverse events were comparable between peginterferon alfa-2a monotherapy and the combination therapy.

 

Conclusions:

Significantly higher post-therapy response rates were achieved with peginterferon alfa-2a (40KD) (PEGASYS(r)) monotherapy or combination therapy than with lamivudine monotherapy in patients with HBeAg-positive CHB. Combining peginterferon alfa-2a and lamivudine did not improve response rates over peginterferon alfa-2a alone. No unexpected adverse events were reported for peginterferon alfa-2a and the addition of lamivudine did not significantly alter the peginterferon alfa-2a safety profile.

 

 

PEGASYS® + placebo (n=271)

PEGASYS® + lamivudine (n=271)

lamivudine (n=272)

Co-primary endpoints

HBeAg seroconversion

32% (P<0.001)*

27% (P=0.023)*

19%

HBV DNA <100,000 copies/ml

32% (P=0.012)*

34% (P=0.003)*

22%

Secondary endpoints

HBeAg loss

34% (P<0.001)*

28% (P=0.043)*

21%

ALT normalization

41% (P=0.002)*

39% (P=0.006)*

28%

* compared with lamivudine therapy

 

 

 


Poster 21

INDUCTION OF HBS-SPECIFIC T CELL RESPONSES IN HBV CHRONIC CARRIERS BY AN HEPATITIS B VIRUS DNA VACCINE

Marie-Louise Michel, Maryline Bourgine, Institut Pasteur, Paris, France; Hélène Fontaine, Hôpital Necker, Paris, France; Daniel Scott-Algara, Institut Pasteur, Paris, France; Christian Brechot, Stanislas Pol, Hôpital Necker, Paris, France.

 

 

Background:

In the 370 millions HBs Ag chronic carriers, the viral persistence is thought to be related to poor HBV-specific T-cell responses.

 

Aim:

To investigate, in a phase I clinical trial, whether specific HBV DNA vaccination could restore T-cell responsiveness.

 

Methods:

Ten patients with chronic active hepatitis B nonresponder to approved treatments for HBV infection were given 4 intramuscular injections of 1 mg of a DNA vaccine encoding HBV envelope proteins. HBV-specific T-cell responses were assessed by proliferation, ELISPOT assays and tetramer staining. Secondary end points included safety and the monitoring of HBV viremia and serological markers.

 

Results:

Proliferative responses to hepatitis B surface antigen were detected in two patients after DNA injections. Few HBV-specific interferon-g secreting T-cells were detectable before immunization, but the frequency of such responses was significantly increased by three DNA injections. Immunization was well tolerated. The mean viremia decreased from 2384 ± 4725 to 1483 ± 1906 pg/ml, with a level lower than 2.5 pg/ml in 2. HBe Ab appeared in 2 patients with an HBe seroconversion in one.

 

Conclusion:

This study provides evidence that HBV DNA vaccination is safe and immunologically effective and demonstrates that DNA vaccination can specifically activate T-cell responses in chronic HBV carriers.

 

 


Poster 22

A DOUBLE-BLIND, PLACEBO-CONTROLLED TRIAL OF EMTRICITABINE (FTC, EMTRIVA) ADMINISTERED ONCE-DAILY FOR TREATMENT OF CHRONIC HEPATITIS B VIRUS (HBV) INFECTION

Mitchell L. Shiffman, Virginia Commonwealth University Health System, Richmond, VA; T. M. Ng, Changi General Hospital, Singapore, Singapore; Z. Krastev, Medical University, Sofia, Bulgaria; I. A. Kotzev, Medical University, Varna, Bulgaria; G. Mechkov, Medical University, Sofia, Bulgaria; N. N. S. Kung, United Christian Hospital, Kowloon, Hong Kong Special Administrative Region of China; S. Chan, New York Hospital at Queens, Flushing, NY; F. Rousseau, J. Anderson, E. Mondou, Gilead Sciences, Inc., Durham, NC; J. Sorbel, Gilead Sciences, Durham, NC; S. G. Lim, National University Hospital, Singapore, Singapore.

 

 

Aims:

The current study was a randomized, double blind, placebo controlled trial to assess the safety and efficacy of FTC in the treatment of chronic HBV. Emtricitabine (FTC) is a cytidine analog that selectively inhibits the HBV DNA polymerase.

 

Methods:

All patients were HBsAg positive, HBeAg positive or negative and HBV DNA positive, had elevated serum ALT within 3 months of enrollment and a necroinflammatory score of >3 (Knodell HAI) on liver biopsy (LBX). Patients were randomized (2:1) to receive either FTC 200 mg QD or placebo for 48 weeks at which time the LBX was repeated. HBV DNA was assessed by the Digene assay (lower limit of detection (LLD) 4700 copies/mL). Patients with detectable HBV DNA at week 48 were typed for presence of YMDD mutation. The primary efficacy parameter was histological response; defined as a reduction of at least two points in the Knodell necroinflammatory score and lack of fibrosis progression. The primary safety parameter was failure to tolerate study medication. Secondary endpoints included virologic, serologic and, biochemical responses and emergence of drug resistant mutations.

 

Results:

The study population consisted of 248 patients with mean age of 40 yrs, 71% male, 59% Asian and 38% Caucasian; 63% were HBeAg positive. After 48 weeks of treatment, serum ALT was normal in 65% vs 25% of FTC and placebo treated patients (p<0.001) and HBV DNA was undetectable in 56% vs 7% (p<0.001) respectively. 43% of patients treated with FTC had both normal ALT and undetectable HBV DNA vs 4% in the placebo group (p<0.001). Similar results were observed in both HBeAg positive and negative patients. 12% of HBeAg positive patients seroconverted and became HBeAg negative and anti-HBe positive. This was similar in both FTC and placebo groups. After 48 weeks, 12.6% of FTC treated patients developed YMDD mutation. Histologic response was observed in 62% vs 25% of FTC and placebo patients respectively (p<0.001). When analyzed by ranked assessment FTC treated patients had a greater improvement in both necroinflammatory activity (p<0.001) and fibrosis (p<0.009) compared to patients treated with placebo. 3% percent of patients discontinued study drug due to adverse events. The most common AEs observed were upper respiratory infection, abdominal pain, fatigue, influenza and headache. These were observed in similar frequency in both the FTC and placebo treatment groups.

 

Conclusions:

FTC at a dose of 200 mg QD produced significant histologic improvement, with both a reduction of inflammation and fibrosis when compared to placebo after 48 weeks of treatment in both HBeAg positive and HBeAg negative chronic HBV patients. FTC demonstrated potent antiviral activity and had a safety and tolerability profile similar to placebo during treatment.

 

 


Poster 23

PHASE I/II DOUBLE-BLIND, RANDOMIZED, PLACEBO-CONTROLLED STUDY OF THE NOVEL ANTI-HBV AGENT LB80380/ANA380 IN PATIENTS WITH CHRONIC HBV INFECTION

M.F. Yuen, The University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China; J. Kim, LG Life Sciences, Seoul, Republic of Korea; D. Averett, Anadys Pharmaceuticals Inc, San Diego, CA; D.K.H. Wong, The University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China; C.R. Kim, LG Life Sciences, Seoul, Republic of Korea; B. Kerr, Anadys Pharmaceuticals Inc, San Diego, CA; V.W.S. Ngai, Y.C.H. Yuen, Ching-Lung Lai, The University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China.

 

 

Background:

LB80380/ANA380 is an orally available drug that is converted after dosing to LB80331 and subsequently to LB80317, a novel guanosine phosphonate nucleotide analogue that exhibits activity against HBV in vitro, including HBV variants resistant to lamivudine. The compound has a favourable toxicity profile in vitro, including low potential for renal toxicity. Animal toxicology studies confirmed the favourable tolerability of LB80380/ANA380, and studies in woodchucks showed reductions in serum viral titre greater than six log after four weeks of dosing. Phase I studies in healthy volunteers demonstrated good safety, tolerability, and pharmacokinetics consistent with once daily dosing.

 

Aims:

We report the final study results of a Phase I/II study designed to assess the safety, pharmacokinetics, and antiviral activity of LB80380/ANA380 in HBeAg positive, HBV DNA positive patients.

 

Methods:

This study was a double-blind, randomized, placebo-controlled, multiple ascending dose evaluation of LB80380/ANA380 dosed once daily for 28 days at 30mg, 60mg, 120mg, and 240 mg. Cohorts of seven patients were randomized (6:1 active: placebo) at each dose level, and safety was established at each dose prior to dose escalation. Patients were followed for 12 weeks after completing the dosing phase.

 

Results:

The median age and male: female ratio was 27.5 years and 20:8 respectively. Serum HBV levels at screening ranged from 1x107 to 5x109. LB80380 was well tolerated, with no serious or moderate adverse events attributed to treatment. Systemic exposure to LB80331 and LB80317 was proportional to LB80380/ANA380 dose. HBV DNA reductions were observed during treatment in all patients receiving LB80380/ANA380, and returned to pre-treatment levels during follow-up. Median log serum HBV reductions on day 28 of treatment were 3 to 4 log.

 

Conclusions:

Treatment with LB80380/ANA380 for 28 days at doses up to 240mg was well tolerated and safe in this study population. Substantial anti-HBV effects were observed at all doses of LB80380/ANA380. These results encourage additional investigation for longer duration and in other study populations.

 

 


 Poster 24

VALTORCITABINE PROVIDES POTENT SUPPRESSION OF HEPATITIS B VIRUS IN PATIENTS WITH CHRONIC HEPATITIS B: RESULTS OF A PHASE I/II CLINICAL TRIAL

Ching Lung Lai, University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China; Nathaniel A. Brown, Maureen Myers, Idenix Pharmaceuticals, Cambridge, MA; Man Fung Yuen, University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China; Chun Tao Wai, National University Hospital, Singapore, Singapore; Deborah Lloyd, Keith Pietropaolo, Xiao Jian Zhou, George Chao, Idenix Pharmaceuticals, Cambridge, MA; Seng Gee Lim, National University Hospital, Singapore, Singapore.

 

 

Background:

Development of more effective monotherapies or combination therapies for chronic hepatitis B (CHB) remains a priority. The L-nucleosides L-deoxycytidine (LdC) and L-thymidine (telbivudine; LdT) are potent inhibitors of hepatitis B virus (HBV) replication in vitro. Telbivudine demonstrated significantly greater viral suppression and serum ALT normalization, compared with lamivudine, in a one-year phase IIb clinical trial in patients with CHB (Lai et al., AASLD 2003). Valtorcitabine is a well-absorbed pro-drug of LdC, and is synergistic with telbivudine for inhibiting HBV replication in vitro and in the woodchuck hepadnavirus model.

 

Aims:

Evaluation of valtorcitabine in patients with CHB has been undertaken as the initial step toward a goal of potentially developing an effective and safe combination therapy with telbivudine.

 

Methods:

A phase I/II dose escalation trial evaluated the antiviral efficacy, safety, and pharmacokinetics of two valyl ester prodrugs of LdC. Initially, a 3',5'-divalyl form was investigated in sequential dose cohorts of 50, 100, 200, and 400 mg/day. The study then switched to a more stable 3'-monovalyl form of valtorcitabine for subsequent cohorts of 300, 600, 900, and 1200 mg/day; dosing with the monovalyl form was initiated at a dosing level (300 mg/day) that approximated the LdC exposure afforded by the last divalyl dose tested (400 mg/day). Each cohort comprised 7 HBeAg+ patients with chronic hepatitis B, randomized 6:1 (drug vs. placebo). Patients were evaluated weekly during 28 days of treatment, with 12 weeks of follow-up.

 

Results: Seven of the eight dose cohorts have completed treatment. Consistent, dose-related HBV DNA reductions have been observed, ranging from a mean 1.63 log10 for the 50 mg/day group at Day 28, to 3.04 log10 copies/mL at the highest completed dose, 900 mg/day. The final dose cohort, 1200 mg/day, is ongoing and results will be reported. Emax modeling of the dose-response data indicate that maximal antiviral effects are achieved with the LdC exposure offered by valtorcitabine doses of 900 mg/day and above. Safety appears comparable to placebo, with no treatment-related pattern of adverse events or laboratory abnormalities.

 

Conclusions:

Valtorcitabine exhibits substantial anti-HBV activity and excellent safety in patients with chronic hepatitis B. Clinical investigation of the efficacy and safety of telbivudine + valtorcitabine in combination is planned.

 

 


Poster 61

VACCINATION WITH AN EXPERIMENTAL ANTI-HBV VACCINE YIELDS HIGH RESPONSE RATES IN LIVER TRANSPLANT PATIENTS AND ALLOWS SAFE DISCONTINUATION OF HEPATITIS B IMMUNOGLOBULINS PROPHYLAXIS

Peter Starkel, Anne Bouvier, St. Luc University Hospital, Brussels, Belgium; Michel Stoffel, Glaxo-Smith-Kline, Rixenart, Belgium; Jan Lerut, Yves Horsmans, St. Luc University Hospital, Brussels, Belgium.

 

 

Background:

Efficient protection against HBV is rarely obtained in liver transplant candidates and transplanted patients with currently available vaccines. Strategies using lamivudine and hepatitis B immunoglobulins (HBIG) for prevention of hepatitis B re-infection after liver transplantation (LT) are expensive since life long treatment is needed.

 

Aim:

To evaluate the possibility to obtain protective anti-HBs titers after LT and to discontinue HBIG prophylaxis after a reinforced course of hepatitis B vaccination using an experimental adjuvanted HbsAg/AS04 vaccine (GSK, Rixensart) in patients transplanted for hepatitis B.

 

Methods:

Fifteen patients on stable low immunosuppression (tacrolimus, cyclosporine monotherapy) were vaccinated with a double dose of the vaccine at 0,1,2,6 and 12 months: 5 patients transplanted for non-viral diseases and 10 consecutive patients transplanted for HBV on HBIG monotherapy. HBIG were continued during baseline vaccination (0,1,2) and then when anti-HBs titres determined every 6 weeks dropped below 150 IU/ml. Follow-up consisted of serum transaminases before and 4 weeks after each injection of the vaccine, anti-HBs titres every 6 weeks, HBs antigen every three months and HBV-DNA at 0,3,6,12 and 18 months. Follow-up period was 18 months. Response to the vaccine was defined as anti-HBs titres > 500 IU/ml 6 weeks after vaccination without prior administration of HBIG in the HBV group. Sustained long-term response was defined as anti-HBs titres > 500 IU/ml during a follow-up period of at least 12 months without further need for HBIG administration in the HBV group.

 

Results:

Overall sustained response to vaccination was 53 % (8/15 patients). 80% (4/5 patients) in the non-viral disease group and 40% (4/10 patients) in the HBV group developed a sustained long-term response (anti-HBs > 1000 IU/ml) and were free of HBIG at the end of the 18 months' follow-up. Three patients had their HBIG requirements reduced by almost 40% during vaccination without meeting the criteria for a sustained long-term response. One patient in the non-viral disease group and 3 patients in the HBV group did not respond to vaccination. No HBV recurrence, rejection or side effects were seen in any of the patients during the 18 months of follow-up.

 

Conclusions:

Protective anti-HBs titers were obtained in a substantial number of LT patients following a reinforced course of HBV vaccination with a vaccine containing a new immuno-stimulating adjuvant. It was possible to withdraw HBIG immunoprophylaxis in almost half of the patients in the HBV group, an interesting and cost effective alternative in prophylactic regimens in the future. Furthermore, vaccination with this vaccine seems well tolerated and safe in LT patients.

 

 


Poster 62

LIVING DONOR LIVER TRANSPLANTATION FROM HEPATITIS B CORE ANTIBODY POSITIVE DONORS

Arzu Celebi, Zeki Karasu, Murat Kilic, Tijen Ozacar, Fatih Tekin, Fulya Gunsar, Galip Ersoz, Yildiray Yuzer, Yaman Tokat, Ege University School of Medicine, Izmir, Turkey.

 

 

Background:

Liver allografts from donors previously exposed to hepatitis B virus (HBV) carries the risk of transmission of HBV infection to the immunosuppressed recipient. However, exclusion of the donor candidates with the serologic evidence of resolved hepatitis B - HBV surface antigen (HBsAg) negative and HBV core antibody (anti-HBc) positive - is not feasible in countries endemic for HBV virus.

 

Aim:

Our aim was to assess the safety and outcome of living donor liver transplantation from anti-HBc positive donors.

 

Methods:

152 consecutive living donor liver transplantations were performed in our institution between June 1999 and April 2003. Among these 152 living donors, 56 (37%) were anti-HBc positive. All of the anti-HBc positive donors had normal liver function tests and liver biopsies. Among 56 recipients, 36 were transplanted for HBV related cirrhosis while 20 of the recipients were transplanted for reasons other than HBV. The recipients who were HBsAg negative, recieved prophylaxis with lamivudine (100 mg/day) alone while those transplanted for HBV cirrhosis received low dose hepatitis B immune globulin combined with lamivudine.

 

Results:

The mean follow-up time for the 56 recipients was 17 (1-56) months. None of the HbsAg negative recipients developed de novo HBV infection under lamivudine monoprophylaxis and only 1 of the recipients transplanted for HBV cirrhosis developed recurrent HBV infection.

 

Conclusion:

The use of liver allografts from anti-HBc positive living donors is reasonably safe even in HbsAg-negative recipients under prophylaxis of lamivudine. These allografts may help to increase the number of available organs in countries with limited number of cadaveric organ donation and high prevalence of HBV. There is no need to administer HBIG in addition to in addition  to lamivudine prophylaxis for the liver recipients who receive anti-HBc positive donor organs.

 

 


Poster 63

THE EFFECT OF HEPATITIS B REPLICATION STATUS ON HEPATITIS B IMMUNOGLOBULIN DOSE REQUIREMENTS AND PHARMACOKINETICS IN LIVER TRANSPLANTATION FOR HEPATITIS B VIRUS

RC Dickson, Mayo Clinic, Jacksonville, FL; Nora Terrault, U. of California, San Francisco, CA; Michael Ishitani, Mayo Clinic, Rochester, MN; Rajender Reddy, U. of Pennsylvania, Philadelphia, PA; Patricia Sheiner, Westchester Med Center, Hawthorn, NY; Consuelo Soldevila-Pico, U. of Florida, Gainesville, FL; Velimir Luketic, Virginia Commonwealth U., Richmond, VA; Richard Brundage, U. of Minnesota, Minneapolis, MN; Donald Jensen, Rush Univ., Chicago, IL; Michael Fried, UNC, Chapel Hill, NC; Robert S. Brown Jr., Columbia, NY, NY; Larry Muenz, Gary Horwith, Nabi, Rockville, MD; Anna Lok, U. of Michigan, Ann Arbor, MI.

 

Background:

Lamivudine combined with Hepatitis B Immune Globulin(HBIG) successfully prevents post liver transplant (LT) HBV recurrence. However, the effect of lamivudine suppression of HBV on the dose requirements and pharmacokinetics (PK) of HBIG is not well understood.

 

Aim:

To assess effect of replication status on the PK, antibody titers and dosage requirements of an intravenous 5% hepatitis B immune globulin (Nabi HBVIg) in the first 36 weeks post LT. We report the final results of our prospective, multicenter trial.

 

Methods:

Adults undergoing LT for chronic HBV received Lamivudine prior to or at time of LT and IV Nabi HBVIg 10,000 IU on day 0 x 2, on days 1-7, weeks 4 and 8, and then 5,000 IU every 4 weeks. Anti-HBs titers were obtained at peak and trough with each dose and at scheduled time points between doses. Additional HBVIg 5,000 IU/L IV was to be given if projected levels were below 500 IU in the first 12 weeks or below 250 IU in weeks 12-36. Replicative status based on serum HBV DNA > or < 5 pg/ml (replicator =R, nonreplicator =N) was determined first at time of Lamivudine initiation (R or N), then at time of LT (r or n), resulting in 3 groups: Nn, Rn, Rr.

 

Results:

30 patients (10 Nn, 13 Rn, 6 Rr, 1 unknown) mean age of 52 years underwent LT from 12/99 to 5/01. 23patientscompleted the 36 week trial and 7 did not due to death in 4 (day 3 to week 8), consent withdrawn in 3 (week 4 to 30). 2patientswere excluded: anti-HBs at time of LT (1), replication status unknown (1). 1 pt had data censored for the first 12 weeks due to reLT on day 2, and 1 pt had data censored post reLT at week 28. Patient and graft survival was 87% and 80% respectively. Analysis of paired serum HBsAg and anti-HBs revealed: HBsAg was present during week 1 in 9/11 with anti-HBs < 300 IU/L vs 1/44 with anti-HBs > 300 IU/L; during weeks 2-12, in 4/7 with anti-HBs < 200 IU/L vs 2/112 with anti-HBs > 200 IU/L; and during weeks 12-36 in 0/4 with anti-HBs <100 and 0/127 with anti-HBs >100 IU/L. No pt had HBsAg present at the end of follow up. The number ofpatientswith trough anti-HBs titers below the target levels and T1/2 of HBVIg for the different replicative groups are listed in the table. a = CMH test, b = One way ANOVA.

 

Conclusions:

Patients with active replication at LT have increased anti-HBs requirements for neutralization of HBsAg during weeks 1-12. However, preLT suppression of HBV replication (Rn) leads to PK similar to native nonreplicators (Nn) within 1 week after LT. This trial provides rational for the use of lower dose HBVIg in Nonreplicators (Nn, Rn) after week 1.

 

 

Period

Nn

Rn

Rr

p value

Titers <300

Day1-2

5/10(50%)

8/11(73%)

6/6(100%)

0.0367a

Titers <300

Day 3-7

1/9(11%)

3/11(27%)

6/6(100%)

0.0012a

Titers <200

Day 8-30

0/8

0/12

4/6(67%)

0.0017a

Titers <200

Day30-Week12

0/8

0/11

2/4(50%)

0.015a

Titers <100

Week 12-36

0/7

0/11

1/3(33%)

0.10a

Half life (hrs)

Day 1-2

59.1

25.7

0.7

<0.0001b

 

Day3-7

83.6

53.9

5.1

<0.0001b

 

Day 8-30

266.2

254

123.4

0.18b

 

Day>30

445.7

512

204.4

0.11b

 

 


Poster 64

TREATMENT OF LAMIVUDINE RESISTANCE HEPATITIS B PATIENTS AFTER LIVER TRANSPLANT: RESULTS OF A SINGLE CENTER EXPERIENCE

Kamran Safdar, Guy Neff, Hideo Yoshida, Jose Nery, Caio Nery, Seigo Nishida, Juan Madariaga, Andreas Tzakis, Eugene Schiff, University of Miami, Miami, FL.

 

 

Background:

The development of therapeutic agents against HBV adefovir dipivoxil and tenofovir disoproxil fumarate, has improved post transplant survival in patients suffering from LAM resistance.

 

Aim:

To analyze the time to HBV DNA suppression and normalization of ALT following the administration of ADV or TNF in liver transplant recipients with HBV suffering from lamivudine resistance.

 

Methods:

We retrospectively analyzed data form our center July 1992-November 2003. During that time 16 patients were found who were HBV DNA positive at the time of surgery. All the patients in post liver transplant period received HBIG and or lamivudine whenever they became available. All patients were closely monitored clinically and biochemically.

 

Results:

The median time to develop LAM resistance was 1,254 days. The evidence of HBV resistance to LAM was documented by rise in ALT and appearance of HBV DNA. None of the patients at that time had acute cellular rejection. Seven patients were started on tenofovir and all but one was maintained on standard lamivudine therapy. Nine patients were given adefovir and all but two were continued with lamivudine. Patients were then monitored for normalization of ALT and disappearance of HBV DNA. Median time for ALT to normalize was 111 days in tenofovir group and 252 days in adefovir group (p value=0.090). The median time for HBV DNA to disappear was 214 days in tenofovir group and 595 in adefovir arm (p value=0.020). Two patients on adefovir never became HBV DNA negative and in addition three broke through with appearance of HBV DNA and was rescued with addition of tenofovir. In contrast one out of seven patients on tenofovir arm had HBV break through and remainder remained HBV negative. The sole patient with tenofovir resistance was started on adefovir. There was no statistical difference in the numbers of viral break through in adefovir and tenofovir arms(p=0.102).

 

Conclusion:

The above data demonstrates the effectiveness of HBV DNA suppression when administering TNF. More importantly is that the period of time to HBV DNA suppression is decrease in the group of patients receiving TNF. Further prospective studies are needed to validate this finding.


Poster 65

LAMIVUDINE PROPHYLAXIS WITH ADEFOVIR SALVAGE IN HEPATITIS B PATIENTS UNDERGOING LIVER TRANSPLANT IS MORE COST EFFECTIVE THAN COMBINATION LAMIVUDINE/HBIG THERAPY

Yock Young Dan, Seng Gee Lim, National University Hospital, Singapore, Singapore.

 

 

Background:

Combination high dose Hepatitis B immunoglobulin with lamivudine prophylaxis (Lam/HBIG) is highly effective in preventing Hepatitis B (Hep B) recurrence post-transplant but is expensive and inconvenient. Lamivudine resistant Hepatitis B, which has limited the usefulness of lamivudine monoprophylaxis in the transplant setting, can now be effectively controlled with adefovir dipivoxil.

 

Aims:

We performed a decision and cost-effectiveness analysis comparing the strategies of lamivudine prophylaxis with adefovir salvage after development of lamivudine resistance (lam/adv), and standard combination Lamivudine/HBIG prophylaxis.

 

Methods:

Markov modeling was performed with TreeAge Pro 2004 software with analysis performed from societal perspective up to 5 years post transplant. Probability rates were derived from systematic review of the literature and cost taken from actual Medicare database. Payoffs were discounted 3% annually. Outcome measures were cost effectiveness ratio (CER) per quality adjusted life year (QALY), incremental cost effectiveness ratio and cost, per incidence of hepatitis B recurrence. All projections and estimates were widely varied in sensitivity analysis to determine validity as well as to study the impact of variables on cost-effectiveness.

 

Results:

Combination Lam/HBIG cost USD $330,000 per patient over 5 years with 8% hepatitis B recurrence and 5% mortality. This translates to USD $109,000/ QALY saved. Lamivudine/adefovir therapy cost USD $27,000 and has 19% recurrence but only slightly higher mortality of 9%. The cost effectiveness ratio using lam/adv is only USD $10,070/ QALY saved. As the incremental benefit of lam/HBIG is marginal, the incremental cost effectiveness of lam/HBIG over lam/adv treatment is a staggering USD2 million per QALY. Cost to prevent each Hep B recurrence is USD $3 million using Lam/HBIG compared to USD $145,000 for lam/adv. Cost-effectiveness is most sensitive to cost of HBIG. If the price of HBIG were to drop to USD $40,000 per year, CER will go below USD $50,000 per QALY. Hep B recurrence rate and mortality rate post liver transplant has minimal impact on cost effectiveness.

 

Conclusion:

Adefovir dipivoxil in the post-transplant setting, has been shown to retard disease progression due to Hepatitis B recurrence and has fairly good outcome. Lamivudine prophylaxis followed by adefovir salvage after the emergence of lamivudine resistance appears to offer a far cheaper option for Hep B patients undergoing liver transplant compared to Lam/HBIG prophylaxis. Long term data and prospective trials are needed to define the optimal treatment strategy.

 

 


Poster 66

RELATION BETWEEN HBV GENOTYPES AND HBV VARIANTS AND INDICATIONS FOR LIVER TRANSPLANT (LT) IN HEPATITIS B PATIENTS IN THE U.S.*

Anna S. Lok, University of Michigan, Ann Arbor, MI; A. Regev, University of Miami, Miami, FL; E. B. Keeffe, Stanford University, Palo Alto, CA; S. H. Han, UCLA, Los Angeles, CA; S. Emre, Mount Sinai Medical Center, New York, NY; M. Ishitani, Mayo Clinic, Rochester, MN; V. Luketic, Virginia Commonwealth University, Richmond, VA; R. Brown, Columbia University, New York, NY; Scott K. Fung, M. Hussain, HBV-OLT Study Group, University of Michigan, Ann Arbor, MI.

 

 

Background:

HBV genotype C and core promoter variants have been reported to be associated with an increased risk of hepatocellular carcinoma [HCC], but the data remain controversial and most of the studies were conducted in Asia.

 

Aims:

To determine the relation between HBV genotypes, core promoter [CP] (A1762T and G1764A) and precore [PC] (G1896A) variants and indications for LT in a cohort of hepatitis B patients in the U.S.

 

Methods:

Sera from patients enrolled in the U.S. HBV-OLT Study with detectable HBV DNA by PCR were tested for HBV genotype, CP and PC variants using InnoLipa assay.

 

Results:

82 patients, mean age 50.4± 10.0 years, 58 M/24 F were included; 33 were Asians, 30 whites, 4 blacks, 7 Hispanics, and 8 others. Indications for LT were cirrhosis (n=54), HCC (n=22) and acute liver failure (n=6). Distribution of HBV genotypes was: A 31%, B 15%, C 35%, D 17% and F 2%. CP variant was detected in 67 (82%) patients and PC variant in 34 (41%) patients. Compared to patients listed for cirrhosis, patients listed for HCC were more likely to be Asians (p=0.04) and had a slightly higher prevalence of genotype C (50% vs. 32%) but there was no difference in prevalence of PC or CP variants. Asian patients were younger than non-Asians (mean age 46.8 years vs. 52.7 years, p=0.008) and more likely to have HCC as indication for LT (42% vs. 16%, p=0.02). Distribution of HBV genotypes among Asians was: A 3%, B 21%, C 73% and D 3%; and among non-Asians: A 50%, B 10%, C 8%, D 27% and F 4%. HCC was the indication for LT in 43% of Asians with genotype B and 42% of those with genotype C; and in 21% of non-Asians with genotype A and 15% of those with genotype D. All 6 patients listed for acute liver failure were non-Asians, 5 had genotype A, 3 had CP and 2 had PC variants.

 

Conclusions:

Asians comprised 40% of hepatitis B patients listed for LT in the U.S. Asian patients were younger and more likely to have HCC. There was no association between HBV genotype, PC and CP variants and HCC as an indication for LT among Asians as well as non-Asians. Patients with acute liver failure were non-Asians, younger, and more likely to be female and to be infected with genotype A. Further studies are required to confirm the relationship of genotype on the clinical outcome of HBV infection.

 

*This study was supported by NIDDK, NIH.

 

 


Cirrhosis (n=54)


HCC (n=22)


P
-Value

Mean Age ± SD (yrs)

51.9 ± 8.2

49.8 ± 11.1

0.4

% Asians

35

64

0.04

% Genotype A

26

27

0.8

% Genotype B

17

14

0.9

% Genotype C

32

50

0.2

% Genotype D

21

9

0.4

% Genotype F

4

0

0.9

% PC variant

42

41

0.8

% CP variant

85

82

0.9

 

 


Poster 70

ENTECAVIR IS SUPERIOR TO LAMIVUDINE FOR THE TREATMENT OF HBEAG(+) CHRONIC HEPATITIS B: RESULTS OF PHASE III STUDY ETV-022 IN NUCLEOSIDE-NAïVE PATIENTS

TT Chang, National Cheng Kung University Hospital, Tainan, Taiwan Republic of China; R Gish, California Pacific Medical Center, San Francisco, CA; R de Man, Erasmus University Hospital, Rotterdam, Netherlands; A Gadano, Hospital Italiano, Buenos Aires, Argentina; J Sollano, University of Santo Tomas, Manila, Philippines; KH Han, Severance Hospital, Yonsei University College of Medicine, Seoul, Democratic People's Republic of Korea; Z Goodman, Armed Forces Institute of Pathology, Washington, WA; J Zhu, A Cross, D DeHertogh, D Apelian, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT; the BEHoLD Study Group.

 

 

Background:

Entecavir (ETV) is a potent and selective inhibitor of hepatitis B virus (HBV) polymerase.

 

Aim:

This Phase III trial compared the efficacy and safety of treatment with ETV 0.5 mg QD to treatment with lamivudine (LVD) 100 mg QD for 48 weeks in nucleoside-naïve (< 12 weeks of prior nucleoside therapy), HBeAg(+) chronic hepatitis B patients.

 

Methods:

ETV-022 is a multinational, double-blind, comparative Phase III trial in which 715 patients were randomized 1:1 to receive ETV 0.5 mg QD or LVD 100 mg QD. Eligible patients were HBeAg(+) and had HBV DNA > 3 MEq/mL by bDNA assay, ALT 1.3 -10 x ULN, and compensated liver function. The primary endpoint, Histologic Improvement, was the proportion of patients having a > 2-point decrease in the Knodell necroinflammatory score and no worsening of fibrosis (worsening: > 1-point increase in Knodell fibrosis score). Results: Mean baseline viral loads by PCR were ETV: 9.61 log10 c/mL; LVD: 9.69 log10 c/mL. Mean baseline ALTs were ETV: 141 U/L; LVD: 146 U/L. Results of primary and major secondary endpoints are listed below:

 

Primary and Major Secondary Endpoints, Week 48 (non-completer = failure)

Endpoint

ETV 0.5 mg
N=354 (treated)

LVD 100 mg N=355 (treated)

p-value

*Histologic Improvement (%)

72%

62%

0.0085

†HBV DNA
Mean change from baseline by PCR
(log10 c/mL)

− 6.98

− 5.46

<0.0001

HBV bDNA
< 0.7 MEq/mL (%)

91%

65%

<0.0001

HBV DNA
< 400 copies/mL (PCR) (%)

69%

38%

<0.0001

ALT Normalization (<1.25 x ULN) (%)

78%

70%

0.0136

*n = 314 with evaluable histology in each treatment group
†n = 340 for ETV and 324 for LVD with baseline and Week 48 results

 

At Week 48, HBeAg seroconversion (loss of HBeAg and gain of HBeAb) occurred in 21% of ETV and 18% of LVD patients. A greater proportion of LVD than ETV patients were observed to have virologic non-response (HBV DNA > 0.7 MEq/mL) at Week 48, and met protocol criteria to discontinue study treatment. No mutations associated with resistance to ETV were detected. Safety was comparable between the two groups: 7% of patients in both groups had serious adverse events.

 

Conclusion:

Entecavir achieves superior histologic, virologic, and biochemical improvement in HBeAg(+) chronic hepatitis B patients, with a comparable safety profile to LVD. Primary treatment with entecavir provided superior benefit over LVD in nucleoside-naïve, HBeAg(+) chronic hepatitis B patients.

 

 

Poster 181

IN VITRO CROSS-RESISTANCE TESTING OF ADEFOVIR, LAMIVUDINE, TELBIVUDINE (L-DT), ENTECAVIR AND OTHER ANTI-HBV COMPOUNDS AGAINST FOUR MAJOR MUTATIONAL PATTERNS OF LAMIVUDINE-RESISTANT HBV

William Delaney IV, Huiling Yang, Xiaoping Qi, A Sabogal, Michael Miller, Shelly Xiong, Gilead Sciences, Inc., Foster City, CA.

 

 

Background:

Four major mutational patterns of lamivudine-resistant HBV are observed in patients who fail lamivudine therapy. The L180M+M204V mutant HBV is observed most frequently in ~ 60% of lamivudine-resistant patients. Three other patterns (V173L+L180M+M204V, M204I, and L180M + M204I) are also frequently observed. All four mutant HBV strains are highly resistant to lamivudine. However, other anti-HBV agents may display differential antiviral efficacy against different patterns of lamivudine-resistant HBV.

 

Aim:

To determine the cross-resistance profiles of clinical anti-HBV candidates against different patterns of lamivudine-resistant HBV.

 

Methods:

Five stable cell lines expressing high levels of wild-type or lamivudine-resistant HBV (L180M+M204V, V173L+L180M+M204V, M204I, and L180M+M204I) were used for drug susceptibility testing. Eleven anti-HBV compounds (including entecavir, L-dT, and emtricitabine that are in phase 3 clinical trials) were tested in all five cell lines. For EC50 determination, cells were seeded in 96 well plates and treated with compounds for one week. HBV DNA was quantified in cytoplasmic extracts by real-time PCR as described previously (Weinberger. Virol. Meth. 85:75).

 

Results:

L-nucleoside analogs lamivudine, emtricitabine, L-dT, L-dC, L-dA, and clevudine demonstrated >100-fold cross resistance in the four cell lines expressing the four different patterns of lamivudine-resistant HBV. Acyclic phosphonate nucleotides adefovir, tenofovir, and alamifovir (MCC-478) retained near wild-type efficacy (0.7 to 3.8-fold increases in EC50) in cell lines expressing the four patterns of lamivudine-resistant HBV. Entecavir demonstrated variable efficacy in the different cell lines, however all patterns of lamivudine-resistant HBV were significantly less susceptible to entecavir compared to wild-type HBV. While the M204I mutant HBV showed the greatest fold reduced susceptibility (471-fold) to entecavir, the other patterns showed 37- to 164-fold reduced susceptibility. DXG showed marginal anti-HBV activity with EC50 of 63 micromolar against wild-type HBV. The M204I (±L180M) mutant HBV displayed >5-fold resistance to DXG.

 

Conclusions:

Adefovir, tenofovir, and alamifovir demonstrated consistent efficacy against wild-type and different patterns of lamivudine-resistant HBV. All patterns of lamivudine-resistant HBV were highly resistant to the L-nucleosides lamivudine, emtricitabine, L-dT, L-C, L-dA, and clevudine. Entecavir and DXG demonstrated varying levels of resistance to the four different patterns of lamivudine-resistant HBV.

 

 

Poster 182

HBV MUTANTS ASSOCIATED WITH CLINICAL RESISTANCE TO ADEFOVIR DIPIVOXIL DISPLAY ONLY SMALL DECREASES IN ANTIVIRAL SENSITIVITY IN VITRO

Stephen Locarnini, Tim Shaw, Tina Sozzi, Rosalind Edwards, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Vic, Australia; Graeme Currie, Carol Brosgart, Shelly Xiong, Gilead Sciences, Foster City, CA.

 

 

Background/Aims:

Emergence of drug resistant hepatitis B virus (HBV) is the main factor limiting the efficacy of antiviral therapy CH-B. Viral resistance to lamivudine (LMV) develops rapidly in CH-B patients; by contrast, development of resistance to adefovir dipivoxil (ADV) is slow, becoming detectable in only a small proportion (2-3%) of treated patients after 96 weeks treatment. Sequencing of clinical isolates of HBV from patients who showed sub-optimal viral load suppression during long-term treatment with ADV revealed mutations in the viral polymerase (pol) gene which resulted in rtN236T or rtA181V substitutions in the viral polymerase (Angus et al. Gastroenterology 2003;125:292-7). The aim of this study was to further characterise the effects of rt236T and rtA181V on antiviral sensitivity by means of in vitro assays.

 

Methods:

A panel of HBV mutants were generated by site-directed mutagenesis from a wild type HBV isolate (genotype D). The mutants encoded either WT HBV pol or pol with rtN236T or rtA181 substitutions in either HBeAg positive (WT) or HBeAg negative (precore (PC) G1896A stop codon mutant) background. Replicate HepG2 cell cultures were transduced with WT or mutant HBVs by means of baculovirus vectors, then exposed continuously to different concentrations (range: 1 - 10,000 nanoMol/L) of LMV, adefovir, or tenofovir (TFV) for 7 days, when intracellular HBV DNA was extracted and quantified by Southern blotting and autoradiography. Each assay was performed in duplicate on three separate occasions.

 

Results:

Viral replication in drug-treated cultures was expressed as a fraction of the amount measured in replicate drug-free controls. Statistical and graphical analyses of the resulting data revealed that both rtN236T and rtA181V confer small decreases in sensitivity to LMV, ADV and TFV. The rtN236T had more effect than rtA181V (increases in EC50 of up to about 4- and 7- fold respectively). HBeAg negativity either had no effect or was associated with a further small decrease in drug sensitivity.

 

Polymerase

HBeAg

Resistance Factor (Fold)*

 

 

LMV

ADV

TFV

None (WT)

WT (+)

1.0

1.0

1.0

 

PC (-)

1.2

1.0

1.4

rtA181V

WT (+)

1.3

1.6

1.0

 

PC (-)

2.2

3.6

1.6

rtN236T

WT (+)

2.1

6.7

3.1

 

PC (-)

4.9

6.7

3.7

 

Conclusions:

In contrast to the effects of mutations that confer LMV resistance, those associated with clinical ADV resistance cause only small decreases in drug sensitivity (1.3 - 3.6 fold and 2.1-6.7 fold for rtA181V and rtN236T respectively, based on EC50 estimates). Furthermore, rtN236T, which confers the greatest resistance to ADV confers less resistance to LMV and TFV. In general, mutants associated with ADV resistance remain relatively sensitive to LMV and TFV.

 

 

Poster 183

MOLECULAR MODELLING OF ENTECAVIR RESISTANT MUTATIONS IN THE HEPATITIS B VIRUS POLYMERASE SELECTED DURING THERAPY

Nadia Warner, Stephen A. Locarnini, Danni Colledge, Rosalind Edwards, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Vic, Australia; Peter Angus, Austin Hospital, Heidelberg, Vic, Australia; William Sievert, Monash Medical Centre, Clayton, Vic, Australia; Michael Kuiper, Victorian Partnership for Advanced Computing, Carlton South, Vic, Australia; David Chalmers, Victorian College of Pharmacy, Parkville, Vic, Australia; Angeline Bartholomeusz, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Vic, Australia.

 

 

Background/Aims:

Entecavir (ETV) resistance was found in two highly antiviral experienced patients and was confirmed by in vitro phenotypic testing [Tenney et al., Antimicrob Agents Chemother (ACC) 2004; in press]. In the first patient isolate, the mutation(s) at rtM250V +/- rtI169T in combination with the Lamivudine (LMV) resistant mutations (rtM204V + rtL180M) resulted in the ETV resistant phenotype. In the second patient isolate, the rtT184G and rtS202I alone resulted in the ETV resistant phenotype. The aims of this study were 1) to determine the molecular basis for ETV resistance by modelling of the HBV pol with ETV selected mutations in combination with the LMV resistance mutations and 2) correlate these molecular changes to functional phenotypic characteristics of the drug resistant HBV pol.

 

Methods:

A three dimensional model of the HBV pol was developed based on the HIV reverse transcriptase structures [Bartholomeusz et al., Antiviral Therapy 2004; 9: 149] and the ETV resistance mutations were then located on this model. In vitro phenotypic analysis of specific HBV mutations was performed by site-directed mutagenesis and transfection of infectious clones of HBV in the presence of either ETV or LMV using standard methods.

 

Results:

Modelling of rtM250V mutation showed the methionine interacts with the terminal two bases in the DNA and can alter the interaction with the DNA. The rtI169T mutation is located near the nucleobase moiety of the dNTP. The in vitro phenotypic testing demonstrated that the rtI169T alone is associated with only low level ETV resistance [Tenney et al., AAC 2004; in press]. Both the rtI169T and rtM250V mutations may affect dNTP binding. The mutations at rtT184G (B domain) and rtS202I (C domain) are predicted to alter the conformation of the region around the YMDD loop (the catalytic domain of the enzyme), resulting in ETV resistance.

 

Conclusion:

Two distinct classes of ETV resistance mutations were identified with different mechanisms of action. The first class of mutations (rtM250V) appear to alter the binding interaction between the primer strand of the DNA and the incoming dNTP. The rtI169T interacts with the dNTP and may work co-operatively with the mutation at rtM250V. The second class of resistance mutations (rtT184G and rtS202I) resulted in an altered geometry of the nucleotide binding pocket of polymerase near the YMDD site. Both classes of ETV resistance mutations interact co-operatively with the LMV resistance mutations at rtM204I/V providing a structural basis for the observed cross resistance between the two antiviral drugs.

 

 

Poster 184

HEPATITIS B VIRUS RESISTANCE TO ENTECAVIR INVOLVES NOVEL CHANGES IN THE VIRAL POLYMERASE

DJ Tenney, DR Langley, AJ Oliver, RE Rose, SM Levine, CF Yu, AW Walsh, RJ Colonno, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT.

Background:

Entecavir (ETV) is a potent and selective antiviral agent with demonstrated efficacy in patients with chronic hepatitis B virus (HBV), including those infected with lamivudine (LVD) refractory HBV. While LVD resistance (LVDR) substitutions decrease ETV susceptibility in vitro, sufficient intracellular levels of ETV-TP are achieved to inhibit LVDR HBV.

Aims:

Two patients with virologic rebound in phase II ETV trials revealed that additional substitutions at positions rtT184, rtS202 and rtM250 in pre-existing LVDR HBV reverse transcriptase (RT) were required for expression of ETV resistance. These substitutions did not reduce ETV susceptibility in the absence of LVDR changes.

Methods:

Analysis of additional isolates from prolonged therapy in ETV clinical trials identified further substitutions at residues rt184, rt202 and rt250 that emerged in pre-existing LVDR virus. Phenotypic analysis showed that only certain combinations of the various substitutions resulted in reduced ETV susceptibility and that at least four combined substitutions were required for maximal resistance. Likewise, only selected combinations resulted in subsequent virologic rebounds. While all LVDR viruses were also resistant to telbivudine (L-dT), all viruses, including those with ETV signature substitutions, remained susceptible to adefovir.

Results:

Despite treatment of 432 nucleoside naive subjects for at least one year with ETV, no unique ETV-resistance substitutions have been found, suggesting that prior LVDR is required to develop ETV-resistance. A unique molecular model of HBV RT was developed based on the HIV RT structure and used along with phenotypic analysis of recombinant viruses and enzymes to explore the mechanism of ETV resistance. These studies identified two unique pathways for ETV resistance with the emergence of substitutions at either residues rt184 and rt202 or at residue rt250 in LVDR HBV. In accordance with the phenotype, the model predicts modest steric hindrance to ETV-triphosphate binding in LVDR HBV. The substitutions at residues rt184 and rt202 are predicted to increase the impact of LVDR changes in the YMDD loop, affecting substrate discrimination upon nucleotide-loading and addition to the primer terminus. Changes at position rt250 impact the “primer grip” region of the RT which discriminates nucleotides in the growing DNA chain. The relationship between these substitutions and effects on each of the functional polymerase steps inhibited by ETV-TP (priming, reverse transcription and DNA synthesis) was also explored. Conclusion:

ETV phenotypic resistance emerges in a small number of subjects who have pre-existing LVDR substitutions after prolonged ETV therapy. To date, two distinct resistance pathways have been identified, requiring multiple unique RT substitutions.

 

 

Poster 185

MOLECULAR MODELLING OF HEPATITIS B VIRUS POLYMERASE AND ADEFOVIR RESISTANCE IDENTIFIES THREE CLUSTERS OF MUTATIONS

Angeline Bartholomeusz, Stephen A. Locarnini, Anna Ayres, Geoff Thompson, Vitina Sozzi, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Vic, Australia; Peter Angus, Austin Hospital, Heidelberg, Vic, Australia; William Sievert, Monash Medical Centre, Clayton, Vic, Australia; Joe Sasadeusz, Victorian Infectious Diseases Service, Parkville, Vic, Australia; David Chalmers, Victorian College of Pharmacy, Parkville, Vic, Australia; Michael Cooper, Victorian College of Pathology, Parkville, Vic, Australia.

 

Background:

An understanding of resistance to Lamivudine (LMV) or Adefovir (ADV) is crucial for development of more effective therapeutic protocols. Resistance to ADV was originally found in the D domain of the HBV polymerase (pol) at rtN236T [Angus et al., Gastro 2003: 125:292].

 

Aims:

We have now detected further HBV polymerase mutations from patients during ADV therapy. The aim of this study was to analyse the mechanism of ADV resistance and cross-sensitivity profiles using molecular modelling together with in vitro functional analysis of these newly described mutations.

 

Methods:

The HBV pol gene was amplified by PCR and sequenced from patients who failed ADV therapy. A software program that detects specific HBV mutations (SeqHepB) was used to analyse therapy-associated unique mutations. A three dimensional model of the HBV pol was developed based on the HIV reverse transcriptase crystal structures [Bartholomeusz et al., Antiviral Therapy 2004; 9: 149]. In vitro phenotypic analysis of specific HBV mutations was performed using transient transfection of infectious clones using standard techniques.

 

Results:

The ADV resistance mutations clustered into three distinct regions in the polymerase; (a) The D and A domains (rtN236T, rtP237H, rtN238T/D, rtV84M and rtS85A) (b) The B Domain (rtA181T/V) (c) The C-D inter-domain (rtV214A, rtQ215S). In vitro antiviral testing of the A and D domain HBV mutants (rtN236T, rtV84M or rtS85A) demonstrated a decrease in sensitivity to ADV. HBV encoding the B domain mutations rtA181T/V showed a reduction in sensitivity to ADV as well as a to LMV. Molecular modelling of the A and D domain mutations revealed the rtS85 directly interacts with the gamma triphosphate of ADV-TP whilst the other mutations indirectly perturb this interaction. The rtA181T/V mutations alter the position of codon rtM204, thus resulting in an indirect steric hindrance. The third group of mutations in the C-D inter-domain do not directly interact with the nucleotide binding pocket nor the primer-template DNA and may be involved in conformational interactions within the polymerase.

 

Conclusion:

We have identified three distinct regions within the HBV polymerase that are associated with ADV resistance. Molecular modelling of these mutations in association with in vitro antiviral drug resistance testing can assist in determining their significance and may provide for the physician a rational basis in the choice of the next therapeutic agent.

 

 

Poster 186

VIRAL DYNAMICS FOR LDT (TELBIVUDINE)-TREATED HEPATITIS B PATIENTS: HIGH DEGREE OF INITIAL INHIBITION WITH DOSE-DEPENDENT SECOND-PHASE HBV CLEARANCE SUGGESTS A NEW MODEL FOR HBV DYNAMICS

Avidan U. Neumann, Bar-Ilan University, Ramat-Gan 52900, Israel; Xiao Jian Zhou, Richard E. Boehme, George Chao, Christopher Fang, Nathaniel A. Brown, Idenix Pharmaceuticals, Cambridge, MA; Ching Lung Lai, University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China.

 

 

Background:

LdT (telbivudine) is a potent HBV polymerase inhibitor that rapidly suppresses viremia in patients with chronic hepatitis B (CHB) (Lai, AASLD 2001). A mathematical viral dynamics model (Neumann, Science 1998) with one population of infected cells has been used to describe the biphasic decline of HBV viremia during antiviral treatment.

 

Aims:

Here we report analyses of changes in HBV viremia during a 4-week LdT phase I/II trial, which suggest the presence of additional efficacy mechanisms, not accommodated by the current model, during second-phase HBV decline.

Methods:

Viremia in 36 treatment-naïve HBeAg-positive CHB patients was quantified weekly during 4 weeks of treatment and 8 weeks post-treatment in a phase I/II dose escalation study of LdT (25, 50, 100, 200, 400, or 800 mg/day). Serum HBV DNA was quantified by Roche COBAS PCR, and data were fitted to the viral dynamics model. Correlations between parameters were tested with the Spearman non-parametric test; differences between dose groups were tested by Mann-Whitney.

 

Results:

A biphasic decline in HBV viremia, with inflection during the second week, was observed with LdT treatment, as expected. The Week 1 slope (0.66 ± 0.14/day) is consistent with a half-life of free HBV virions of 12-24 hrs, and provides a minimal estimate of LdT effectiveness in blocking production of e = 98.8% for doses 25-200 mg and 99.4% for doses 400-800 mg (P=NS). Surprisingly, despite the high degree of initial inhibition, second phase decline was clearly dose-dependent with a mean decline of 0.27±0.08 log/wk for doses 25-100 mg and 0.41±0.14 log/wk for doses 200-800 mg (P<0.01). Post-treatment viral relapse was delayed and had slower slope in the 200-800 mg dose groups, compared to the lower doses (P<0.002). A dose dependence for second-phase viral clearance and for post-treatment viral relapse cannot be explained by the current viral dynamics model when initial effectiveness is = 96%, as was observed with LdT for all dose groups. These observations suggest that, for very potent anti-HBV agents, the viral dynamics model needs to be extended to consider a third functional compartment of latently infected cells, that contribute to a slower second-phase decline unless its activation is highly suppressed by the drug.

 

Conclusion:

HBV decline during LdT treatment is biphasic, with first-phase effectiveness consistently >99% at doses = 200 mg/day. Contrary to results with previous agents and model predictions, second phase clearance and post-treatment relapse were clearly dose dependent. These results, revealed by the LdT response data, suggest the presence of additional efficacy mechanisms pertaining to an additional functional compartment of infected cells.

 

 


Poster 212

IMPROVED SURVIVAL WITH SCREENING FOR HEPATOCELLULAR CARCINOMA IN CHRONIC HEPATITIS B - PRELIMINARY RESULTS OF A NATIONAL SURVEILLANCE PROGRAM

James Fung, Edward Gane, New Zealand Liver Transplant Unit, Auckland, New Zealand; Chris Bullen, Auckland Healthcare, Auckland, New Zealand; John Hornell, New Zealand Hepatitis Foundation, Whakatane, New Zealand; John McCall, New Zealand Liver Transplant Unit, Auckland, New Zealand.

 

 

Background:

Despite universal neonatal vaccination, almost 300 million adults currently have chronic hepatitis B virus (HBV) infection, of whom more than 500,000 die annually from hepatocellular carcinoma (HCC). Poor outcomes associated with this complication reflect late symptomatic presentation when few therapeutic options are available.

 

Aims:

Screening for HCC should enable earlier diagnosis. We report the preliminary results from an HCC screening programme and compare outcomes of screen-detected HCCs to those of symptomatic HBV-related cases diagnosed during the same period.

 

Methods:

In August 1999, a national HBV screening programme was commenced, targeting adult Maori, Pacific Islanders and Asians. All identified HbsAg carriers were offered long-term surveillance for HCC with 6 monthly measurement of serum alpha foetoprotein (AFP). Cirrhotic patients and those with a family history of HCC also underwent 6 monthly abdominal ultrasounds.

 

Results:

Between August 1999 and December 2002, 177,507 New Zealanders were screened, of whom 11,936 were HbsAg+ (6.7%). To-date, 425 patients (2.4%) have had at least one elevated AFP (>20ng/ml) during follow-up, one third of which were pregnancy-related. Of the remainder, 48 had confirmed HCC. During the same time period, 63 symptomatic HBV-related HCCs were diagnosed in the non-screened population. Of the screen-detected HCC cases, 42 had cirrhosis (88%) and tumour size was <5cm in 77%, 5-10cm in 19% and >10cm in 4%. Of the nonscreen-detected HCCs, 52 had cirrhosis (83%) and tumour size was <5cm in 25%, 5-10cm in 32% and >10cm in 43%. Treatment was possible in 76% of screen-detected HCCs (transplantation in 33%, resection in 25%, radiofrequency ablation/chemoembolisation in 18%), compared to only 15% of symptomatic cases (transplantation in 6%, resection in 9%). Median survival from diagnosis was 1284 days in screen-detected cases and 153 days in non-screened cases. One and five year survival rates were 84% and 44% in the screen-detected cases, compared to 21% and 13% in the non-screened cases (p <0.0001; Logrank Test, see Fig 1).

 


 

Conclusion:

These preliminary results suggest that screening for HCC in a population with endemic HBV may improve survival, provided liver transplantation is available. However, this is not a randomised-controlled study of screening versus non-screening and longer follow-up is needed to exclude lead-time and length biases and also to determine whether HCC screening is cost-effective.

 

 

Poster 229

SUSCEPTIBILITY TO ANTIVIRALS OF AN HBV STRAIN HARBORING POLYMERASE MUTATIONS CONFERRING RESISTANCE TO BOTH LAMIVUDINE AND ADEFOVIR

Marie-Noelle Brunelle, Anne-Carole Jacquard, Christian Pichoud, INSERM, Lyon, France; Jean-Pierre Villeneuve, Centre Hospitalier Universitaire de Montreal, Montreal, PQ, Canada; Christian Trépo, Fabien Zoulim, INSERM, Lyon, France.

 

 

Background /Aim:

Long-term lamivudine (3TC) or adefovir (PMEA) treatments lead to the emergence of resistant strains characterized by mutations within the HBV polymerase gene. As there are no other approved drugs against HBV, treatments with 3TC or PMEA are used either sequentially or in addition depending on treatment response or failure. In view of the future use of combination therapy, we have investigated the possibility of emergence of an HBV strain harboring polymerase mutations conferring resistance to both 3TC and PMEA.

 

Methods:

We constructed such an HBV strain, and assessed its genome replication capacity, and its susceptibility to 3TC, PMEA, Interferon and other drugs in development (FTC, L-FD4C, L-FMAU, PMPA) in hepatoma cell lines (Huh7 and HepG2). The N236T mutation that confers resistance to PMEA was introduced by site-directed mutagenesis into a plasmid containing 1.1 unit genome length already harboring the 3TC-resistant mutations L180M/M204V within the polymerase gene. The resulting L180M/M204V/N236T construct was transfected into hepatoma cells lines in parallel to wild-type (wt), N236T and L180M/M204V strains to compare the levels of replication of the different HBV strains, and their susceptibility to different drugs after a 5-days treatment. Intracellular DNA was purified and subjected to Southern blot analysis. The levels of viral DNA synthesis, and the inhibitory concentations 50 (IC50) were determined by phosphorimager analysis.

 

Results:

The L180M/M204V/N236T HBV strain replicates in Huh7 cells, but 3- to 3.5-fold lower than the other tested strains. Its level of replication in HepG2 cells is 4-fold lower compared to Huh7 cells. This triple mutant was 6-fold less susceptible to PMEA than the wt, and was resistant to 3TC (> 40-fold increase in lamivudine IC50), and to a 3TC + PMEA therapy (60-fold increase in bitherapy IC50). In agreement with the observed resistance to 3TC, FTC and L-FD4C, two others L-deoxycytidine analogs, had no effect on the replication of the L180M/M204V/N236T mutant, as well as L-FMAU. Interferon inhibited wild type and all 3 viral genome mutant replication equally. PMPA appeared to be the more promising nucleoside analog since the triple mutant had only a 4.5-fold reduced susceptibility to PMPA compared to wt. Interestingly, during the study, a L180M/M204V/N236T strain was identified in the viral quasi-species from a liver transplanted patient who became resistant to the combination of 3TC and PMEA, after 42 months of combination therapy for lamivudine resistance.

 

Conclusion:

The L180M/M204V/N236T mutant can replicate its genome in hepatoma cell lines and may emerge in patients who received a combination of 3TC and PMEA. PMPA was the most active compound against this multiple drug resistant strain of HBV.

 

 

Poster 230

MECHANISMS OF HYPORESPONSIVENESS OF HBV-SPECIFIC CD4+ T CELLS IN CHRONIC HEPATITIS B

Yasuteru Kondo, Koju Kobayashi, Yoshiyuki Ueno, Tohoku University, Sendai, Japan; Masaaki Shiina, Sendai National Hospital, Sendai, Japan; Hirofumi Niitsuma, Tohoku University, Sendai, Japan; Tomoo Kobayashi, Furukawa city Hospital, Furukawa, Japan; Noriatu Kanno, Tooru Shimosegawa, Tohoku University, Sendai, Japan.

 

 

Background:

We have reported impaired function of HBV-specific CTLs, namely the lack in recovery of HBcAg-specific Th1 during lamivudine therapy. Recently, increasing evidence has suggested that not only cytokine balance including IFN-?and IL-4 but direct signaling through T cell receptor is important for Th1/Th2 commitment. There have been also many reports about the possibility of inducing anergy by CD4+ CD25+ T cells (Tregs).

 

Aim:

To identify the potential therapeutic targets of chronic hepatitis B (CHB), we investigate the mechanisms involved in the Th1 hyporesponsiveness.

 

Method:

Eight patients with CHB were studied. As controls, 2 patients with acute hepatitis B and 10 healthy subjects were studied. Cytokines in PBMC culture supernatant were measured with cytokine bead array after stimulation with HBsAg and HBcAg (cytokines). Simultaneously, levels of mRNA for IL-12 receptor (IL-12R) ß1 and ß2 chains, interferon (IFN)-?, IL-4, GATA3 and T-bet in stimulated CD4+ T cells were quantified by realtime PCR. (Th polarization). IL-10-producing cells were identified by IL-10-secretion assay and antibodies to CD4, CD25, CD3 and CD14 (IL-10 assay). Role of Tregs in Th1 polarization was analyzed by IFN-?-secretion assay using Tregs-depleted cells. (Tregs depletion). Repolarization of CD4+ T cells was attempted by siRNA against GATA-3 in MOLT-4 (Re-skewing Th polarization).

 

Results:

Cytokines: cytokines in response to HBcAg in CHB (cytokines with HBcAg stimulation minus without HBcAg, pg/ml): IFN-Y (0), TNF-a (53), IL-10 (298), IL-4 (1). Cytokines in response to HBsAg: IFN-? (50), TNF-a (318), IL-10 (688), IL-4 (0). Th polarization: Changes in mRNA level in response to HBcAg (ratio of mRNA level in response to HBcAg and that without stimulation, mean stimulation index ± SEM): T-bet (0.5 ± 0.1), IFN-Y (0.8 ± 0.3), IL-12R ß2 (0.9 ± 0.1), GATA-3 (0.9 ± 0.2), IL-4 (0.5 ± 0.2). Changes in mRNA level in response to HBsAg: T-bet (1.1 ± 0.5), IFN-? (1.1 ± 0.4), IL-12R ß2 (1.5 ± 0.5), GATA-3 (2.0 ± 0.9), IL-4 (0.4 ± 0.1). IL-10 assay: Tregs secreting IL-10 was 1 % in patients with CHB and <0.1 % in normal subjects. Tregs depletion: IFN-Y secreting CD4+ cells increased to 3-fold of control. Re-skewing Th polarization: Introduction of siRNA against GATA-3 resulted in reduction of GATA-3 after 24 h and up-regulation of T-Bet after 48 h.

 

Conclusion:

In CHB, polarization towards Th1 is suppressed by both Tregs at HBcAg stimulation and polarization towards Th2 at HBsAg stimulation. Depletion of Tregs or introduction of siRNA against GATA-3 may intervene in these mechanisms of suppression of Th1 polarization.

 

 

Poster 231

VARIABILITY OF HEPATITIS B VIRUS SURFACE ANTIGEN IN CHRONIC HEPATITIS B VIRUS CARRIERS WHO PRESENT BOTH HBS AG AND ANTI-HBS ANTIBODY

Olivier Lada, Henri Agut, Thierry Poynard, Vincent Thibault, GH Pitie Salpetriere, Paris, France.

 

 

Background:

Chronic hepatitis B (CHB) is usually characterized by the persistence of hepatitis B virus surface antigen (HBsAg) in infected patients. In some cases, persistence of HBs Ag is associated with antibodies to hepatitis B surface antigen (anti-HBs).

 

Aims:

The mechanism underlying the emergence of anti-HBs in CHB is unknown but one reason might be the selection of HBs Ag immune escape variants. To assess this hypothesis, we studied the variability of the S gene of 15 chronic hepatitis B carriers who were selected on the basis of persistent concomitant HBs Ag and anti-HBs.

 

Methods:

Among 864 CHB patients followed in our laboratory, 77 (8.9%) carried both HBs Ag and anti-HBs on a same sample. Fifteen patients who presented both markers on several consecutive samples were selected. After purification of HBV DNA from the serum, whole HBV genome of each patient was PCR-amplified and cloned. The S region of 15 to 19 clones per patient was sequenced and analysed.

 

Results:

Mean anti-HBs antibody titre was 135 UI/L (range 38-434). Analysis of the a-determinant of HBs Ag has been performed so far on 11 patients (129 clones) (genotype D n=1; A n=3, C n=4, E n=2, one coinfection A and C). Eight patients (72%) presented at least 2 clones carrying at least 1 residue change within the a-determinant region (124-147) and among them 7 patients carried more than one substitution. The most frequent changes were located at position s145 (35/129, 27%), s129 (13/129, 11%) and s126 (35/129, 27%). The variant G145R was found in 3 patients with a clone frequency of 85, 80 and 31%; three patients (38%) carried the residue change I126S in 86, 87 and 37% of the clones. Three patients (38%) carried residue changes at position 129: two had a substitution Q129N in respectively 33 and 31% of their clones and one had a substitution Q129P in 13% of the clones. One patient carried a residue change D144R in 35% of the clones. Interestingly, less described mutations were also found in the a-determinant region. In two patients the substitution T125M/A and T143L/S were found with a clone frequency of 100 and 11%, respectively. Less frequent variant present in at least 2 clones included the following changes T123N, P127T, G130E, S132Y, P135H, S136F and C138Y.

 

Conclusion:

In a population of chronic hepatitis B patients carrying both HBs Ag and anti-HBs antibodies, a high frequency of residue changes within the a-determinant of HBs Ag was detected. This data suggest the existence of immune escape mechanisms that result in the selection of HBV variants less efficiently neutralized by anti-HBs antibodies.

 

 

Poster 232

IMPAIRED CAPACITY OF LIVER DENDRITIC CELLS FROM MURINE HEPATITIS B VIRUS (HBV) CARRIER TO INDUCE AND SUSTAIN HBV-SPECIFIC ANTIVIRAL IMMUNITY: RELEVANCE TO HBV PERSISTENCY

Aki Hasebe, Sk. Md. Fazle Akbar, Shinya Furukawa, Norio Horiike, Morikazu Onji, Ehime University School of Medicine, Shitukawa, Japan.

 

 

Background:

In spite of ongoing replication of hepatitis B virus (HBV) and availability of abundant amounts of HBV-related antigens in the liver, chronic HBV carriers exhibit only very weak and narrowly-focused HBV-specific immune responses. Accordingly, some defects in priming and maintenance of HBV-specific immunity are presumed in these subjects.

 

Aim:

As antigen-presenting dendritic cells (DCs) in the tissues are responsible for capturing, processing and presenting viruses for the induction of antiviral immunity, we evaluated the functions of fresh liver DCs from HBV transgenic mouse (HBV-Tg), an animal model of HBV carrier state.

 

Methods:

Murine liver was perfused with phosphate-buffered saline through portal vein. Cell suspensions containing hepatocytes and liver nonparenchymal cells (NPCs) were prepared by cutting and pressing the liver against steel mesh. Hepatocytes were depleted from the cell suspensions by density centrifugation and liver NPCs were retrieved from cultures. CD11c+ liver DCs were isolated from NPCs by two positive selection steps using CD11c-coated immunomagnetic beads in magnetic-activated cell sorter (MACS). The purity of liver DC was more than 95%. Highly purified populations of T cells were isolated by using MACS. Lymphoproliferative assays were done by culturing DCs and T cells for 4 days and [3H]-thymidine was added to the cultures during last 12 hours. The levels of blastogenesis were expressed as counts per minute (CPM). The capacity of liver DCs to produce proinflammatory cytokines was estimated by stimulating liver DCs with herpes simplex virus-1 (HSV-1) and lipopolysachcharide (LPS).

 

Results:

(1) The allostimulatory capacity of liver DCs from HBV-Tg were significantly lower than that of liver DCs from normal C57BL/6 (14,689±6,308 CPM versus 101,299±13,293 CPM, n=5, p<0.01). (2) When cultured with HSV-1 or LPS, liver DCs from HBV-Tg produced significantly lower levels of TNF-alpha and interleukin-6 compared to those produced from liver DCs from normal mouse (p<0.05). (3) In spite of being in the microenvironment of HBV, liver DCs from HBV-Tg could not produce interferon-alpha. (4) Finally, in comparison to liver DCs from normal mouse, liver DCs from HBV-Tg had significantly decreased capacity to induce proliferation of HBsAg-specific memory T lymphocytes (p<0.05).

 

Conclusions:

This is the first study that showed the functional impairment of fresh liver DCs from chronic HBV carrier. These impaired antigen handling properties of liver DCs of HBV-Tg explain the impaired priming and maintenance of HBV-specific immune response in chronic HBV carriers. Potential immune therapy for chronic HBV carrier might be developed by upregulating the function of liver DCs in vivo.

 

 

Poster 233

LOW LIVER CELL PROLIFERATION AND HIGH APOPTOSIS RATES IN PATIENTS WITH HBeAG-NEGATIVE CHRONIC HEPATITIS B RESPONDING TO INTERFERON

Maria Francesca Donato, Eliana Arosio, Pietro Lampertico, Ospedale Maggiore IRCCS Policlinico, University of Milan, Milano, Italy; Davide Trerè, University of Bologna, Bologna, Italy; Mauro Viganò, Massimo Iavarone, Ospedale Maggiore IRCCS Policlinico, University of Milan, Milano, Italy; Massimo Derenzini, University of Bologna, Bologna, Italy; Massimo Colombo, Ospedale Maggiore IRCCS Policlinico, University of Milan, Milano, Italy.

 

Background:

Patients with high liver cell proliferation and low apoptotic rates are at increased risk of developing hepatocellular carcinoma (HCC).

 

Aims:

To assess the effect of interferon (IFN) therapy on liver cell proliferation and apoptosis.

 

Methods:

We studied baseline and end-treatment liver biopsies of 39 patients with HBe-Ag negative chronic hepatitis B treated with 6 MU IFN a thrice weekly for 24 months (36 men, mean age 42 + 10 yr, ALT 252 + 218 U/l, Ishak grading 9 + 3, 20% with cirrhosis). Two liver cell proliferation and one apoptosis indices were assessed using an automated image analysis system (Proliferating Cell Nuclear Antigen immunoperoxidase staining, PCNA-LI; nucleolar silver staining, Nucleolar index; TUNEL method, APO-I). Histological activity was graded according to Ishak. End-treatment responders (ETR) were patients with normal ALT and undetectable serum HBV-DNA by Digene assay; non responders (NR) had high ALT and HBV-DNA positivity throughout treatment; sustained viral responders (SVR) had normal ALT and undetectable HBV-DNA during post-treatment follow-up. RESULTS. At the end of treatment, PCNA-LI, Nucleolar-index and Ishak grading were significantly reduced and APO-I was significantly increased (Table).

 

Results:

Significantly changes of proliferation and apoptosis scores were observed only in 24 ETR (61%) whereas Ishak grading significantly decreased in both groups of patients (Table). During 6 + 3 yr of post-treatment follow-up, 3 patients (1 SVR, 1 NR, 1 relapse) developed HCC with an annual incidence of 0.9%. The proliferative indexes decreased during treatment in all 3 patients and apoptosis increased in 2 patients.

 

Conclusions:

The reduction of liver cell proliferation and the increase of apoptosis during treatment encourage long-term administration of IFN to prevent HCC in HBe-Ag patients.

 

 

 

All (n=39)

 

 

ETR (n=24)

 

 

NR (n=15)

 

 

Baseline

End Therapy

P

Baseline

End Therapy

P

Baseline

End Therapy

P

PCNA-LI(%) (mean+SD)

1.0 + 1.1

0.5 + 0.6

=0.04

1.2 + 1.2

0.5 + 0.7

=0.01

0.5 + 0.4

0.5 + 0.4

ns

Nucleolar-index (%)(mean+SD)

0.4 + 0.4

0.2 + 0.2

=0.001

0.4 + 0.5

0.1 + 0.1

=0.001

0.3 + 0.4

0.2 + 0.3

ns

APO-I (%) (mean+SD)

0.4 + 0.2

0.5 + 0.3

=0.03

0.4 + 0.2

0.6 + 0.3

=0.03

0.3 + 0.2

0.4 + 0.3

ns

Ishak grading (mean+SD)

9.1 + 3.4

3.6 + 2.6

<0.0001

9.6 + 3.5

3.3 + 2.9

<0.0001

8.3 + 3.2

4.1 + 3.0

=0.001

 

 

Poster 234

CYTOPATHIC EFFECTS OF HEPATITIS B VIRUS INFECTION IN LONG-TERM INFECTED CHIMERIC UPA-SCID MICE

Philip Meuleman, Center for Vaccinology, Ghent University and Hospital, Ghent, Belgium; Louis Libbrecht, Rita De Vos, Tania Roskams, Laboratory of Morphology and Molecular Pathology, University of Leuven, Leuven, Belgium; Geert Leroux-Roels, Center for Vaccinology, Ghent University and Hospital, Ghent, Belgium.

 

 

Background:

Hepatocellular damage in acute and chronic HBV infections is considered to be caused by the host's immune response rather than by the virus itself.

 

Aim:

uPASCID mice transplanted with human hepatocytes were infected with HBV to examine whether HBV causes hepatocellular damage in a severely immune-deficient host.

 

Methods:

Five uPASCID mice were transplanted with human hepatocytes and subsequently infected with HBV. At several time-points post infection (p.i.), human albumin, HBsAg, HBeAg, and HBV DNA concentrations were measured in plasma. HBV infection and hepatocellular changes were monitored using immunohistochemistry and electron microscopy.

 

Results:

HBsAg, HBeAg, HBV DNA levels progressively rose in the weeks p.i. to reach peak values around week 4 (HBsAg >20,000 IU/ml; HBeAg >750 PEIU/ml; HBV DNA >5x1010 cop/ml). Thereafter, the concentrations of these markers declined. A significant correlation was observed between HBsAg and HBeAg (r=0.9808, P<0.0001), HBsAg and HBV DNA (r=0.8710, P<0.0001) and HBeAg and HBV DNA (r=0.9344, P<0.0001). There was no correlation between human albumin and any viral marker. Albumin levels remained constant for several weeks but also declined around week 4 p.i., which coincided with the start of a progressive clinical deterioration, ultimately leading to the death of the animals. Mice succumbed on average 50 days p.i., whereas uninfected animals survived for at least an additional 150 days.

 

The livers of 2 long-term infected mice (73 and 88 days p.i.) were morphologically compared with that of an animal sacrificed 4 weeks p.i. (referred to as short-term infection). Compared with short-term infection, human hepatocytes within long-term infected animals had a typical 'ground-glass aspect', showed much more pronounced staining for HBsAg and HBcAg and stained poorly for albumin. Moreover, these human hepatocytes did not proliferate and signs of considerable cell damage and loss were present, which was not the case in the short-term infected mouse liver. Ultrastructural analysis of long-term infected human hepatocytes showed an abundant presence of cylindrical HBsAg structures and Dane particles, while these structures were much less frequent in short-term infected hepatocytes.

 

Conclusion:

Long-term infection with HBV of human hepatocytes residing in a chimeric uPA-SCID mouse causes dramatic intracellular changes and hepatocellular damage, indicating that HBV has endogenous cytopathic effects. The overwhelming staining of human hepatocytes for HBcAg and HBsAg in an immune deficient host is partly reminiscent of fibrosing cholestatic hepatitis in immunosuppressed patients and suggests that cytopathic effects of HBV do not develop in an immune competent host in whom the pathogenesis of liver damage is predominantly immune-mediated.

 

 


Poster: 329

CHANGE OF HEPATITIS B VIRUS ACTIVITIES WITH TRANSCATHETER ARTERIAL CHEMOEMBOLIZATION THERAPY IN PATIENTS WITH HBV-RELATED HEPATOCELLULAR CARCINOMA

Kyung Woo Park, Joong-Won Park, Sang Hyung Cho, Hong Suk Park, Woo Jin Lee, Chang-Min Kim, Do Hoon Lee, National Cancer Center, Goyang, Republic of Korea.

 

 

Background:

Hepatitis B virus (HBV) reactivation is known to be related with host immune state. Immune suppression, such as systemic chemotherapy could reactivate HBV infection and prophylatic anti-viral therapy before chemotherapy may be necessary in cancer patients with chronic hepatitis B.

 

Background:

Transcatheter arterial chemoembolization (TACE) is one of important therapeutic modalities for unresectable hepatocellular carcinoma (HCC). TACE is not a systemic chemotherapy, but HBV reactivation after a single session of TACE was reported. However, no any case-control studies about HBV reactivation after TACE were performed.

 

Aims:

The aim of this study is to evaluate whether TACE aggravates the viral hepatitis in patients with HBV-related HCC.

 

Methods:

One hundred thirteen patients with HBV-related HCC and no history of anti-viral therapy who attended National Cancer Center between October 2003 to March 2004 were studied prospectively. Patients treated with TACE were enrolled in the case group (n=69), and patients , who had no history of TACE, waiting for the management were enrolled in the control group (n=20). Baseline and follow-up evaluation including serum HBV DNA quantification using hybridization method, viral marker study, liver function study and tumor staging study were done in both group. TACE was performed according to National Cancer Center protocol, by doxorubicin (20-60mg) and lipiodol (2-20ml), and/or gelfoam embolization.

 

Results:

Mean follow-up period was 56 days in case group, 59 days in control group. Three (4.3%) patients of case group and 2 (10%) patients of control group showed HBV reactivation (p=0.334). A Twofold or more increase of serum HBV DNA (copies/ml) was detected in 21 (30.4%) patients of case group and 4 (20%) patients of control (p=0.361). Exacerbation of viral hepatitis B, defined by the twofold or more rise of transaminase activities and HBV DNA copies, was found only in 4 (5.8%) patients of the case group but there is no statistical significance (p=0.271). Three patients of them were in spontaneous recovery within further 4-8 weeks, but one patient who had tumor progression showed continuous exacerbation. In univariate analysis on the baseline studies including HBV DNA quantification, HBeAg positivity, elevated transaminase, presence of precore mutant, and tumor characteristics, there were no any significant factors affecting a rise of HBV DNA.

 

Conclusion:

There was no differences in a change of HBV DNA activities and in the exacerbation of viral hepatitis between the case and the control groups. This study suggests that TACE using doxorubicin and lipiodol probably does not aggravate HBV hepatitis in patients with HBV-related HCC, although further proof with a large-scale study is necessary.

 

 


Poster 457

HBSAG LEVEL AT TIME OF LIVER TRANSPLANTATION PREDEFINES EARLY HBSAG AND ANTI-HBS KINETICS AND AFFECTS HBV-DNA DECREASE DURING IMMUNOGLOBULIN ADMINISTRATION

Jens Rosenau, Matthias Kujawa, Therese Solga, Matthias J. Bahr, Medizinische Hochschule Hannover, Hannover, Germany; Gerd Michel, Abbott GmbH, Wiesbaden, Germany; Ernst R. Kuse, Jürgen Klempnauer, Hans L. Tillmann, Michael P. Manns, Medizinische Hochschule Hannover, Hannover, Germany.

 

 

Background:

The administration of high doses of hepatitis-B-immunoglobulin (HBIG) during the first days after liver transplantation (OLT) of hepatitis B patients is considered important to prevent hepatitis-B-reinfections even with simultaneous application of lamivudine. However, dosing schedules differ considerably between transplant centers.

 

Aims:

We measured quantitative kinetics of HBsAg, anti-HBs and HBV-DNA to create a rational basis for dosing schemes.

 

Methods:

During the initial phase after OLT 13 patients received daily doses of 10000 IU HBIG until HBsAg became negative (group 1), whereas 5 patients received boluses of 5000 IU HBIG every 30 minutes (group 2). HBsAg was measured with the Architect assay (lower detection limit 0.05 IU/ml), anti-HBs with the Axsym assay (Abbott laboratories, Wiesbaden, Germany). HBV-DNA was determined using the COBAS TaqMan PCR-assay (Roche Molecular Systems, lower detection limit 35 copies/ml).

 

Results:

Initial HBsAg levels ranged from 0.12 to 12990 IU/ml at time of OLT. All patients became HBsAg negative during HBIG infusions (dose range 10000 to 80000 IU). A close correlation between initial HBsAg and required HBIG dose to decrease HBsAg level below 1 IU/ml was found in groups 1 and 2 (r=0.97 / p<0.001, r=1.0 / p<0.001). A close correlation was also seen between initial HBsAg and required HBIG to raise anti-HBs above 1000 IU/l (r=0.94 / p<0.001, r=1.0 / p<0.001). At time of OLT HBV-DNA was positive (range 36 to 1.8x108, median 7330 copies/ml) in 9 of 18 patients despite lamivudine pre-treatment. 5 of 9 patients became HBV-DNA negative during HBIG infusions, 4 patients remained HBV-DNA positive (range 70 to 1.1x104, median 3860 copies/ml). 17 of 18 patients were HBV-DNA and HBsAg negative at week 12 post OLT. A close correlation between HBsAg and HBV-DNA decrease could be demonstrated in a patient with high HBV-DNA and high HBsAg at time of OLT. In one patient who did not become HBV-DNA negative HBsAg reappeared 8 days after OLT.

 

Conclusions:

In conclusion, required HBIG doses to achieve HBsAg negativity and certain anti-HBs-titers differ between individuals and are essentially determined by the HBsAg serum level at time of OLT. A significant HBV-DNA decline is seen during HBIG infusions in most HBV-DNA positive patients. Therefore HBIG should be administered individualized under monitoring of HBsAg and HBV-DNA.

 

 


Poster:458

LIVER TRANSPLANTATION IN LAMIVUDINE-RESISTANT YMDD-MUTANT CARRIERS: ROLE OF ADEFOVIR-DIPIVOXIL IN PREVENTION OF HEPATITIS B VIRUS RECURRENCE

Alfredo Marzano, Molinette, Turin, Italy; Pietro Lampertico, IRCCS Maggiore Policlinico, Milan, Italy; Silvia Gaia, Molinette, Turin, Italy; Mauro Vigano, IRCCS Maggiore Policlinico, Milan, Italy; Raffaele Romito, Enrico Regalia, Vincenzo Mazzaferro, National Cancer Institute, Milan, Italy; Massimo Colombo, IRCCS Maggiore Policlinico, Milan, Italy; Alessandro Franchello, Mauro Salizzoni, Mario Rizzetto, Molinette, Turin, Italy.

 

 

Background:

Lamivudine (LAM) and Hepatitis B immunoglobulins (HBIG) in combination reduce the risk of hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT).

 

Aims:

Adefovir Dipivoxil (ADV) could be useful to prevent HBV recurrence in presence of YMDD mutants.

 

Methods:

This not controlled, pilot study includes 22 HBV-carriers who selected YMDD variant during preemptive LAM therapy before OLT.

 

Results:

Thirteen had a significant viremic breakthrough (HBV DNA>10E5 copies/ml), (group A, phenotypic mutation). Two of them continued LAM and 11 were treated with a double therapy (ADV and LAM) before OLT.

 

Moreover, 9 patients selected YMDD mutant without a significant viremic breakthrough (<10E5 copies/ml) and continued LAM therapy without ADV addition (group B, genotypic mutation).

 

Results. In group A (mean follow-up: 23 months after OLT), hepatitis B recurred in both two patients transplanted with significant viral load, treated with LAM before OLT and prophylaxed with LAM and HBIG after surgery. None of the 11 patients with HBV DNA <10E5 copies/ml, inducted by ADV therapy before OLT, and treated long-term with a double or a triple prophylaxis after surgery, developed HBV recurrence. In group B (mean follow-up: 29 months after OLT) the HBV recurred in 2 subjects who suspended HBIG post-surgery, and in none of those treated with a combined prophylaxis post-OLT.

 

Conclusions:

OLT waiting list suspension and ADV addition are needed in patients with YMDD phenotypic mutation, but no in those with the genotypic mutation. Post OLT long-term combined prophylaxis with HBIG and LAM is effective in both groups and triple prophylaxis could be the surest option in case of phenotypic mutation.

 

 


Poster: 459

LAMIVUDINE MONO PROPHYLAXIS FOR LIVER TRANSPLANT RECIPIENTS WITH HEPATITIS B-SINGLE CENTER EXPERIENCE OVER 10 YEARS

HIDEO YOSHIDA, TOMOAKI KATO, JUAN R. MADARIAGA, DAVID M. LEVI, SEIGO NISHIDA, JANG I. MOON, GENNARO SELVAGGI, KAMRAN SAFDAR, ENRIQUE MARTINEZ, EUGENE R. SCHIFF, ANDREAS G. TZAKIS, UNIVERSITY OF MIAMI, Miami, FL.

 

 

Background:

With the administration of hepatitis B immune globulin (HBIG) and lamivudine (LAM), outcome of orthotopic liver transplantation (OLT) for patients with hepatitis B virus (HBV) has significantly improved.

 

Aims:

The aim of this study is to evaluate the lamivudine mono prophylaxis post OLT for HBV infection, especially for patients with HBV-DNA negative at OLT.

 

Methods:

OLT recipients with chronic HBV infection received LAM mono prophylaxis without HBIG were analyzed. At our center, patients with HBV-DNA negative at OLT were elected to receive LAM monotherapy since 1994. LAM was given 150mg/day. Liver function tests, HBV-DNA, and serological markers of HBV of these patients were collected. The efficacies of LAM monotherapy on survival after OLT and on prevention of recurrent hepatitis B were analyzed.

 

Results:

Among 1378 patients who received OLT from January 1994 to April 2004, 130 patients (9.4%) had hepatitis B infection. 67 patients were HBV-DNA negative at OLT without co-infection with hepatitis C virus or Human Immunodeficiency Virus. Of those, 38 patients received LAM mono prophylaxis without HBIG post OLT. Median age was 54 (range 27-68), male/female was 29/9. Median follow up after OLT was 63 months (range 5-116). Five patients needed re-OLT, 3 were due to primary non function, 1 hepatic artery thrombosis, 1 delayed graft function. Five patients died from the reasons other than recurrent hepatitis at 5 to 64 month after OLT. One and 5 year patient /graft survival were 95/84% and 87/77% respectively. HBV-DNA was re-detected in 5 patients (13%) at 1 to 29 month. Recurrent hepatitis B was seen in a patient (3%) at 27 month. This patient is currently on Tenofovir therapy with partial response. There was no significant side effect with lamivudine use.

 

Conclusions:

Lamivudine mono prophylaxis after OLT is effective and safe treatment for hepatitis B patients with negative HBV-DNA. Monotherapy without HBIG may provide patients more cost-effective and more comfortable treatment.

 


Poster: 460

LIVER TRANSPLANTAION FOR HEPATITIS B ASSOCIATED END STAGE LIVER DISEASE IN KOREA

Nam-Joon Yi, Kyung-Suk Suh, Seoul national university, College of medicine, Seoul, Republic of Korea; Dong Goo Kim, Catholic medical center, Seoul, Republic of Korea; Jae Won Joh, Samsung medical center, Seoul, Republic of Korea; Soon Il Kim, Yonsei medical center, Seoul, Republic of Korea.

 

 

Background;

During the past 15 years, outcomes after liver transplantation (LT) of hepatitis B related liver cirrhosis (HBV-LC) were improved with the use of hepatitis B immune globulin (HBIG), and later, lamivudine.

 

Aims:

The aim of this study was to evaluate the current outcomes of HBV-LC after LT in Korea.

 

Methods:

From Jan. 1999 to Dec. 2002, 334 among 532 cases of LT (62.8%) were collected from four centers.

 

Results:

Preoperative active viral replication was noted in 221 cases (66.2%) who had HBeAg (+) or HBV DNA (+). For prophylaxis of HBV recurrence, combination therapy of HBIG plus lamivudine was most frequently used in 210 cases (62.9%), and secondly, HBIG monotherapy in 124 cases (37.1%). Prophylaxis consisted of HBIG infusion of 10000~20000 IU intraoperatively, 10000 IU per day for seven days postoperatively, and following infusion of 10000 IU per week for three times. After that, hepatitis B antibody titer was checked and maintained > 500 IU/L for HBIG monotherapy or > 350 IU/L for combination therapy. For combination therapy, lamivudine 100 mg po daily was added for one year or more. Immunosuppressant was calcineurin inhibitor based protocol. One-year survival rate was 81% and three-year survival rate was 69%. Overall recurrence rate was 6.6 % (22/334). The mean time interval to recurrence after transplant was 15.8 (2-40) months. The HBV recurrence rate was 8.3% with preoperative lamivudine therapy vs 5.5% with no therapy, 8.5% with whole grafts vs 6.2% with partial grafts, and 7.4% in active viral replicators vs 4.0% in inactive replicators (p>0.05). The recurrence rate in HBIG monotherapy was 4.0% and 8.1% in combination therapy (p>0.05). The recurrence rate of HB after LT in Korea was 6.6%.

 

Conclusions:

These results suggest that LT for HBV-LC was safe with low recurrence rate if providing adequate prophylaxis even in active viral replicators.

 

 


Poster LB-07

ENTECAVIR DEMONSTRATES SUPERIOR HISTOLOGIC AND VIROLOGIC EFFICACY OVER LAMIVUDINE IN NUCLEOSIDE-NAIVE HBEAG(-) CHRONIC HEPATITIS B: RESULTS OF PHASE III TRIAL ETV-027

Daniel Shouval, Hadassah-Hebrew University Hospital, Jerusalem, Israel; Ching-Lung Lai, Queen Mary Hospital, Hong Kong, China; Hugo Cheinquer, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil; Anna Lok, University of Michigan, Ann Arbor, MI; Deborah DeHertogh, University of Connecticut, Farmington, CT; Richard Wilber, Anne Cross, Richard Zink, Lori Fernandes, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT.

Background:

HbeAg-negative (HBeAg(-))chronic hepatitis B can be characterized by a progressive course of severe liver disease activity and has a generally poor long term prognosis. Entecavir (ETV) is a potent and selective inhibitor of hepatitis B virus (HBV) polymerase.

Aims:

ETV-027 is a multinational, randomized, double-blind Phase III trial comparing ETV to lamivudine (LVD) for up to 96 weeks of blinded dosing in patients with HBeAg(-) chronic hepatitis B.

Methods:

648 patients were randomized 1:1 to ETV 0.5 mg QD or LVD 100 mg QD. Eligible patients were nucleoside-naïve, HBeAg(-) and anti-HBe(+), and had HBV DNA = 0.7 MEq/mL by bDNA, and ALT levels 1.3-10 x ULN. The primary efficacy endpoint, histologic improvement, was the proportion of patients at Week 48 having a > 2-point decrease in Knodell necroinflammatory score and no worsening of fibrosis (worsening: >1 point increase in Knodell fibrosis score). Secondary efficacy endpoints included proportions with improvement in Ishak fibrosis score, mean reduction of HBV DNA, and proportions achieving the Composite Endpoint (HBV DNA <0.7 MEq/mL by bDNA and ALT normalization (<1.25 x ULN)). Results: Mean baseline HBV DNA was 7.6 log10 copies/mL by PCR in both groups; mean ALT was 141 and 143 U/L in the ETV and LVD groups, respectively. Results of primary and key secondary endpoints are given below:

 

Week 48 Study Endpoints (treated patients, non-completer = failure)

Endpoint

ETV
0.5 mg
N= 325

LVD
100 mg
N= 313

p-value

*†Histologic Improvement

70%

61%

0.0143

†Ishak Fibrosis Score Improvement

36%

38%

NS

‡HBV DNA
Mean change from baseline by PCR
(log10 copies/mL)

−5.20

−4.66

<0.0001

HBV DNA
<400 copies/mL by PCR

91%

73%

<0.0001

Composite Endpoint Response

84%

78%

0.0401

*Primary Endpoint
n = 296 for ETV and 287 for LVD with evaluable baseline histology
n = 314 for ETV and 295 for LVD with Week 48 results

 

Results:

There was no evidence of emergence of ETV resistance in any ETV-treated patients at 48 weeks. Safety was comparable between treatment groups: 6% and 8% of patients in the ETV and LVD groups, respectively, had serious adverse events. At 48 weeks, a greater proportion of ETV than LVD patients achieved the Composite Endpoint and were followed off-treatment for sustained response.

Conclusion:

Primary treatment with entecavir was superior to LVD for histologic and virologic response and achievement of the Composite Endpoint, with a comparable safety profile to LVD, in nucleoside-naïve, HBeAg(-) chronic hepatitis B patients.

 

 


Poster LB-11

ONE YEAR RESULTS FROM A MULTI-CENTRE, DOUBLE-BLIND, PLACEBO (PLA)-CONTROLLED 5 YEAR STUDY OF ADEFOVIR DIPIVOXIL (ADV) IN CHINESE PATIENTS WITH HBEAG POSITIVE CHRONIC HEPATITIS B (CHB)

MD Zeng, Renji Hospital, Shanghai, China; GB Yao, Jing-An Qu Central Hospital, Shanghai, China; YZ Wang, Infectious Diseases Hospital, Jinan, China; JL Hou, NanFang Hospital, Guangzhou, China; Susan Scott, GlaxoSmithKline R&D, London, United Kingdom; Chai-Ni Chang, GlaxoSmithKline R&D, Durham, NC; Keith Barker, GlaxoSmithKline R&D, London, United Kingdom.

Background:

Chronic hepatitis B (CHB) is an important global health issue and a leading cause of cirrhosis and hepatocellular carcinoma. ADV is a nucleotide analogue with potent in vitro and in vivo activity against wild-type and lamivudine-resistant HBV.

Aims:

To evaluate the efficacy and safety of ADV 10 mg once daily (OD) in Chinese patients with HBeAg+ve CHB and to assess health-related quality of life (HrQOL) improvements associated with treatment using the 36-item Short Form Health Survey (SF-36).

Methods:

480 patients with HBeAg +ve CHB, HBV DNA = 106 copies/mL and ALT > 1 x upper limit of normal were randomised in 3 phases: double-blind ADV vs PLA (3:1) for 12 weeks, then open label ADV (OL-ADV) for 28 weeks for all patients, followed by double-blind re-randomisation (for those who had ADV in the 1st 12 weeks) to ADV or PLA (2:1) for 12 weeks. The primary efficacy measure was log10 reduction in serum HBV DNA (Roche COBAS PCR) from baseline at week 12 between ADV (n=360) and PLA (n=120). Secondary endpoints at week 52 included HBV DNA, HBeAg and ALT responses.

Results:

All 480 patients were HBsAg positive with HBV DNA =106 copies/mL at screening; 83% males; median age 30 years. There was a significant difference in the median log10 HBV DNA reduction at week 12 between patients treated with PLA and ADV (-0.1 and -3.4 log10 copies/mL respectively, p<0.001). Further reductions in serum HBV DNA were observed with ADV therapy at week 52 (median reduction 4.5 log10 copies/mL). At week 52, the ADV + OL-ADV + ADV treatment group showed statistically significant (p<0.05) HrQOL improvements compared to the ADV+OL-ADV+PLA treatment group for the following SF-36 scores: role limitations due to physical problems, role limitations due to emotional problems, social function, vitality and general health perceptions. Adverse events were of similar low frequency on ADV and PLA.

Conclusion:

ADV10mg OD is effective in Chinese patients with HBeAg+ve CHB and resulted in significant reductions in serum HBV DNA and ALT normalisation rates. Discontinuation of ADV led to increased HBV DNA and ALT levels in most patients, thus monitoring is needed after stopping therapy. ADV treatment improves HrQOL in Chinese patients with HBeAg+ve CHB. This study is ongoing and will provide data on the long-term efficacy and safety of ADV in patients with HBeAg+ve CHB.

 

Summary of results

Endpoint

Week

PLA+OL-ADV+ADV
(n=120)

ADV+OL-ADV+ADV
(n=240)

ADV+OL-ADV+PLA
(n=120)

Pts with ALT normalisation (%)

40

69/106 (65.1)

163/223 (73.1)

81/109 (74.3)

 

52

74/107 (69.2)

176/224 (78.6)

23/109 (21.1)

Median reduction in HBV DNA (log10 copies/mL)

40

-4.6

-4.2

-4

 

52

-5

-4.5

-0.2

Patient s with HBV DNA response (%)1

40

110/115 (95.7)

227/231 (98.3)

111/115 (96.5)

 

52

112/115 (97.4)

218/231 (94.4)

25/115 (21.7)

1 HBV DNA response defined as HBV DNA level =105 copies/mL or a = 2 log10 copies/mL decrease from baseline.

 

 


Poster 990

HEPATITIS B VIRUS-RELATED INSERTIONAL MUTAGENESIS IN PATIENTS WITH CHRONIC HEPATITIS B AS AN EARLY DRASTIC GENETIC CHANGE LEADING TO HEPATOCARCINOGENESIS

Masahito Minami, Kojiro Mori, Hidetaka Takashima, Kota Fujii, Tomoki Nakajima, Yoshito Itoh, Takeshi Okanoue, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan.

 

 

Background:

Growing evidence demonstrates that hepatitis B virus (HBV) integration and resulting insertional mutagenesis play an important role in cell growth or maintenance in hepatocellular carcinomas (HCCs). Accumulating data on the human genome permitted the identification of several HBV integration-related genes. In particular, several groups have independently reported HBV integration into the human TERT gene in HCCs as well as in hepatoma derived cell lines, proving that viral integration activates hTERT expression and, probably, maintenance of telomere length. We previously reported that HBV integration occurs early during HBV infection, even after acute self-limiting hepatitis. Insertional mutagenesis may, therefore, represent the first drastic genetic change preceding the development of HCC by a few decades.

 

Aims:

To determine if HBV integration occurs and affects cellular genes at such a stage of infection, we analyzed viral-host junctions in chronic hepatitis tissues without HCC.

 

Methods:

Human sequences adjacent to HBV integration were cloned from six liver tissues with chronic hepatitis type B after PCR amplification using primers specific to human Alu-repeat and HBV. Chromosomal location and genes near the integration sites were examined by the database search. Results: We obtained 42 independent viral-host junctions from all of the patients examined and identified chromosomal locations in 20 subjects. Twelve of the 20 integrants interrupted cellular genes or hypothetical proteins. In six clones, each integration apparently affected a single gene because it occurred within the gene coding region and no other gene existed within 250 kb. These six candidate genes, possibly related to tumorigenesis, included one known tumor suppressor gene (AXIN1), three human homologues of drosophila genes that are critical for organ development (EYA3, BBX, and ODZ2), one putative oncogene (CTNND2), and one recently found chemokine (TAFA-1). Our data, together with previously reported HBV integrants, revealed preferential HBV integration into chromosome 3 (p = 0.027).

 

Conclusions:

Our virus-tagging approach provided (a) firm evidence of HBV integration in hepatocytes at an early stage of chronic infection and (b) revealed cellular genes possibly affected by HBV integration and related to liver tumorigenesis. In contrast to the previous consensus, we demonstrated a preferred chromosome for HBV integration which implies a selection mechanism for some integrants.

 

 

Poster 991

CHANGES IN THE STABILITY OF HBV-SPECIFIC CTL EPITOPES BY YMDD MUTATIONS MAY DETERMINE THE OCURRENCE OF BREAKTHROUGH HEPATITIS

Masami Minemura, Yukihiro Shimizu, Katsuharu Hirano, Yasuhiro Nakayama, Yoshiharu Tokimitsu, Kazuto Tajiri, Yutaka Yata, Yoshinari Atarashi, Satoshi Yasumura, Toyama Medical and Pharmaceutical University, Toyama, Japan.

 

 

Background:

 Long-term lamivudine (LAM) treatment is associated with an increasing rate of LAM-resistant mutations in patients with chronic hepatitis B (CH-B). Clinical manifestations of LAM-resistance range from normal ALT to hepatitis flares or hepatic decompensation. Changes in the nucleotide and amino acid sequence including YMDD motif could affect immunogenic viral epitopes, but the mechanism of breakthrough hepatitis is still unclear.

 

Aims:
The aim of this study was to identify the changes in HLA-restricted T cell epitope repertoires after the emergence of LAM-resistant mutations and analyze its association to the degree of hepatic inflammation.

 

Methods:

Eleven patients with CH-B who had breakthrough of serum HBV DNA during LAM treatment were studied. Five of them had hepatitis flares (>2x upper limit of normal ALT, (ALT 448±333 IU/L, Mean±SD); group I, and the others were without hepatitis flares (ALT 48±6); group II. Serum HBeAg, anti-HBe, ALT, and HBV DNA levels were tested subsequently. Full-length sequences of HBV before LAM treatment and after the emergence of YMDD variants were determined by direct sequencing. HLA types were determined by lymphocyte cytotoxicity test. HLA-restricted HBV-specific CTL epitopes were identified by reverse immunogenetics, a strategy that identifies rank potential of CTL epitopes from a given amino acid sequence based on HLA class I binding peptide motifs and predicted half-time of dissociation (DT1/2) to HLA class I molecules. We used a database called BioInfomatics & Molecular analysis Section for this purpose.

 

Results:

Mutations(nt 1762/1764) in core promoter were found in all patients and a stop codon mutation in precore was found in only one patient. The LMA resistant mutations, rtM204I (YIDD) and rtL180M+rtM204V (LMA+YVDD), were detected in 2 and 9 of the 11 patients, respectively. There was no significant difference between the two groups in viral loads and frequency of mutations. HLA-A2 was detected only in patients of group II (67%), and HLA-A11 was detected only in group I (40%). DT1/2 (62.3 min) of a predicted epitope peptide YMDDVVLGA (pol 552-560) was remarkably shortened after a mutation to YIDD (DT1/2: 11.9) or YVDD (DT1/2: 7.5) only in HLA-A2 patients, suggesting that the peptide binding became unstable by those mutations. On the other hand, DT1/2 of another peptide YMDDVVLGAK (pol 552-561) was elongated by the YVDD mutation only in HLA-A11 patients, suggesting that the peptide binding became more stable by the mutation.

 

Conclusion:

Our results suggest that changes in the binding stability of the immunogenic viral epitopes to HLA class I molecules by YMDD mutations rather than a resurgence of viral load is closely associated with the exacerbation of CH-B with the emergence of LAM-resistant mutations.

 

 

Poster 992

A SHORT SEQUENCE IN THE 3' END OF CORE GENE IS CRITICAL FOR HEPATITIS B VIRUS GENOME REPLICATION, E ANTIGEN PROCESSING, AND CORE PROTEIN NUCLEAR LOCALIZATION

Kyun-Hwan Kim, Michael Guarnieri, Adrienne Tsai, Jack R. Wands, Shuping Tong, Liver Research Center, Rhode Island Hospital/Brown Medical School, Providence, RI.

 

 

Background:

The core gene of hepatitis B virus (HBV) codes for both core protein and e antigen (HBeAg), a secreted protein translated from an upstream initiation codon. HBeAg maturation requires removal of the N-terminal signal peptide and C-terminal arginine-rich sequence.

 

Aims:

A recent study suggested role of a furin-like endopeptidase in the C-terminal cleavage through the 151-RRGR-154 motif. Due to a dipeptide insertion, this motif becomes 151-RRDRGR-156 in genotype A. We previously identified clone 4B of genotype A as high replicating and low in HBeAg secretion. During the mapping experiments, we found that the 3' end of 4B core gene reduced HBeAg expression and surprisingly, also markedly impaired viral replication.

 

Methods:

Chimeric constructs and site-directed mutants were made and converted into tandem dimer. After transfection into Huh7 cells, genome replication, HBeAg secretion, and core protein localization were investigated. Levels of secreted HBeAg in culture supernatant were determined by enzyme immunoassay and immunoprecipitation. Genome replication was monitored by southern blot analysis. Core protein localization was determined by western blot analysis following subcellular fractionation. The transcriptional levels of precore and pregenomic RNA were determined by primer extension analysis.

Results:

Clone 4B contained 4 amino acid substitutions in the C-terminus of core protein. Introduction of this fragment into a wild-type clone reduced HBeAg expression by 60%, and also markedly impaired viral replication. Further site-directed mutagenesis revealed that the R151C mutation was primarily responsible for the reduction in HBeAg expression and genome replication. Although this point mutation did not affect RNA transcription, it reduced levels of encapsidated pregenomic RNA. The wild-type construct generated two HBeAg species of 18-kd and 17-kd. The R151C mutation (creating a CRDRGR motif) increased 18-kd species, whereas the D153G mutation (with RRGRGR motif) generated an extra band of 16.5kd. The D153G mutation, but not R151C/D153G double mutation, greatly enhanced the nuclear localization of core protein, suggesting that the net positive charges promote nuclear localization.

 

Conclusion:

 D153 in the core protein of genotype A negatively regulates core protein nuclear localization, while the nearby R151 is critical for pregenome packaging and HBeAg expression (or antigenicity). Mutations inside the RRDRGR motif can alter the site of HBeAg processing and possibly the enzyme involved.

 

 

Poster 993

INTERACTION OF HBV VARIANTS DETERMINES THE PHENOTYPE OF A HETEROGENEOUS VIRAL POPULATION

Martina Sterneck, Lutz Fischer, UKE/ University Hospital Hamburg, Hamburg, Germany; Anna Garcia, Hans Will, Tatjana Kalinina, Heinrich Pette Institut Hamburg, Hamburg, Germany.

 

 

Background:

Due to the particularly high mutation rate of HBV heterogeneous viral populations are usually found in infected patients.

 

Aims:

Therefore interaction between variants may determine selection and spread of viral strains. Previously, HBV variants with an almost complete secretion defect were found to represent the dominant viral population in the serum of two patients with fulminant hepatitis. In both cases the secretion block resulted from several mutations within the S-gene of the variants. In HBV the S-gene is expressed as the small (S)-protein and also as part of the large (L)-envelope protein. Therefore S-gene mutations may affect S- and L-protein which are both required for virion formation and secretion. Here, we investigated the interaction of the defective variants with Wt to understand the mechanisms that allow a secretion defective variant to emerge as the dominant viral strain in the patients' blood stream. In addition, we studied the consequences of S-gene mutations for the function of S- and L-protein.

 

Methods:

Cotransfection experiments were performed using varying amounts of the defective variants 1 or 2 and Wt or a secretion competent mutant HBV genome. In addition, viral constructs expressing only mutant or Wt S- or L-protein were cotransfected with Wt or the variants 1 and 2, respectively. Northern, Western and Southern blot analyses were performed to study viral production and secretion in cell culture.

 

Results:

A small amount of Wt or of a secretion competent mutant was able to rescue secretion of both defective strains. Cotransfection of mutants with viral constructs expressing only Wt S-protein or only Wt L-protein revealed that only Wt S-protein, but not L-protein was able to rescue secretion. Notably, Wt S-protein increased the intracellular level of mutant L-protein suggesting that Wt S-protein stabilizes mutant L-protein and prevents its degradation. On the other hand, only mutant S-, but not L-protein had a transdominant negative effect on secretion of Wt.

 

Conclusion:

(i) Interaction with a small amount of Wt reversed the secretion defect of variants. These data apply that interaction between different HBV quasispecies determines the biological properties and spreading of a viral population in vivo. (ii) Structure and correct folding of S-protein, but not of L-protein determined the process of virion particle formation and secretion. We speculate that S-protein plays a chaperone-like role in the folding of L-protein.

 

 

Poster 994

EDITING OF HEPATITIS B VIRUS DNA BY THE CYTIDINE DEAMINASE APOBEC3G

Christine Rosler, Josef Kock, Hubert E. Blum, Fritz von Weizsacker, University of Freiburg, Freiburg, Germany.

 

 

Background:

The cytidine deaminase APOBEC3G (A3G) was recently identified as a natural resistance gene restricting propagation of HIV and other retroviruses. The enzyme induces massive C-to-U deamination of single stranded viral DNA resulting in DNA degradation or lethal G-to-A hypermutation. Hepadnaviruses, including hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) replicate via reverse transcription of a pregenomic RNA intermediate and thus represent potential targets for APOBEC3G as well.

 

Aims:

To address this issue, the potential effect A3G on hepadnaviral replication and sequence diversity was investigated.

 

Methods:

Replication competent HBV or DHBV constructs were co-transfected with a CMV-driven A3G expression construct into the human hepatoma cells Huh-7 and HepG2 or the avian hepatoma cell line LMH. Transfected cells were analyzed for viral RNA, DNA and protein synthesis as well as pregenomic RNA packaging. In addition, newly synthesized HBV DNA produced in transfected cells was cloned and a total of 430 individual clones was sequenced.

 

Results:

A3G strongly suppressed RNA pregenome packaging of both, HBV and DHBV, resulting in a marked suppression of replication. This antiviral effect was not modulated by abrogating or enhancing expression of the viral X protein. In Huh-7 cells newly synthesized HBV DNA rarely displayed G-to-A mutations, irrespective of the absence or presence of A3G, confirming previous findings by Turelli et al. (Science, 2004). In A3G-expressing HepG2 cells, the majority of recovered sequences were wild-type as well. Strikingly, however, extensive G-to-A mutations were identified in individual clones, while other nucleotide substitutions were rare. Targeted sequence motifs matched well with the hallmarks of A3G action and mutations were identified in various regions of mutated HBV clones, suggesting that they were indeed caused by processive enzymatic activity rather than by global imbalances in the cellular nucleotide pool.

 

Conclusions:

HBV represents a dual target for the cytidine deaminase APOBEC3G: interference with RNA pregenome packaging results in a direct suppression of viral DNA synthesis while induction of G-to-A mutations in individual clones may contribute to the emergence of variants.

 

 

Poster 995

BMI IS AN INDEPENDENT PREDICTOR OF DISEASE PROGRESSION IN PATIENTS WITH CHRONIC HEPATITIS B

Supriya Joshi, University of Toronto, Toronto, ON, Canada; Maha Guindi, University of Toronto - Toronto General Hospital, Toronto, ON, Canada; E Jenny Heathcote, University of Toronto, Toronto, ON, Canada.

 

 

Background:

More than 350 million people worldwide are hepatitis B (HBV) carriers and 25% will develop cirrhosis. Worldwide obesity rates are increasing, however the effect of obesity related liver disease on fibrosis and its progression in those with HBV is unknown.

 

Aim:

To determine if obesity and/or nonalcoholic fatty liver (NAFL) is associated with fibrosis and its progression in patients with HBV.

 

Methods:

Following approval by the local REB, patients with chronic HBV who had a liver biopsy since 1990 were reviewed. Charts were reviewed for other causes of progressive liver disease and patients were excluded if they had evidence of co-existing alcoholic, metabolic, autoimmune, HCV or HDV liver disease, had received therapy with TPN or corticosteroids or there was inadequate information available. Body mass index (BMI) at first liver biopsy was recorded. High BMI was considered BMI > 30 (non-Asian) and BMI > 28 (Asian). All liver biopsies were reviewed and scored by Laennec for activity and fibrosis, and by Brunt for grade of steatosis. To assess degree of nonalcoholic steatohepatitis (NASH) presence of ballooning, Mallory and perisinusoidal fibrosis was recorded. Statistical analyses included chi-square, Fisher's exact test and analysis of covariance.

 

Results:

Of 315 patients with HBV who had a liver biopsy, 102 met exclusion criteria. Records of 213 patients were assessed; 52 had more than 1 liver biopsy, 162 (76%) were male, 151 (71%) were Asian, 101 (47%) were eAg positive, 128 (60%) had indications to receive antiviral therapy and 44 (21%) had high BMI. Of patients who had only one liver biopsy performed, significant associations were found between Brunt score, % fat, features of NASH and BMI (p< 0.0001) but not with fibrosis. However in patients who underwent more than 1 liver biopsy, high BMI was found to be significantly associated with greater progression of fibrosis when compared to those with normal BMI (p=.0345) after adjusting for eAg status, age, ALT, % fat, Brunt steatosis grade, evidence of NASH, treatment with antivirals and effect of estimated duration of disease using analysis of covariance.

 

Conclusion:

Our study indicates that patients with HBV and high BMI had a greater rate of progression of fibrosis independent of other factors known to cause progressive liver disease in chronic HBV. The majority of those with more than 1 liver biopsy were on antiviral therapy for HBV yet still had progression of fibrosis, which was BMI dependent, but not associated with NASH suggesting another mechanism. The role of weight based antiviral therapy in chronic HBV should be investigated to evaluate whether progression of fibrosis in patients with high BMI is due to inadequate dosing.

 

 

Poster 996

VIRAL LOAD AS A PREDICTOR OF LIVER DISEASE IN CHRONIC HEPATITIS B INFECTION

G Chen, Fox Chase Cancer Center, Philadelphia, PA; WY Lin, Haimen City Center for Disease Control, Haimen City, China; FM Shen, Fudan University School of Public Health, Shanghai, China; UH Iloeje, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT; WT London, AA Evans, Fox Chase Cancer Center, Philadelphia, PA.

 

 

Background and Aims:

This prospective cohort study, conducted over a 10-year period in adult residents of Haimen City, China, assessed the relationship between past hepatitis B (HBV) viral load and current liver disease.

 

Methods:

The cohort was recruited in 1992-1993. Ten years later, 1520 surviving individuals identified as chronically infected with HBV at study entry were invited for rescreening which included viral markers, liver function tests, physical examination, and liver ultrasound. Liver disease was classified according to criteria adapted from the Dionysos Study. Viral load was assayed by real-time PCR using banked samples from cohort entry in 1992-1993. For this analysis, viral load was classified as: undetectable (LOQ: 3x104 copies/mL ); low titer (<105 copies/mL); high titer (>105 copies/mL). Nominal logistic regression was used to estimate the odds ratio (OR) (95% CI) for mild/moderate liver disease or cirrhosis/HCC for each viral load category vs. the reference category (no liver disease with undetectable HBV DNA).

 

Results:

At time of rescreening, 897 subjects (59.0%) had no evidence of liver disease other than HBsAg positivity; 281 (18.4%) had mild/moderate liver disease; 328 (21.6%) had evidence of cirrhosis, and 14 (0.9%) had hepatocellular carcinoma (HCC). The population was 54.7% male with a mean age of 51.2 (+8.6) years in 2003. In the mild/moderate group, the ORs for low and high titer HBV DNA were 1.4 (1.0-2.0) and 1.9 (1.4-2.6), respectively. In the cirrhosis/HCC group, they were 1.0 (0.7-1.5) and 2.5 (1.9-3.4).

 

Viral load

No Liver Disease
(N=897)

Mild/Moderate Liver Disease (N=281)

Cirrhosis/HCC
(N=342)

Undetectable
(N=569)

393 (43.8%)
reference

83 (29.5%):
--

93 (27.2%)
--

Low
(N=357)

226 (25.2%)
--

71 (25.3%)
OR 1.4 (1.0-2.0)

60 (17.5%):
OR 1.0 (0.7-1.5)

High
(N=594)

278 (31.0%)
--

127 (49.2%)
OR 1.9 (1.4-2.6)

189 (55.3%)
OR 2.5(1.9-3.4)

 

*Odds Ratio (OR), adjusted for age and sex

 

Conclusion:

Although liver disease status was not assessed at cohort entry in 1992-1993, these findings suggest that high viral load was strongly associated (OR: 1.9-2.5) with increased risk of liver disease a decade later. In both the mild/moderate and cirrhosis/HCC groups, low-titer viral load was not associated with increased risk compared to the undetectable group. Nevertheless, 307 (33.2%) of the 926 individuals with undetectable or low-titer viral load at cohort entry had liver disease on examination ten years later. Based on these results, this group cannot truly be classified as low-risk for disease progression.

 

 

Poster 997

MBL2 GENOTYPES ARE IMPORTANT DETERMINANTS OF HBV CLEARANCE OR PERSISTENCE

Chloe L. Thio, Timothy Mosbruger, Jacquie Astemborski, Spencer Greer, Johns Hopkins, Baltimore, MD; David Vlahov, New York Academy of Medicine, New York, NY; Stephen O'Brien, National Cancer Institute, Frederick, MD; David Thomas, Johns Hopkins, Baltimore, MD.

 

 

Background:

The innate immune response is important for the clearance of a hepatitis B virus (HBV) infection. A central component of this response is mannose binding lectin (MBL), which either serves as an opsonin or activates the classical complement cascade. MBL binds mannose-rich oligosaccharides, which are contained in the HBV envelope. Several single nucleotide polymorphisms (SNPs), which alter the functional levels of MBL, have been described in its gene (mbl2) and have been associated with outcomes of various viral infections.

 

Aims:

We hypothesized that SNPs in mbl2 may be a determinant of HBV clearance or persistence.

 

Methods:

Utilizing a nested case-control study design from an injection drug using cohort and cohort of men who have sex with men, we matched subjects with HBV persistence (HBsAg positive on two occasions separated by a minimum of 6 months) to two individuals who cleared a HBV infection (anti-HBc and anti-HBs positive) based on HIV status, race, age, and gender. Two promoter SNPs at -550 and -221 and three exon 1 SNPs in mbl2 were genotyped using a single base extension method. We constructed haplotypes between the promoter and exon 1 SNPs using PHASE and grouped them based on the amount of functional MBL produced. The individual SNPs and the grouped haplotypes were compared between those with viral clearance and persistence using conditional logistic regression.

 

Results:

Included in this study were 189 and 338 subjects with either HBV persistence or clearance, respectively. The majority of the study cohort was White (75%) with the remaining 22% Black, and 3% Other. The promoter mutant -221 C allele, which produces less MBL, was associated with viral persistence (OR 1.38, 95% CI 1.01-1.89, P=0.04) and was only in a haplotype with wild type alleles at the other SNPs. The wild type -221G was distributed among five haplotypes, and only the one consisting of all wild type alleles was significantly associated with viral clearance (OR 0.75, 95% CI 0.57-0.99, P=0.04). When the haplotypes were grouped according to the amount of MBL produced, those homozygous for the genotype leading to the highest MBL concentration had a significantly increased odds of viral clearance (OR 0.55, 95% CI 0.37-0.84, P=0.005). Conversely, those with the genotype producing the lowest amount of MBL were more likely to have viral persistence (OR 1.76, 95% CI 1.02-3.01, P=0.04). None of these relationships were altered when the data were stratified by HIV or ethnicity.

 

Conclusions:

Persons with mbl2 genotypes leading to maximal MBL production are nearly two times more likely to experience HBV clearance. These data are consistent with the hypothesis that functional MBP is an important part of the innate immune response that results in recovery from an acute HBV infection.

 

 

Poster 998

IMPACT OF HAART ON HEPATITIS B VIRUS CO-INFECTION: ASSOCIATION BETWEEN IMMUNORESTORATION AND HBS/E ANTIGEN SEROCONVERSION

Patrick Miailhes, Hotel-Dieu, Lyon, France; Mary-Anne Traubaud Faculte Rockefeller, Lyon, France; Bertrand Lebouche, Francois Bailly, Pierre Pradat, Hotel-Dieu, Lyon, France; Michele Chevallier, Laboratoire Marcel Merieux, Lyon, France; Philippe Chevallier, Faculte Rockefeller, Lyon, France; Marianne Maynard, Fabien Zoulim, Christian Trepo, Hotel-Dieu, Lyon, France.

 

 

Background:

In Western countries, co-infection with HBV occurs in 5% to 10% of HIV-infected patients. Before the era of HAART, chronic hepatitis B (CHB) was often in a state of immunotolerance. Under HAART, the natural history of CHB co-infection was bound to change.

 

Methods:

Baseline characteristics were assessed before HAART and lamivudine therapy. The impact of HAART on the natural history of CHB was studied by a retrospective analysis of 85 HIV-HBV co-infected patients.

 

Results:

Mean age was 41 years (21-65); 82% were males; 58% were homo/bisexuals, 33% heterosexuals, and 9% IVDU; CDC stage was A in 47%, B in 24% and C in 29%. Mean duration of HIV and CHB infections were 8.5 and 8.2 years, respectively. Median CD4 cell count was 274 cells/mm3; median plasma HIV RNA was 4.49 log10 copies/mL and median serum HBV DNA 8.4 log10 copies/mL; 54 patients (65%) had normal ALT level; 57 (68%) were HBeAg(+). During follow-up, 77 patients (91%) had ARV therapy, of whom 76 had HAART with a median duration of 62 and 49 months, respectively. Lamivudine was given in 72 patients (94%) with a median duration of 32 months. Twenty-four patients out of 72 (33%) had a YMDD mutation under lamivudine therapy. Presence of YMDD mutation was associated with a longer lamivudine therapy (56 vs 30 months, respectively; p<0.001) and a higher baseline HBV DNA level (8.68 vs 6.92 log10 copies/mL; p=0.005). Under ARV, 10/55 HBeAg(+) patients (18%) developed anti-HBe. Among them, 3 lost HBsAg and 2/3 developed anti-HBs. 2/27 HBeAg(-) (7%) lost HBsAg and developed anti-HBs. HBe or HBs seronversion was only obtained in cases with HIV virological response (p=0.001), defined as an undetectable HIV RNA (<50 copies/mL) during at least 80% of HIV treatment duration, and was related to duration of HAART (p=0.04). The seroconversion rate did not differ between HBeAg(+) and HBeAg(-) patients (p=0.32). Remarkably, the impact of ARV on HBs/e antigen clearance was inversely related to the severity of HIV disease (3%, 20% and 29% for CDC stage A, B and C, respectively; p=0.02). After excluding patients with YMDD mutation, HBV response was associated with longer lamivudine therapy (p=0.01).

 

Conclusion:

In HBV-HIV co-infected patients, HBe or HBs seroconversion is associated with the quality and pace of HAART immunorestoration. Longer lamivudine therapy is also associated with better HBV response in patients without YMDD mutation.

 

 

Poster 999

NATURALLY OCCURRING MUTATIONS IN HEPATITIS B VIRUS CORE GENE MODULATE CORE PROTEIN AND e ANTIGEN EXPRESSION OR THEIR ANTIGENICITY

Kyun-Hwan Kim, Sang Hoon Ahn, Michael Guarnieri, Jac, R. Wands, Liver Research Center, Rhode Island Hospital/Brown Medical School, Providence, RI.

 

 

Background:

The hepatitis B virus (HBV) causes liver cirrhosis and hepatocellular carcinoma. Expression of core protein and its secreted variant form, hepatitis B e antigen (HBeAg), is achieved by two separate mRNA species, the precore mRNA for HBeAg and the pregenomic RNA for core protein.

 

Aims:

We recently cloned and characterized several naturally occurring HBV genomes such as 2A and 3.4 (Parekh et al., 2003, J. Virol., 77: 6601). In the present study, we investigated how the mutations in the coding sequence of core gene modulate levels of HBeAg and core protein.

 

Methods:

Different regions of the core gene of clone 3.4 were introduced into clone 2A and tandem HBV dimers were generated. After transfection into the human hepatoma cells, secreted HBeAg level in culture supernatant was determined by both enzyme immunoassay and immunoprecipitation, and normalized against HBsAg levels. Genome replication was analyzed by southern blot analysis of intracellular core particles. The expression of core protein and HBsAg was determined by western blot analysis. The transcription levels of precore and pregenomic RNA were determined by primer extension analysis. For mechanistic studies, CMV-driven core protein expression constructs were made.

 

Results:

Consistent with the localization of the major B cell epitope of the core protein to residues 74-84, we found that a single E77Q mutation completely abolished core protein and HBeAg recognition by a rabbit polyclonal antibody. Interestingly, this point mutation occurs frequently in genotype A clones subsequent to core promoter mutations. Of the remaining mutations, a G63V substitution reduced levels of both core protein and HBeAg, whereas a Q184R mutation moderately enhanced both proteins as well as viral replication. An A36T mutation reduced viral replication. Interestingly, primer extension analysis suggested that the G63V mutation was associated with a reduction in precore and pregenomic RNA levels, while the Q184R mutation elevated both RNA species. When core protein was expressed from CMV promoter, similar levels of core protein were generated, suggesting that the mutations in the core protein do not affect protein stability.

 

Conclusion:

An immune escape mutation in the antigenic epitope of core protein often arises subsequent to core promoter mutations, suggesting a possible role of anti-HBc humoral immunity in viral clearance. Other missense mutations in the core gene affect levels of core protein and HBeAg, possibly at the transcriptional level.

 

 


Poster 1000

OCCULT HEPATITIS B VIRUS INFECTION IN A NORTH AMERICAN ADULT HEMODIALYSIS PATIENT POPULATION

Manna Zhang, Dongfeng Sun, Rebecca Greenberg, Kim Hawkins, Julia Uhanova, Adam Gutkin, Keevin Bernstein, University of Manitoba, Winnipeg, MB, Canada; Antonio Giluivi, Health Canada, Ottawa, ON, Canada; Carla Osiowy, Canadian Science Centre for Human and Animal Health, Winnipeg, MB, Canada; Gerald Y. Minuk, University of Manitoba, Winnipeg, MB, Canada.

Background:

Despite extensive infection control guidelines and the availability of effective hepatitis B virus (HBV) immunoprophylaxis, HBV infections continue to occur in adult hemodialysis units.

Aims:

A possible contributing factor is the presence of occult HBV (serum hepatitis B surface antigen (HBsAg) negative but HBV-DNA positive). To date, the prevalence of occult HBV infection has not been documented in a North American adult hemodialysis unit.

Methods:

Two hundred and forty-one adult hemodialysis patients were screened for occult HBV. HBV-DNA testing was performed by real time polymerase chain reactions (PCR) with two independent primer sets (core promoter and surface) and a sensitivity of 10 viral copies/ml. Results: Two (0.8%) of the 241 patients were HBsAg positive. Of the remaining 239 HBsAg negative patients, nine (3.8%) were HBV-DNA positive. Viral loads in these individuals were low (103-105 viral copies/ml). Seven of the 9 (78%) were nt 587 mutation (S-mutant) positive. Demographic, biochemical and HBV serologic testing did not help to identify those with occult HBV.

Conclusions:

The results of this study indicate that in this North American urban centre, the prevalence of occult HBV in adult hemodialysis patients is approximately 4-5 times higher than standard HBsAg testing would suggest. The results also indicate the majority of these infections are associated with low viral loads and a high prevalence of the S-mutant. Finally, the demographic, biochemical and serologic features of HBV-DNA positive subjects do not distinguish these individuals from the remainder of the dialysis patient population. Additional studies are required to determine the infectivity and natural history of occult HBV. Until such data are available, consideration should be given to screening dialysis patients with sensitive PCR-based assays for HBV-DNA and possible segregation of those who test positive.

 

 


OCCULT HEPATITIS B VIRUS INFECTION IN A COMMUNITY-BASED POPULATION

Dongfeng Sun, Julia Uhanova, Manna Zhang, Shaunna Caouette, Adam Gutkin, Bruce Martin, University of Manitoba, Winnipeg, MB, Canada; Antonio Giulivi, Health Canada, Ottawa, ON, Canada; Gerald Y. Minuk, University of Manitoba, Winnipeg, MB, Canada.

Background:

Occult hepatitis B virus (HBV) infections [HBV-DNA detection in hepatitis B surface antigen (HBsAg)-negative individuals] have been implicated as a cause for ongoing viral transmission, fulminant hepatic failure, acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Although the prevalences of occult HBV have been documented in specific donor and patient populations, to date, data from community-based populations have yet to be reported.

Aims:

The principal objective of this study was to document the prevalence of occult HBV in the inhabitants of an isolated, North American community.

Methods:

Stored sera from 544 (64%) residents of a Northern Canadian Inuit (Eskimo) community with a total population of 850 were available for testing. Thirty-nine sera were known to be HBsAg positive. Of the remaining 515 samples, 108 (Group 1) had a serologic profile in keeping with resolved HBV infection [HBsAg negative, antibody to hepatitis B core antigen (anti-HBc) and HBsAg (anti-HBs) positive] and 407 (Group 2) were seronegative for all HBV markers. Samples were tested for HBV-DNA and nt 587 "S-variants" by real time PCR (sensitivity 10 viral copies/ml) with two independent primer sets (core promoter and surface).

Results:

The mean (+ SD) age of Group 1 was 46.2 + 15.7 and Group 2; 17.4 + 10.7 years. Forty five percent of Group 1 and 52% of Group 2 were male. HBV-DNA was present in 14/108 (13%) of Group 1 and 33/407 (8.1%) of Group 2. In all cases (Groups 1 and 2) viral loads were low (<106 viral copies/ml). S variants were present in 12/14 (86%) and 17/33 (52%) of HBV-DNA positive individuals in Groups 1 and 2 respectively. The age, gender and extent of serum alanine aminotransferase (ALT) abnormalities in HBV-DNA positive individuals from Groups 1 and 2 were similar to those who were HBV-DNA negative

Conclusions:.

The results of this study indicate that in this North American community-based population; 1) the prevalence of occult HBV infection is 13% in those with serologic evidence of previous HBV infection and 8.1% in those who are seronegative for HBV, 2) S-variants are present in the majority of individuals with occult HBV and 3) age, gender and serum ALT levels do not distinguish between those with and without occult HBV infections.

 

 


Poster 1002

REGULATORY T CELLS CONTRIBUTE TO IMMUNOLOGIC HYPORESPONSIVENESS IN CHRONIC HBV PATIENTS

Jeroen N. Stoop, Renate G. van der Molen, Carla C. Baan, Luc J. W. van der Laan, Ernst J. Kuipers, Solko W. Schalm, Johannes G. Kusters, Harry L. A. Janssen, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands.

 

Background:

Chronic hepatitis B virus (HBV) infection is characterized by a weak immune response to HBV.

 

Aims:

Since CD4+CD25+ regulatory T cells (Treg) are capable of suppressing the proliferation and the IFN-? production of a wide range of effector T cells and NK cells, we investigated whether Treg contribute to this impaired immune response.

 

Methods:

Peripheral blood samples were collected from 50 patients, 23 healthy controls and 9 persons with a resolved HBV infection. The percentage of Treg was determined using flowcytometry with specific antibodies (CD4, CD25, CD45RO and CTLA-4). The FoxP3 expression, which is a specific transcription factor expressed by CD4+CD25+ Tregs, was determined in PBMCs by real time RT-PCR. The inhibition of the immune response against HBV core antigen by Treg was tested by isolation of CD4+ CD25+ T cells using a negative selection for CD4 and positive selection for CD25. The CD4- and the CD4+CD25- cells were pooled and used as a Treg (CD25) depleted cell fraction. The CD25 depleted, non-depleted PBMCs and CD25 depleted cells reconstituted with 10%, 20% or 30% CD4+CD25+ Tregs from chronically infected patients were stimulated with HBV core antigen and after 6 days their proliferation was determined by incorporation of [3H]-thymidine. IFN- ý production was determined in the supernatant of the proliferation assays by ELISA.

 

Findings:

In chronic HBV infected patients, a higher percentage of Treg was found within the CD4+ population in peripheral blood compared to healthy controls (4.1% vs. 2.6%, p= 0.002) and compared to persons with a resolved HBV infection (4.1% vs. 1.5%, P<0.001). In a subgroup of 18 chronic HBV patients FoxP3 expression, corrected for the percentage of CD4+ cells present in the sample, was increased as compared to 8 healthy controls (195.51 copies/ ng RNA vs. 99.16 copies / ng RNA, p= 0.030). Depletion of the CD25+ cells from PBMCs of 12 chronic HBV patients resulted in a significantly enhanced proliferation (p= 0.034) after stimulation with HBV core antigen. Reconstitution with 10% 20% and 30% of CD4+CD25+ Tregs with cells from six chronic HBV patients resulted in a dose dependent inhibition of the proliferation and IFN-ý production upon stimulation with HBV core antigen.

 

Conclusion:

In conclusion, patients with a chronic HBV infection display a significantly increased percentage of CD4+CD25+ Treg in peripheral blood. These cells have an HBV-specific suppressive effect, as was shown both by depletion of CD25+ cells and by reconstitution of CD4+CD25+ T cells. The presence of CD4+CD25+ Treg in chronic HBV patients may explain the inadequate immune response against the virus.


 

 

Poster 1003

A CASE-CONTROL STUDY FOR DIFFERENCES AMONG HEPATITIS B VIRUS INFECTIONS OF GENOTYPES A (SUBTYPES AA AND AE) AND D

Yasuhito Tanaka, Izumi Hasegawa, Takanobu Kato, Etsuro Orito, Noboru Hirashima, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; Subrat K. Acharya, All India Institute of Medical Sciences, New Delhi, India; Robert G. Gish, California Pacific Medical Center, San Francisco, CA; Anna Kramvis, Michael C. Kew, University of the Witwatersrand, Johannesburg, South Africa; Santosh Man Shrestha, Liver Foundation Nepal, Nepal, Nepal; Mobin Khan, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh; Yuzo Miyakawa, Miyakawa Memorial Research Foundation, Tokyo, Japan; Masashi Mizokami, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.

 

Background:

There are two subtypes of hepatitis B virus genotype A (HBV/A) and they are provisionally designated Aa ("a" standing for Africa/Asia) and Ae ("e" for Europe/US). There have been some lines of evidence for virological and clinical differences between subtype Aa in Africa and subtype Ae in Europe and the US. Infection with subtype Aa is associated with low serum levels of HBV DNA as well as low prevalence of hepatitis B e antigen (HBeAg) in serum, and is implicated in the high incidence of HBV-induced hepatocellular carcinoma (HCC) in Africa.

 

Aim:

Our purpose is to determine clinical and virological differences between subtypes Aa and Ae infections, in comparison to genotype D infection prevalent along with genotype A in the Western countries.

 

Methods:

Sera were obtained from 234 patients infected with subtype Aa (78 patients) or Ae (78 patients) of genotype A, or genotype D (78 patients) from several countries, who were controlled for sex, age and severity of liver disease: 9 were from Bangladesh, 84 India, 29 Japan, 7 Nepal, 14 South Africa, 10 Tanzania and 81 the US. Sequence analyses for mutations affecting the synthesis of HBeAg on HBV DNA from these patients were conducted.

 

Results:

In a case-control study between 78 HBV/Aa, 78 HBV/Ae and 78 HBV/D carriers from several countries, the prevalence of HBeAg in serum was significantly lower in carriers of HBV/Aa than HBV/Ae (31% vs. 49%, P = .033), with a difference more obvious in the carriers aged 30 years or younger (34% vs. 67%, P = .029). HBV DNA levels in the carriers of HBV/Aa (median, 3.46 log copies/mL [95% Confidence Interval, 2.93-3.95]) were significantly lower than those of HBV/Ae (6.09 [4.24-7.64]) or HBV/D (5.48 [4.06-7.02]), regardless of the HBeAg status (P < .001). The most specific and frequent substitutions in 54 HBV/Aa isolates were double substitutions for T1809 (100%) and T1812 (96%) immediately upstream of the precore initiation codon, which would interfere with the translation of HBeAg in HBV/Aa infections. They were not detected in 57 HBV/Ae or 61 HBV/D isolates examined. The double mutation in the core promoter (T1762/A1764) was more frequent in both HBV/Aa (50%) and HBV/Ae (44%) than in HBV/D isolates (25%, P < .01), whereas the precore mutation (A1896) occurred in HBV/D isolates only (48%, P < .0001).

 

Conclusions:

The clearance of HBeAg from serum might occur by different mechanisms in HBV/Aa, HBV/Ae and HBV/D infections, which may influence clinical manifestations in the Western countries where both genotypes A and D are prevalent.

 

 


Poster 1004

ANALYSIS OF INTRAHEPATIC IMMUNE RESPONSE IDENTIFIES FOUR STAGES OF CHRONIC HBV INFECTION

Dave Sprengers, Renate G. van der Molen, Johannes G. Kusters, Solko W. Schalm, Harry L. A. Janssen, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands.

 

 

Background:

Based on virologic and biochemical parameters chronic HBV (CHB) patients are divided into 3 groups: 1) the immune tolerance phase (IT) (HBV DNA high, HBeAg+, ALT N), 2) the immune clearance phase (IC) (ALT high, HBeAg +/-.), and 3) the inactive phase (INA) (HBV DNA low, HBeAg-, ALT N).

 

Aims:

Unclear is whether these phases can be characterized by intrahepatic immune response, as most studies have concentrated on peripheral blood (PB).

 

Methods:

Fine-Needle-Aspiration-Biopsy (FNAB) of the liver represents an atraumatic method suitable for cytological monitoring. We aimed to characterise the composition of key populations of immune effector cells in the different phases of CHB, both in the liver by FNAB and in PB. In 47 patients (IT n=7, IC n=24, INA n=16) the percentage of CD3-CD56+ NK cells, CD8+ cytotoxic T cells (CTL) and CD4+ T helper cells (Th) were determined by flow cytometry. Additionally, in 15 HLA A2+ patients (IT n=3, IC n=9, INA n=3) HBc 18-27-tetramers were used to identify HBV-specific CTL.

 

Results:

No significant differences were found between the three phases in proportion of Th cells and CTL in the liver or PB. The proportion NK cells in the liver was higher in IT than in IC-patients and INA-patients (mean % 41.1, 29.8 and 35 resp; IT vs IC and IC vs. INA p<0.05). However, when IC-patients with similar ALT levels were divided according to viral load: HBV DNA >107 geq/ml (n=14) vs. HBV DNA <105 geq/ml (n=10), further discrimination could be made. Among IC-patients Th cells in the liver but not PB were higher in those with low HBV DNA vs. high HBV DNA (mean % 30.4 vs. 22.5 resp., p<0.05). In contrast, the intrahepatic but not PB proportion of CTL was increased in IC-patients with high HBV DNA vs. low HBV DNA (mean % 40.3 vs. 31.7, p<0.05). In 3 of 15 HLA A2+ patients HBV-specific CTL were detected in the liver. All of them were IC-patients and intrahepatic HBV specific CTL concentration was 10-100 fold higher than in PB. Interestingly, in the only patient with high HBV DNA and detectable intrahepatic HBV-specific CTL, viral load permanently decreased to <105 geq/ml within 4 weeks after sampling.

 

Conclusion:

In conclusion, analysis of intrahepatic immunology suggests that CHB can be divided into 4 stages, and that during the course of infection patients may evolve through these immunological stages to inactive disease.

 

 


Poster 1005

IMPAIRED TOLL-LIKE RECEPTOR EXPRESSION IN CHRONIC HEPATITIS B: IMPLICATIONS FOR PATHOGENESIS AND THERAPY

Kumar Visvanathan, Narelle Skinner, Murdoch Children's Research Institute, Parkville, Vic, Australia; Stephen Riordan, The Prince of Wales Hospital, Sydney, NSW, Australia; Vitina Sozzi, Rosalind Edwards, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Vic, Australia; Judy Chang, Alfred Hospital, Prahran , Vic, Australia; Sharon Ruth Lewin, Alfred Hospital, Prahran, Vic, Australia; Stephen Locarnini, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Vic, Australia.

 

 

Background:

Mechanisms by which hepatitis B virus (HBV) establishes persistent infection remain unclear. In particular, expression of Toll-like receptors (TLR's), increasingly recognized as critically involved in the innate immune response to bacterial and viral pathogens, has not been investigated.

 

Methods:

The TLR2 and TLR4 expression on hepatocytes from fresh liver biopsies of ten patients with untreated chronic hepatitis B (CH-B) was measured by flow cytometry using anti-CD14 (Becton Dickinson) and anti-TLR2 and anti-TLR4 (eBioscience, USA) monoclonal antibodies. Hepatic cell lines including HepG2 cells were infected with recombinant HBV baculovirus expressing wildtype HBV, precore stop codon (G1896A) mutant HBV or the mock baculovirus control and grown for 7 days prior to harvesting and staining for subsequent flow cytometry. Aliquots of cells were also reserved for total RNA extraction using the RNeasy mini kit (Qiagen) in order to measure TLR2 and TLR4 specific mRNA transcripts by realtime PCR. The TLR2 and TLR4 expression on HepG2 was also measured by flow cytometry and analysed for specific mRNA levels.

 

Results:

The expression of TLR2 on hepatocytes (expressed as a ratio to matched isotype controls using mean fluorescence) was significantly reduced in patients with CH-B compared with controls whilst TLR4 expression did not differ significantly between the two groups. Down regulation of TLR2 expression was also found to occur in HepG2 cell cultures infected with wild type HBV (HBeAg-positive). The level of TLR4 expression did not change. In contrast, there was an increase in TLR2 and TLR4 expression in the HepG-2 cells infected with pre-core mutant (HBeAg-negative) HBV. TLR2 and TLR4 mRNA analysis confirmed these findings.

 

Conclusions:

Chronic infection with HBV down-regulates expression of TLR2 on hepatocytes. This down-regulation can be demonstrated in HepG2 cells infected with wildtype HBV but not in cells infected with the pre-core stop-codon mutant virus. This study highlights the importance of the precore protein in the innate immune response to HBV and suggests that this virus-induced defect in innate immunity may contribute to the development of persistent infection.

 

 

Poster 1006

PREDICTION OF LONG-TERM PROGNOSIS OF HEPATITIS B VIRUS CARRIERS WITH NEGATIVE HBe ANTIGEN AND NORMAL TRANSAMINASE LEVEL

Kenji Ikeda, Masahiro Kobayashi, Yasuji Arase, Satoshi Saitoh, Takashi Someya, Tetsuya Hosaka, Hitomi Sezaki, Norio Akuta, Yoshiyuki Suzuki, Fumitaka Suzuki, Hiromitsu Kumada, Toranomon Hospital, Tokyo, Japan.

 

 

Background and Aims:

To elucidate the incidence of hepatitis activation and hepatocellular carcinogenesis in patients with negative HBe antigen and normal aminotransferase over long-term follow-up.

 

Methods:

Among 116 consecutive patients with normal aminotransferase and negative HBe antigen at the time of liver biopsy, sequential frozen sera for initial 5 years were available for 95 patients. There were 75 men and 20 women aged 21 to 67 years with a median of 39 years. The reasons for liver biopsy in spite of "stable hepatitis" included history of ALT elevation in the past (n=41), patient's apprehension based on family history of advanced liver diseases (n=25), possible advanced liver disease on ultrasonography or liver function tests (n=8), or simple desire for thorough examination including biopsy (n=21). HBV DNA assay (sensitivity >400 copy/ml) was annually examined in the initial 5 years after biopsy, and aminotransferase was examined every three month for 5 years or longer after biopsy.

 

Results:

Liver biopsy showed minimal hepatitis (F0) in 9, F1 in 53, F2 in 21, F3 in 6, and F4 and cirrhosis in 6. Initial HBV DNA concentration was low (less than 104copies/ml) in 33, intermediate (104 - 106) in 53, and high (106 or more) in 9. Hepatitis activation rates with twice as high as normal aminotransferase at the end of the third year were 12.1% in the low DNA group, 43.4% in the intermediate group, and 66.7% in the high DNA group. Initial HBV DNA values were significantly associated with future increase in aminotransferase (P<0.0001). Relationship between carcinogenesis rate and laboratory data in early years was also assessed. Carcinogenesis rate in patients with and without high DNA of 106copies/ml during the initial 3 years were 6.9 and 0% at the end of 5th year, and 11.5 and 1.8% at the 10th year, respectively (P=0.021).

 

Conclusion:

Advanced stages of hepatitis were sometimes found in HBe antigen-negative, aminotransferase (ALT)-normal HBV carriers. Serial HBV DNA assessment in the early three years predicted future hepatitis activation and carcinogenesis.

 

 

Poster 1007

HBX AND THE CELLULAR ACETYLTRANSFERASE PCAF ARE CO-RECRUITED IN VIVO ONTO THE HBV CCCDNA MINICHROMOSOME TO REGULATE THE COUPLED VIRAL TRANSCRIPTION/REPLICATION PROCESS

Teresa Pollicino, University of Messina, Messina, Italy; Laura Belloni, Fond. A. Cesalpino - CRS-IRE, Rome, Italy; Francesca Di Padova, Regina Elena Cancer Institute, Rome, Italy; Giuseppina Raffa, Giovanni Squadrito, University of Messina, Messina, Italy; Maurizio Fanciulli, Regina Elena Cancer Institute, Rome, Italy; Massimo Levrero, Fond. A. Cesalpino - CRS-IRE, Rome, Italy.

 

 

Background:

The replicative intermediate of hepatitis B virus (HBV), the covalently closed, circular DNA (cccDNA), is organized into minichromosomes in the nucleus of the infected cell by histone and non-histone proteins.

 

Aims:

We have recently developed an innovative chromatin immunoprecipitation-based technique to study how transcription from the cccDNA minichromosome couples with HBV replication. We found that HBV replication is regulated in vivo, both in a cell based replication system and in the liver of HBV chronically infected patients by the acetylation status of H3/H4 histones bound to the viral cccDNA in the nuclei of HBV infected cells. HBV X protein (HBx) is a multifunctional regulatory protein known to activate transcription, to affect DNA repair processes and to modulate cell growth and death. X protein is expressed at all stages of viral infection in vivo and is implicated in the establishment of the infection in woodchucks. Although many putative HBx targets have been shown to regulate HBV transcription in vitro, the connection between HBx and HBV transcription/replication in vivo remains elusive.

 

Methods:

Using the chromatin immuno-precipitation technique approach we investigated the recruitment on HBV-cccDNA of the viral transactivator HBx, coactivators and co-repressors showing deacetylase or acetyltransferase activities (HDAc(s), p300, CBP, PCAF). Using anti-PCAF and anti-acetyl-H3 antibodies to immunoprecipitate transcriptionally active chromatin and a PCR-based method that discriminates between viral cccDNA and rcDNA, we found that cccDNA-bound H3 acetylation parallels both PCAF recruitment on cccDNA and viral replication. Conversely, the recruitment of HDAc1 paralles the decline of viral replication. By co-immunoprecipitation experiments, we found that PCAF interacts with and stabilizes HBx protein. In addition, HBx is recruited onto the replicating ccc-DNA and its expression potentiates HBV replication and cccDNA accumulation. The kinetics of HBx recruitment onto cccDNA closely matches that of PCAF and parallels the increase of cccDNA-bound H3 acetylation. An HBV mutant lacking HBx expression displays impaired replication capacity and a rapid decline kinetics that is mirrored in a lower acetylation of cccDNA-bound H3 histones. Exogenously expressed HBx complement the replication defect of the HBx-minus virus in trans. On the other hand, abrogation of PCAF expression by specific siRNAs reduces HBV replication and acetylation of cccDNA bound histones.

 

Conclusion:

Our results support a model in which the recruitment of a PCAF-HBx complex onto the cccDNA leads to a prolonged half-life of HBx and the formation of a transcriptionally active "hyperacetylated" HBV chromatin requires the cooperation of HBx and PCAF.

 

 

Poster 1008

SUBTYPES OF HBV GENOTYPE A IN THE UNITED STATES - CLINICAL SIGNIFICANCE AND IDENTIFICATION OF A NOVEL SUBTYPE

Scott K. Fung, C. J. Chu, University of Michigan, Ann Arbor, MI; R. P. Perillo, Ochsner Clinic, New Orleans, LA; A. D. Min, Beth Israel Medical Center, New York, NY; M. Hussain, A. S. Lok, US HBV Epidemiology Study Group, University of Michigan, Ann Arbor, MI.

 

 

Background:

HBV genotypes and subtypes may account for differences in the natural history and response to treatment of chronic hepatitis B. HBV genotype A (HBV/A) has been subclassified into Aa (Asia and Africa) and Ae (Europe) based on differences in 3 nucleotide positions in the core promoter and precore regions.

 

Aims:

(1) To determine the prevalence of subtypes of HBV/A among U.S. patients with chronic HBV infection. (2) To compare clinical and virologic characteristics among patients with various subtypes of HBV/A.

 

Methods:

Stored serum samples from 159 patients with HBV/A who participated in a study on HBV genotypes in the U.S. were analyzed. HBV DNA was extracted; the core promoter, precore and core region (nt 1704-1942) was amplified by PCR and directly sequenced. Sequences were aligned by CLUSTAL W and genetic distances were estimated by the six-parameter method.

 

Results:

Subtype Ae was found in 119 (75%) patients, and Aa in 20 (13%) patients. In addition, a novel subtype, Au (USA), was identified in 20 (13%) patients. Compared to Ae, Au and Aa had nucleotide homologies of 96% and 97%, respectively. Au differed from both Ae and Aa at positions 1802, 1803, 1850 and 1858; while Aa differed from Ae and Au at positions 1809, 1812 and 1862. Patients infected with Ae (72% vs. 15%, p=0.0001) and Au (60% vs. 15%, p=0.009), were more likely to be white than those infected with Aa. The prevalence of HBeAg was higher among patients with Ae (57%), compared to those with Aa (30%, p=0.04) and Au (10%, p=0.0003). Mean HBV DNA levels (log10 copies/ml) were also higher in patients with Ae than in patients with Aa or Au, p=0.01 and p<0.0001, respectively. Patients with Ae were more likely to have elevated ALT compared to those with Au (p=0.004). No patient with Ae or Aa but 3 (15%) patients with Au had the precore G1896A stop codon mutation; this is likely related to the presence of T at nucleotide 1858 in 15 (75%) patients with Au.

 

Conclusions:

A novel subtype Au was identified in 13% of U.S. patients infected with HBV/A. Most patients with Au were white and born in the U.S. Patients with Au had the lowest prevalence of HBeAg and lowest serum HBV DNA levels, and were less likely to have elevated ALT. Au was frequently associated with T at nucleotide 1858, while 100% of Aa and 98% of Ae isolates had C at nucleotide 1858, which prevents selection of the G1896A mutation. Additional studies are required to determine if subtypes of HBV/A are associated with different rates of liver disease progression and response to antiviral therapy.

 

 


Ae
(n=119)


Aa
(n=20)


P value
Ae vs. Aa


Au
(n=20)


P
value
Ae vs Au

% US-Born

84

55

0.01

65

0.09

% HBeAg+ve

57

30

0.04

10

0.0003

Mean HBV DNA± SD
(log10 copies/ml)

7.1 ± 2.4

5.7 ± 2.1

0.01

4.1 ± 1.5

<0.0001

% elevated ALT

75

60

NS

40

0.004

Precore C1858T, %
G1896A, %

2
0

0
0

NS
-

75
15

<0.0001
0.01

 

 

Poster 1009

THE PREVALENCE OF HBV GENOTYPES, VIRAL VARIANTS AND HBeAG-VE CHRONIC HEPATITIS B (E-CHB) IN A CANADIAN CENTER

Scott K. Fung, M. Hussain, University of Michigan, Ann Arbor, MI; F. Wong, D. Wong, University of Toronto, Toronto, ON, Canada; A. S. Lok, University of Michigan, Ann Arbor, MI.

 

 

Background:

HBeAg-negative chronic hepatitis B was initially thought to be common in Asia and Southern Europe but uncommon in North America. The prevalence of e-CHB and the distribution of HBV genotypes and viral variants in North America have not been defined.

 

Aims:

1) To determine the prevalence of e-CHB in Canadian patients with chronic hepatitis B. 2) To determine the distribution of HBV genotypes, core promoter [CP] (A1762T and G1764A) and precore [PC] (G1896A) variants in this population. 3) To compare virologic and clinical characteristics among the major genotypes.

 

Methods:

Adult patients with CHB were enrolled in a cross-sectional study at University Health Network (Toronto, Canada) from 7/03 to 2/04. HBV DNA was quantified using a PCR assay and HBV genotypes, CP and PC variants were determined using the InnoLipa assay.

 

Results:

264 consecutive patients, 159M/105F, with a mean age of 43.4 ± 14.0 years were studied; 90% were Asians, 7 (3%) had received antiviral therapy previously. 93 (35%) patients were HBeAg+ve. Among the HBeAg-ve patients, 20% had ALT >1.5XULN and HBV DNA >5 log10 copies/ml. Genotypes B and C were found in 40% and 50% patients, respectively; while CP and PC variants were detected in 54% and 43% patients, respectively. Compared to HBeAg+ve patients, HBeAg-ve patients were older (47.0 vs. 37.2 years), more likely to have normal ALT (43% vs. 31%) and had lower HBV DNA (4.8± 1.9 vs. 8.2± 1.5 log10 copies/ml). HBeAg-ve patients were also more likely to have CP and PC variants (70% vs. 30%, and 53% vs. 27%, respectively) and a higher prevalence of HBV/C (62% vs. 44%). Compared to HBV/C, HBV/B was associated with a lower prevalence of HBeAg, lower serum HBV DNA level, and a lower prevalence of CP variants but a higher prevalence of PC variants. Among HBeAg+ve patients, male gender (p=0.07) was weakly associated with elevated ALT; while CP variants (p=0.01) were associated with high HBV DNA (>6 log10 copies/ml). Among HBeAg-ve patients, high HBV DNA (p=0.03) was associated with elevated ALT; while male gender (p=0.009), elevated ALT (p=0.002), CP (p=0.005) and PC (p=0.01) variants were associated with high HBV DNA.

 

Conclusions:

In this cross-sectional study, 65% patients presenting to a Canadian tertiary referral center were HBeAg-ve and 20% patients met diagnostic criteria for e-CHB. The major genotypes in this predominantly Asian population were B and C; CP and PC variants were detected in half of the patients. Male gender, presence of CP and PC variants were more predictive of elevated ALT and high HBV DNA than HBV genotypes.

 

 


HBV/B (n=60)


HBV/C (n=75)


P
-Value

Mean age ± SD (yrs)

40± 14

41± 13

0.9

Male gender, %

48

60

0.2

Mean ALT ± SD (IU/L)

58± 54

90± 152

0.08

HBeAg+, %

38

62

0.02

Mean HBV DNA± SD (log10 c/ml)

6.5± 2.0

7.0± 1.8

0.09

CP variant, %

32

63

0.0009

PC variant, %

68

22

<0.0001

 

 

Poster 1010

CHRONIC HBV PATIENTS DISPLAY IMPAIRED MATURATION AND ANTIGEN PRESENTATION OF MYELOID DENDRITIC CELLS AND REDUCED IFN-a PRODUCTION OF PLASMACYTOID DENDRITIC CELLS

Renate G. van der Molen, Dave Sprengers, Rekha S. Binda, Patrick P. C. Boor, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands; Esther C. de Jong, Academic Medical Center, Amsterdam, Netherlands; Johannes G. Kusters, Jaap Kwekkeboom, Harry L. A. Janssen, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands.

 

 

Background:

Dendritic cells (DC) play an important role in the induction of T cell responses.

 

Aims:

We postulate that the hampered anti-viral T cell response in chronic hepatitis B patients (HBV) is the result of impaired DC function.

 

Methods:

To investigate this we focused our research on two major precursor subsets of DC in peripheral blood, the myeloid (mDC) and the plasmacytoid DC (pDC). MDC and pDC were isolated from peripheral blood of chronic HBV patients (n=30, median HBV-DNA 1x108 geq/ml and median ALT 48 U/l) and healthy controls (n=19), using magnetic cell sorting techniques. MDC were isolated with an anti-CD1c (BDCA1) antibody after depletion of CD19+ cells. PDC were isolated with an anti-BDCA4 antibody. Freshly isolated mDC and pDC were matured for 24 hours with IL-1ß and TNF-a.

 

Findings:

The allostimulatory capacity of matured mDC, but not of matured pDC, was significantly decreased in chronic HBV patients compared to healthy controls. Also, mDC of patients displayed a decreased percentage costimulatory molecules CD40, CD80 and CD86 after maturation. The defects in expression of costimulatory molecules and allostimulatory capacity of mDC were not related to viral load or ALT. A significantly decreased TNF-a production by purified mDC of chronic HBV patients was observed after stimulation with poly (I:C) and IFN-?, while no difference was found in IL-6, IL-10 and IL-12 p70 production. TNF-a was significantly reduced in patients with low viral load (HBV-DNA < 105 geq/ml) as compared to patients with high viral load (HBV-DNA > 108 geq/ml) but not related to ALT levels. Compared to controls, purified pDC from patients produced less IFN-a in response to stimulation with Staphylococcus aureus Cowan strain I antigen. No difference was observed in IL-6, IL-10 and TNF-a production. The IFN-a production of pDC was significantly decreased in patients with high ALT (ALT > 2 x upper limit of normal) as compared to patients with normal ALT. The functional impairment of mDC and pDC was not observed in patients chronic inflammatory liver disease of non-viral origin (primary biliary cirrhosis n=2; hemochromatosis n=4) and ALT comparable to HBV patients (median ALT 40 U/l).

 

Conclusions:

Therefore, our findings appear indeed related to HBV infection rather than a general inflammation effect. In conclusion, the functional impairment of mDC and pDC might be an important mechanism for HBV persistence and chronicity.

 

 

Poster 1011

FUNCTIONAL VARIATION IN THE IFNAR2 AND IL10RB GENES LINKED TO PERSISTENT HBV INFECTION

Mohd Taib Nor Azizah, Andrew Durham, Susanne Knapp, Howard C. Thomas, Mark Thursz, Imperial College London, London, United Kingdom; Angela J. Frodsham, Lyna Zhang, Adrian V. S. Hill, University of Oxford, Oxford, United Kingdom; Mary Graves, Hoffman La Roche, Nutley, NJ.

 

 

Background:

Persistent hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma, the most frequent cancer in some developing countries. Up to 15% of those infected fail to clear HBV resulting in approximately 350 million persistent carriers worldwide.

 

Aims:

Via a whole genome scan in Gambian families, we identified a major susceptibility locus as a cluster of class II cytokine receptor genes on chromosome 21q22.

 

Methods:

Fine mapping using high density SNP genotyping in family association studies was used to identify causal variants. Coding changes in two of these, the type I interferon receptor gene, IFNAR2, and the IL10RB gene that encodes a receptor chain for IL-10-related cytokines including the interferon lambdas, are associated with viral clearance; Haplotype P value = 0.0003.

 

Relative transcriptional efficiency of the allelic variants in both receptors was tested: RNA from EBV transformed B cells of normal heterozygous individuals was analysed by a mass spectrometry-based method, to determine the relative levels of the two polymorphic transcripts, using genomic DNA to control for assay efficiency. There was no significant difference in RNA expression in the IFNAR2-F8S variants (P=0.401) but mRNA encoding the E allele of IL10RB-K47E was expressed at significantly higher levels than the K allele (P<0.001).

 

Results:

Using antibody labeling and FACS analysis cell surface expression the IFNAR2*F allele was shown to be significantly higher than the IFNAR2*S allele and the IL-10RB*E allele was higher than that of the IL-10RB*K allele. In U5A cell lines, which lack expression of IFNAR2, transfected with either of the IFNAR2 allelic variants interferon induced MHC class I expression was greater in cells expressing the F allele (P=0.001). Furthermore, interferon a induced anti-viral protection was significantly enhanced in U5A cells transfected with the IFNAR2-F allele (P<0.001).

 

IL10 mediated inhibition of lipopolysaccharide induced TNFa expression was higher in monocytes from individuals homozygous for the IL-10RB*E allele (associated with viral clearance): P =0.015.

 

Conclusions:

Association of a gain-of-function variant in IL-10RB with HBV clearance suggests that signaling of an alternative ligand for IL-10RB may be responsible for the observed genetic effect. The most likely candidates are the anti-viral cytokine IL-28 & 29.

 

 

Poster 1012

SERUM HBV-DNA LEVELS HIGHER THAN 105 CP/ML ARE ASSOCIATED WITH SIGNIFICANT INFLAMMATORY ACTIVITY IN THE LIVER OF HBeAG NEGATIVE HBSAG CARRIERS WITH PERSISTENTLY NORMAL OR ELEVATED ALT

Diego Flichman, Anna Maria Maina, Filippo Oliveri, Barbara Coco, Pietro Ciccorossi, Piero Colombatto, Rodolfo Sacco, Pisa University Hospital, Pisa, Italy; Giuseppe Colucci, Roche Molecular System, Basel, Switzerland, Basel, Switzerland; Maurizia R. Brunetto, Pisa University Hospital, Pisa, Italy.

 

 

Background:

Recently the threshold of 105 cp/ml of HBV-DNA in serum was proposed to discriminate HBV carriers with or without active liver disease. However, viremia fluctuations over time in a significant proportion of carriers may hamper its clinical usefulness.

 

Aims and Methods:

To correlate biochemical and virologic profiles with histological activity in 30 HBV carriers (25 HBeAg negative) ALT and viral load (COBAS Taqman HBV test) were measured in 266 samples (4-19 from each carrier every 1 to 3 months during 20.3±7.9 follow-up); a liver biopsy was performed in 22 cases. Patients were grouped by their biochemical profiles: A-Persistently normal ALT (12), B- Persistently elevated ALT (4), C- ALT flares with biochemical remission (6), D- ALT flares without biochemical remission (8, 5 were HBeAg positive).

 

Results:

Mean viral load were significantly different between groups (A: 5.37 103 cp/ml, B: 2.75 105 cp/ml, C: 1.51 106 cp/ml and D: 2.82 107 cp/ml, p < 0,001). Major (= 2 logs) HBV-DNA fluctuations occurred in many cases, but less frequently in group A patients (8.3%) vs the others [B (25.0%), C (83.3%) and D (57.1%)] (p<0.014). Range of HBV-DNA fluctuations was larger in patients with ALT flares (C and D) (mean 2.57 103 cp/ml) as compared to carriers with stable ALT levels (A and B) (1.74 101 cp/ml) (p< 0.001). Overall 19 patients (including 3 from group A) had significant levels of necro-inflammation at histology (grading >4), all but one had HBV-DNA levels higher than 105 cp/ml in at least one observation; however, 9 of them had levels below 105 cp/ml at least once during follow-up, particularly in group C where viremia levels fall below this cut-off in 4 out of 6 patients, even in 4 consecutive monthly samples. Overall 105 cp/ml cut-off showed 83.8% specificity, 100% sensitivity and 100% PPV to identified necro-inflammatory damage.

 

Conclusion:

HBV-DNA levels >105 cp/ml had a high diagnostic accuracy in the identification of the patients with significant liver necro-inflammation, however the evidence of HBV-DNA levels <105 at the single point observation is not sufficient to role out the presence of liver disease.

 

 

Poster 1013

HEPATITIS B VIRAL LOAD IS ASSOCIATED WITH THE DEVELOPMENT OF HEPATOCELLULAR CARCINOMA (HCC)

AA Evans, RE Fabre, G Chen, L Pasternak, Fox Chase Cancer Center, Philadelphia, PA; UH Iloeje, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT; WT London, Fox Chase Cancer Center, Philadelphia, PA.

 

 

Background and Aim:

This prospective study in a cohort of 3754 Asian-American HBV-infected individuals identified through routine screening programs in the Philadelphia area tested the hypothesis that elevated serum viral load is a positive predictor of HCC development.

 

Methods:

A nested case-control analysis of 37 HCC cases and 61 controls was carried out. Serum samples collected at cohort entry were assayed for HBV viral load by real-time PCR (LOQ 3x104 copies/mL). Viral load was categorized as undetectable (<LOQ), low titer (<105 copies/mL) or high titer (>105 copies/mL). For the HCC cases, cohort entry occurred between <1-17 years (median 4 years) prior to diagnosis with HCC. Each case was matched with up to two controls by age (+ 2 years), sex, and year of cohort entry. Median age was 54 years (range 42-74) in the cases and 56 years (range 44-77) in the controls. 86.5% of both cases and controls were male. Median year of cohort entry was 1991 (range 1983-2000), and median time to death for cases was 4.5 years (range <1-17 years). Two cases were still alive as of last follow-up in 2004.

 

Results:

Five of 37 cases (13.5%) and 32 of 61 controls (52.4%) had no detectable serum HBV. Overall, the relative risk (RR) (95% CI) of HCC for HBV DNA positives was 7.2 (2.1-24.5). With the undetectable group as reference, low titer viral load (<105 copies/mL) conferred a RR of 4.1 (95% CI 1.1-15.5) while higher titer (>105 copies/mL) had a RR of 8.4 (95% CI 2.8-25.7) (p for trend <0.001). Median viral load was 9.3x105 copies/mL in the HCC cases and undetectable (<3x104 copies/ml) in the controls (p<0.001). When subjects negative for HBV DNA were excluded, the median viral loads of the HCC cases and controls (1.2x106 and 1.5x105 copies/mL, respectively) remained significantly different (p=0.005).

Conclusion:

Viral load is strongly associated with risk of future development of HCC in patients chronically infected with HBV. This relationship exhibited a dose-response characteristic, where increasing viral load was associated with increased risk.

 

Category

HCC Cases (n=37)
N (%)

Controls (n=61)
N (%)

RR (95% CI)

Undetectable
HBV DNA

5 (13.5%)

32 (51.6%)

Reference

Low Titer
HBV DNA

7 (18.9%)

11 (18.0%)

4.1 (1.1-15.5)

High Titer
HBV DNA

25 (67.6%)

18 (29.5%)

8.4 (2.8-25.7)

 

 


Poster 245

RANDOMIZED, DOUBLE-BLIND STUDY COMPARING ADEFOVIR DIPIVOXIL (ADV) PLUS EMTRICITABINE (FTC) COMBINATION THERAPY VERSUS ADV ALONE IN HBeAG (+) CHRONIC HEPATITIS B: EFFICACY AND MECHANISMS OF TREATMENT RESPONSE

George Lau, Department of Medicine, Queen Mary Hospital, Hong kong, Hong Kong Special Administrative Region of China; Helen Cooksley, Institute of Hepatology, University College, London, United Kingdom; Ruy M. Ribeiro, Kimberly A. Powers, Los Alamos National Laboratory, Los Alamos, NM; Scott Bowden, Victorian Infectious Diseases Reference Laboratory,, Victoria, Australia; Herve Mommeja-Marin, Elsa Mondou, Gilead sciences, Inc, Durham, NC; Sharon Lewin, Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, Australia; Franck Rousseau, Gilead sciences, Inc, Durham, NC; Alan S. Perelson, Los Alamos National Laboratory, Los Alamos, NM; Stephen Locarnini, Victorian Infectious Diseases Reference Laboratory, Victoria, Australia; Nikolai V. Naoumov, Institute of Hepatology, University College, London, United Kingdom.

 

 

Background:

Improvement of treatment response in chronic hepatitis B requires new antiviral regimens and better understanding of viral and host factors associated with sustained control of HBV replication.

 

Aims:

i) To compare the efficacy of a new combination therapy of ADV plus FTC versus ADV alone; ii) To examine early HBV kinetics and virus-specific T-cell reactivity to gain understanding of the mechanisms of successful HBV control.

 

Methods:

Thirty treatment naïve, HBeAg (+) patients (HBV genotype B, n=9; genotype C, n=21) with ALT> 1.3xULN were randomized to receive ADV 10 mg + FTC 200 mg qd (Group A, n=14) or ADV 10 mg + placebo qd (Group B, n=16) for 48 weeks. Peripheral blood samples were collected prospectively at Day 0, 1, 3, 5, 7, 9, 11, 14; Week 3, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48. HBV DNA was quantitated by ultrasensitive Digene assay and by molecular beacons assay (range 3x102 to 109 copies/mL). Mathematical modeling was employed to determine the patterns of viral kinetics. HBV-specific CD4 and CD8+ T-cells were enumerated by IFN? ELISpot assays and tetramer staining for CD8+ T-cells.

 

Results:

The combination (ADV+FTC) therapy showed greater antiviral activity compared to ADV alone: median log10 reduction of viremia at treatment week 24 (TW24) was 3.14 vs 2.16 (p=0.004); at TW48 - 3.48 vs. 2.22 (p=0.036), respectively. HBeAg seroconversion occurred in 3 patients - Group A (n=2) and Group B (n=1). Mathematical modeling of early HBV kinetics (baseline to TW12) revealed a two-phase decay in most patients with the first phase t1/2 from 0.5 day to 3.3 days (mean: 1.4 ± 0.7 day) and the second phase t1/2 from 3.6 to 112 days (mean: 24.4 ± 22.6 days). The infected cell loss rate (d) was significantly higher in the combination arm (d =0.066 day-1) vs monotherapy ( (d =0.035 day-1 p=0.039). Early HBV kinetics identified two subsets - patients who cleared the virus (<300 copies/mL) by TW12 (fast responders) and those who did not (slow responder). Fast responder had a larger d (0.069 day-1) than slow responder (d =0.029 day-1, p=0.0061). Patients on combination treatment were more likely to be fast responders than those on monotherapy (p=0.01). TW12 clearance of virus (<300 copies/mL) was associated with enhanced T-cell reactivity (for T-cell response to HBcAg p=0.05; and to HBsAg p=0.03). All 3 cases with HBeAg seroconversion were amongst those fast responders.

 

Conclusions:

1.)   ADV plus FTC combination therapy shows faster and greater HBV suppression in comparison with ADV alone.

2.)   This faster suppression is reflected in the second phase viral kinetic parameters; and

3.)   Early HBV suppression during therapy is associated with enhanced antiviral immunity.

 

 


Poster 246

EFFICACY AND SAFETY OF LAMIVUDINE IN LATE PREGNANCY FOR THE PREVENTION OF MOTHER-CHILD TRANSMISSION OF HEPATITIS B; A MULTICENTRE, RANDOMISED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDY

Wei-Min Xu, Shanghai Infectious Disease Hospital, Shanghai, China; Yu-Tao Cui, Beijing United Family Hospital, Beijing, China; Ling Wang, Beijing Ditan Hospital, Beijing, China; Hong Yang, Beijing You'an Hospital, Beijing, China; Zhi-Qing Liang, Xinan Hospital, Chongqing, China; Xiao-Mao Li, 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; Shulin Zhang, The 1st Affiliated Hospital of Xian Communication University, Xian, China; Fu-Yuan Qiao, Wuhan Tongji Hospital, Wuhan, China; Raida Gobenciong Varona, Cebu Doctors Hospital, Cebu City, Philippines; Fiona M. Campbell, GlaxoSmithKline, Greenford, United Kingdom; Chai-Ni Chang, Stephen D. Gardner, GlaxoSmithKline, Research Triangle Park, NC.

 

 

Background/Aims:

This multicentre, randomised, double-blind, placebo-controlled study aimed to evaluate whether lamivudine therapy during late pregnancy, in addition to HBV vaccination plus HBIG for infants, reduces the rate of transmission of HBV from mother to infant compared to vaccination and HBIG alone.

 

Methods:

114 mothers (HBsAg positive, serum HBV DNA >1000 MEq/mL by CHIRON assay) were randomised to receive either lamivudine (LAM, 100mg daily, n=56) or matched placebo (PLA, n=58), administered from week 32 ± 2 weeks of gestation until 4 weeks postpartum. All infants received both recombinant HBV vaccination (VAC, 10g/0.5mL dose, within 24 hours of birth, at week 4 and at week 24) and hepatitis B immunoglobulin (HBIg, 200IU, single dose within 24 hours of birth).

The primary efficacy endpoint was the incidence of HBsAg seropositivity in infants at age 1 year (week 52). Secondary efficacy endpoints included HBsAb seropositivity and serum HBV DNA by PCR (Roche COBAS Amplicor assay) in infants at age 1 year. Safety measures included adverse events (AEs), deaths, and laboratory abnormalities.

 

Results:

Endpoint
(Infants at Week 52)1


LAM+VAC+HBIg
N=56


PLA+VAC+HBIg
N=59
2


p value

HBsAg +ve

10 (18%)

23 (39%)

0.014

HBsAb +ve

47 (84%)

36 (61%)

0.008

HBV DNA +ve3

11 (20%)

27 (46%)

0.003

 

1.All missing values counted as failure

2.Includes one set of twins

3.By Roche COBAS Amplicor assay (LLOD 200 copies/mL)

 

The proportion of mothers with serum HBV DNA levels = 1000 MEq/mL (by CHIRON assay) at the time of delivery, was higher in the lamivudine than the placebo group (55/56 [98%] versus 18/59 [31%], respectively).The incidence of AEs in the mothers was similar during lamivudine and placebo therapy (4/56 [7%] versus 6/58 [10%], respectively).The incidence of AEs in the infants in the LAM+VAC+HBIg and PLA+VAC+HBIg groups was similar (10/56 [18%] versus 12/59 [20%], respectively). The incidence of serious AE's in the infants was also similar in both groups (5/56 [9%] versus 3/59 [5%], respectively).

 

Conclusion:

The rate of HBV vertical transmission was significantly reduced in infants receiving HBV vaccine and HBIG whose mothers received lamivudine in the 8 weeks (+/- 2 weeks) prior to delivery compared to those whose mothers received placebo. No safety concerns were observed in either the mothers or the infants. Lamivudine is a well-tolerated and effective addition to the current management options available to control the vertical transmission of HBV from mothers to infants.

 

 


Poster 1281

QUANTITATIVE DETECTION OF (-) AND (+) STRAND HBV DNA AND THEIR REPLICATIVE INTERMEDIATES IN THE LIVER OF CHRONICALLY INFECTED PATIENTS

Andreas Laras, Ageliki Kostamena, John Koskinas, Athens University School of Medicine, Athens, Greece; Stephanos J. Hadziyannis, Henry Dunant Hospital, Athens, Greece.

 

 

Background:

Replication of HBV occurs via a covalently closed circular (ccc) DNA intermediate responsible for the production of pregenomic (pg) RNA. Reverse transcription of pgRNA and (-) strand DNA synthesis in the nucleocapsid is followed by (+)strand synthesis and formation of the relaxed circular (RC) DNA genome. The molecular events of HBV replication are well understood, however, there is little information regarding the stoichiometry of its different nucleic acid components in vivo.

 

Aims:

a) To develop sensitive assays for the quantification of intrahepatic (-) and (+) strand HBV DNA. b) To utilize this method in conjunction with our previously developed assays for cccDNA and pgRNA detection, to determine the in vivo concentration and stoichiometry of these molecules.

 

Methods:

Intracellular HBV cccDNA, pgRNA, (-) and (+)strand DNA levels were quantified by real-time PCR. Total DNA and RNA were extracted from liver biopsy samples of 15 pts with HBV infection. HBV DNA copy numbers were determined using primers that specifically detect cccDNA, (-) and (+)strand DNA. For the quantification of pgRNA we utilized a transcript-specific RT-PCR assay that monitors HBV core promoter activity. Results were normalized to cellular beta-globin levels.

 

Results:

We detected HBV replicative intermediates and genomic DNAs in all liver samples. HBV cccDNA levels had a median value of 1.59 copies per cell (range, 0.09 - 280), pgRNA levels 9.63 cp/cell (0.02 - 486), (-)strand DNA levels 200 cp/cell (4.21 - 32,000) and (+)strand DNA levels 99 cp/cell (0.7 - 20,700). The levels of (-) and (+)strand DNA correlate with the levels of HBV replicative intermediates (cccDNA and pgRNA) measured in the liver. Levels of (-)strand were higher than (+)strand DNA levels, reflecting the presence of (-)strand in both immature and mature HBV particles. Plus-strand HBV DNA, present in mature particles, represents on the average 38% of the total encapsidated HBV. The relative amount of (+)strand DNA increases with the total intracellular HBV DNA.

 

Conclusions:

  1. We have developed a sensitive method for the quantification of (-) and (+)strand HBV DNA in the liver.
  2. Although the levels of these molecules, as well as those of cccDNA and pgRNA, correlate with viral activity, they were 10- to 100-fold higher than those of either cccDNA or pgRNA, measuring intracellular HBV replication with higher sensitivity.
  3. The ratio of (+)/(-) strand increases with viral activity reflecting higher rate of production of mature particles. 4. The in vivo quantification of HBV replicative intermediates and genomic DNA molecules, provides direct information on HBV genome formation and closely monitors HBV particle maturation. This method may be used to evaluate the activity and efficacy of new antiviral agents.

 

 


Poster 1282

ACCUMULATION OF BASIC CORE PROMOTER MUTATIONS DURING THE NATURAL COURSE OF HBV INFECTION

Evangelini Dimou, Henry Dunant Hospital, Athens, Greece; Andreas Laras, Athens University School of Medicine, Athens, Greece; Stephanos J. Hadziyannis, Henry Dunant Hospital, Athens, Greece.

 

 

Background:

Mutations in the HBV core promoter (CP) are common in HBeAg(-) patients with chronic HBV infection but their role in the natural course of HBV infection remains controversial. The double nucleotide mutation A1762T/G1764A (T/A) in the basic CP (BCP) is frequently observed and most studies focus on this mutation alone. Other mutations in this region have not been sufficiently studied and their prevalence and role remain undetermined.

 

Aims:

The objective of this study was to evaluate the distribution of all mutations in the CP/BCP region and their evolution during the natural course of HBV infection.

 

Methods:

Hepatitis B virus CP sequences were determined in 126 chronically HBV genotype D infected patients. All patients had HBeAg(-)/anti-HBe(+) infection. Twenty-two were inactive carriers [F/M: 6/16, median age 46.5 yrs (30-55)], 78 had CHB [F/M: 15/73, median age 53 yrs (30-74)], 10 had decompensated cirrhosis [F/M: 4/6, median age 62.5 yrs (50-80)] and 16 hepatocellular carcinoma [F/M:2/8, median age 66.5 yrs (53-76)]. All CP sequences were analyzed, sequences in the nt 1160-1770 BCP region were classified according to mutations mutations found: wild type (wt) - no mutations; I - only the double T/A mutation found; II - the double T/A plus additional mutations; III - mutations other than T/A.

 

Results:

Mutations detected in the CP were found to cluster in the BCP region between nts 1760-1770 and at nt 1753. In the group of inactive carriers wt sequences were identified in 8 (36,3%), type I mutations in 9 (40,9%), type II in 2 (9,1%) and type III in 3 (13,6%) pts. In CHB pts wt sequences were found in 17 (21,8%), type I mutations in 34 (43,6%), type II in 5 (5,4%) and type III in 22 (28,2%). In pts with decompensated cirrhosis wt sequences were found in 2 (20%), type I mutations in 4 (40%), type II in 0 (0%) and type III in 4 (40%). HCC patients with wt sequences were not found. Type I mutations were detected in 12 (75%), type II in 2 (12,5%) and type III in 2 (12,5%) HCC pts. Type III mutations often involve more than one nt position: in inactive carriers we found 4 point mutations in 3 pts (1,33 mutations/pt), in CHB 27 in 18 pts (1,5mutations/pt) and in HCC 8 in 4 pts (2 mutations/pt). At nucleotide position 1753, in inactive carriers we found mutations in 12 (54,5%), in CHB in 45 (58%), in decompensated cirrhosis in 4 (40%) and in HCC pts in 13 (81,3%).

Conclusions:

  1. The majority (79%) of HBeAg(-) patients harbor BCP mutations and one-third of them (31%) have mutations other than T/A.
  2. The proportion of wt BCP sequences decreases with the duration of infection and the progression of liver disease; one third (36%) of inactive carriers but none (0%) of the HCC patients had wt BCP sequences.
  3. These findings show that in addition to the very common double T/A mutation (predominating in 80% of HCC patients), other mutations frequently develop and accumulate in the BCP region and they may play an important role during the course of HBV infection.

 

 


Poster 1285

DYNAMIC RANGE AND REPRODUCIBILITY OF COBAS TAQMAN DETECTION OF HEPATITIS B VIRUS DNA

Magnus Lindh, Göteborg University, Göteborg, Sweden; Giuseppe Colucci, Roche Molecular Systems, Rotkreutz, Switzerland; Charles Hannoun, Göteborg University, Göteborg, Sweden.

 

 

Background/Aim:

Real-time polymerase chain reaction (PCR) analysis of hepatitis B virus (HBV) DNA in serum using the Cobas Taqman assay was evaluated.

 

Methods:

Analysis of serially diluted samples representing genotypes A-D showed a linear detection over 7 logs, from 10 IU/mL to 100 million IU/mL.

 

Results:

Reproducibility testing of 6-replicates analyzed on 4 occasions showed a coefficient of variation (CV) of 22.0% for HBV DNA values around 10 IU/mL, and 1.2% for levels at or above 1000 IU/ml. Comparison of quantification of 100 clinical samples by Cobas Amplicor and Cobas Taqman showed a good correlation with an R2 of 0.96. However, at very high levels the viremia was overestimated by Amplicor as compared to Cobas Taqman. Monitoring of patients after liver transplantation and during therapy showed that HBV DNA levels as low as 1 IU/mL, i.e. below the linear detection range, could be detected. Using this assay the half-life of HBV DNA after liver transplantation was found to be 7 days.

 

Conclusion:

The high reproducibility and wide detection range should be of value for clinical assessment of chronic carriers and for monitoring the response to antiviral treatment.

 

 


Poster 1287

LONGITUDINAL EVALUATION OF VIROLOGICAL PROFILES IN HBV/HCV COINFECETED PATIENTS, AN ITALIAN MULTICENTRE STUDY

Giovanni Raimondo, University of Messina, Messina, Italy; Maurizia Rossana Brunetto, Santa Chiara Hospital, Pisa, Italy; Patrizia Pontisso, University of Padova, Padova, Italy; Antonina Smedile, University of Torino, Torino, Italy; Anna Maria Maina, SANTA CHIARA HOSPITAL, Pisa, Italy; Carlo Saitta, Giovanni Squadrito, University of Messina, Messina, Italy; Natascia Tono, University of Padova, Padova, Italy; A.I.S.F. Cooperative Group.

 

 

Background:

Much evidence suggests that dual HBV and HCV infection has major clinical relevance in terms of low rate of response to therapy, progression to cirrhosis and risk of hepatocellular carcinoma development.

 

Aims:

However, exhaustive studies in this field are still lacking. Particularly, very scarce information is at present available about the virological behaviour of HBV/HCV coinfected individuals over time.

 

Methods:

This is an Italian multicentre study that enrolled 133 untreated HBsAg/anti-HCV positive patients (M/F=102/31; 51 years median age, range 22-83; HBeAg/anti-HBe=12/121) longitudinally followed up for one year with bimonthly evaluation (seven time points for each patient) of HBV/HCV viremia levels (COBAS Amplicore, Roche Diagnostics) and liver biochemistry. One hundred three of them were negative (group-A) and 30 positive (group-B) for HDV coinfection.

 

At baseline, in group-A, active infection of both HBV and HCV (HBV DNA >105 copies/ml, HCV RNA >600 IU/ml) appeared to be present in 12 cases, inactive infection by both viruses (HBV DNA <105 copies/ml, HCV RNA <600 IU/ml) in 20 cases, active HBV and inactive HCV in 14 cases, inactive HBV and active HCV in 57 cases. During the follow-up, however, 32 of the 103 cases (31%) showed fluctuation of HBV and/or HCV viremia levels that at different time points were over or under the cut off limits. Such virological profiles did not parallel analogous fluctuation of aminotransferase values. Considering that cases with flares of viremia must be classified as carriers of active infection, at the end of the study the virological behaviour led us to diagnose 24 cases as HBV/HCV active infections, only 15 confirmed to have both viruses inactive, 15 had active HBV and inactive HCV, 49 had inactive HBV and active HCV.

 

Results:

Univariate and multivariate statistical evaluation of several clinical/biochemical/histological features were performed, and significant results were found in the association between HCV genotype 1b and double active infection (p<0.05) and in the association of abnormal aminotransferase values with double active infection (p<0.05) and active HBV/inactive HCV infection (p=0.015).

Among the 30 group-B subjects, 15 had both HBV and HCV persistently inactive, while the other 15 HDV positive cases showed fluctuating patterns of either HBV or HCV active infection.

 

Conclusions:

This study, longitudinally examining the largest series of HBV/HCV coinfected patients analysed so far, shows that the virological patterns in cases of coinfection may be widely divergent and have dynamic profiles. The longitudinal evaluation of the viremia levels of both viruses is essential for correct etiologic diagnosis and proper therapeutic choices.

 

 


Poster 279

HEPATITIS B VIRUS LAMIVUDINE RESISTANT MUTATIONS ABROGATE SECRETION OF HEPATITIS DELTA VIRUS

Angeline Bartholomeusz, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Vic, Australia; Patricia Vietheer, Hans Netter, Monash University, Clayton, Vic, Australia; Vitina Sozzi, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Vic, Australia.

 

 

Background:

Infections with Hepatitis delta virus (HDV) is dependent on a concurrent hepatitis B virus (HBV) infection. HDV requires the envelope proteins (HBsAg) of HBV for assembly and for subsequent rounds of infection. Lamivudine (LMV) is currently used for the treatment of HBV infections and is also used in patients co-infected with HDV. Prolonged therapy with LMV results in the selection of mutations in the HBV polymerase at rtM204I/V which are associated with concomitant changes in the overlapping envelope gene at positions sI195M or sW196L/S/stop.

 

Aims:

The aim of this study was to investigate the affect of mutant HBsAg proteins encoded by LMV-resistant HBV variants on HDV secretion.

 

Methods:

The LMV resistance mutations were introduced into the HBsAg cDNA by site directed mutagenesis. Plasmids (pCI) that express modified HBsAg proteins and a pSVL-construct encoding for a replication-competent HDV were co-transfected into HuH-7 cells. HBsAg was measured using the chemiluminescence Abbott Prism HBsAg assay. HDV replicative RNA-intermediates and the presence of secreted HDV particles were analysed by Northern blot. The presence of the HDV-specific delta antigen in the cell culture supernatant was assayed by immunoblot.

 

Results:

Northern analysis of HDV-specific RNA in the supernatant versus the cell lysate demonstrated that the HBsAg with the mutation sI195M (rtM204V) was able to support HDV secretion. In contrast the HBsAg mutants sW196S (rtM204I) or sW196L (rtM204I) did not efficiently support HDV secretion. In addition, there was a comparable decrease in the amount of the delta-antigen in the presence of the HBsAg mutants with sW196S/L amino acid exchanges.

 

Conclusion:

An understanding how newly selected antiviral resistant mutants alter viral interactions is important for the treatment of patients co-infected with HBV and HDV. Clinically relevant HBV mutants selected during antiviral therapy have a differential profile to support HDV secretion, in particular the data confirmed that amino acid position HBsAg 196 is critical for HDV secretion and hence, may indirectly abrogate an underlying HDV infection. The HBsAg mutation at amino acid sI195M was shown to have a minimal effect of HDV packaging and release. Differential support for HDV secretion may have consequences for clinical prognosis as co-infected patients are treated with antiviral agents.

 

 


Poster 281

INVOLVEMENT OF HEPATITIS B VIRUS (HBV) COVALENTLY CLOSED CIRCULAR DNA IN SUSTAINED HBV REPLICATION IN IMMUNOCOMPROMISED MICE AFTER HYDRODYNAMIC INJECTION OF A NAKED PLASMID ENCODING HBV DNA

Takahiro Suzuki, Tetsuo Takehara, Kazuyoshi Ohkawa, Atsushi Housui, Masashi Jinushi, Tomohide Tatsumi, Kanazawa Yoshiyuki, Norio Hayashi, Oasaka University Graduate School Of Medicine, Suita, Japan.

 

 

Background and Aims:

The lack of a small animal model of HBV infection hampers the elucidation of pathophysiological mechanisms of this disease. Although transgenic mice which contain HBV DNA produce virus, this production is derived from integrated viral transcription template which is clearly different from the natural infection.

 

Methods:

We injected a plasmid encoding HBV DNA into nude mice to establish an episomal HBV replication system. The injection led to high levels of HBV replication in the murine liver for over 1 year. Of note is that the transfected liver clearly produced HBV covalently closed circular (CCC) DNA, which appeared to be required for long-term production of HBV in this system. Materials and Methods: pHBV1.5 plasmid contains 1.5-fold-overlength recombinant HBV DNA. A plasmid containing mutant HBV DNA carrying stop codon instead of 54Trp of the pol gene was generated from pHBV1.5 and verified by sequencing. Wild-type or mutated pHBV1.5 was injected into adult nude mice through the tail vein with acute circulatory overload. Expression of HBV was examined by Northern blot, immunohistochemistry and CLIA-based detection of serum HBsAg. To examine the levels of virus-associated HBV DNA, serum were treated with DNaseI and then subjected to real-time PCR analysis. PCR detection of CCC DNA was performed according to the procedure of Jun-Bin et al. with some modification.

 

Results:

Tail vein injection of pHBV1.5 resulted in expression of the 2.1 kb and 3.5 kb viral transcripts exclusively in the liver and expression of HBc in around 3% of the hepatocytes at 3 days; the expression persisted over 1 year in all mice injected. HBV was secreted into the blood, evidenced by the presence of DNase I-resistant HBV sequence. The density of the positive fraction for HBV DNA was determined 1.21 g/ml. The titers of the virus produced into the circulation are around 107 copies per milliliter at 3 days after the injection and gradually decreased 2 log throughout the observation. Of note is the finding that CCC DNA, the template of viral replication in natural infection, was produced in the livers and critically involved in the long term HBV production, because disruption of the pol gene of the inoculated DNA resulted in transient expression of both HBs and HBc antigens.

 

Conclusions:

Hydrodynamics-based transfection of HBV DNA in nude mice provides us a long-term HBV DNA replication system in vivo. HBV expression and production is generated at lest in part form CCC DNA, which recapitulates human infection. Intentional mutation could be easily introduced in inoculated DNA and mice with various different genetic background could be used; so, this model could provide unique opportunity to investigate HBV biology as well as anti-HBV therapy.

 

 


Poster 1271

HIGH PREVALENCE OF CHRONIC HEPATITIS B (HBV) INFECTION IN ADULT CHINESE AMERICANS LIVING IN CALIFORNIA

Stephanie Chao, P V. Le, W Prapong, J Su, S So, Stanford University, Stanford, CA.

 

 

Background:

The incidence of chronic hepatitis B virus (HBV) infection in the U.S. population was estimated at 0.3% based on data collected through 1994. Since then, the Asian American population has doubled and in the state of California, it has surpassed that of African-American. Nearly 88% of Asian Americans are either foreign-born themselves or have at least one foreign-born parent. Many came from areas where HBV is endemic and may not be aware that they themselves have been infected.

 

Aim:

In this study, we report the incidence of chronic HBV infection in adult Chinese Americans living in California.

 

Methods:

In 2001, a large-scale culturally targeted, multifaceted outreach campaign, collectively called the Jade Ribbon Campaign (JRC) was initiated in California to build awareness and preventive action against HBV and liver cancer in the Asian American communities. The JRC builds visibility and familiarity by combining mass media with the neighborhood accessibility of local outreach. This includes a community-based component of local seminars and health fairs, and working with and through hundreds of local community partners ranging from churches, schools, and health clinics to professional organizations and large corporations. As part of JRC, large-scale, free hepatitis B surface antigen (HBsAg) screening of the adult Chinese-American community were conducted in the San Francisco Bay Area, Los Angeles, and Orange County between July 2001-June 2003.

 

Results:

152 of 1311 adult Chinese-Americans or 11.5% who participated in the screenings were tested positive for HBsAg. The incidence was 12.3% in the Bay Area, 12.5% in Los Angeles, and 9.3% in Orange County. In a cohort of 486 people screened, a follow-up telephone survey was conducted after one year. Of those who were screened positive for HBsAg, 67% reported following the advice to see their physicians, and one reported being placed on the liver transplant list. 71% reported never talked to their doctors about HBV before the screening even though 89% had a regular family physician, and 80% were highly educated with college or postgraduate degrees. 19% reported that other members of the family (60% siblings) were later tested positive for HBsAg after they follow the advice to urge others in the family to be tested. Among all respondents, 21% reported having their unvaccinated children vaccinated for HBV after the screening.

 

Conclusion:

The incidence of chronic HBV infection in adult Chinese Americans living in California mirrors the high rates reported in overseas Chinese living in Asia. Routine screening, and counseling about the risks and prevention of HBV is warranted in Chinese Americans and other Asian-American ethnic groups with high rates of HBV infection.

 

 


Poster 1272

INPATIENT COSTS OF LIVER CIRRHOSIS AND HEPATOCELLULAR CARCINOMA IN THE UNITED STATES, 1993-2001

Kaafee Billah, Susan Goldstein, William Bower, Harold Margolis, CDC, Atlanta, GA.

 

 

Background:

In the United States, liver cirrhosis and hepatocellular carcinoma (HCC) cause >95,000 hospitalizations and >35,000 deaths annually. Despite the high disease burden, there are few national data on the economic burden of these diseases.

 

Aims:

To determine the inpatient costs of hospitalization and care for cirrhosis and HCC in the United States.

 

Methods:

Hospital inpatient admission records in an employment-based health insurance claims database (MarketScan(r) Database), which includes 3.5-5.0 million enrollees annually, were analyzed for 1993-2001. All patients >18 years old with a primary diagnosis of cirrhosis (ICD-9-CM code 571.2 [alcoholic cirrhosis] or 571.5 [non-alcoholic cirrhosis]) or HCC (ICD-9-CM 155.0) were included. For each patient, all cirrhosis- and HCC-related admissions were identified by review of both primary and secondary diagnoses and procedures for each admission. Admissions due to liver transplantation were excluded. Cost estimates were calculated from actual paid claims, adjusted for inflation using the medical care component of the consumer price index, and are reported in 2002 US dollars.

 

Results:

A total of 2,073 cirrhosis patients (alcoholic cirrhosis: 1,177; non-alcoholic cirrhosis: 896) and 272 HCC patients were identified during the 9-year period. Patients were predominantly male (cirrhosis: 66%; HCC: 67%). The median age was 54 years for cirrhosis patients (range: 19-94 years) and 55 years for HCC patients (range: 18-73 years). The average annual number of admissions per patient was 1.5 for both cirrhosis patients (95% confidence intervals [CI]: 1.5-1.6), and HCC patients (95% CI: 1.3-1.6). The average annual length of hospital stay was 11.1 days (95% CI: 10.1-12.1) for cirrhosis patients and 10.7 days (95% CI: 9.0-12.4) for HCC patients. For cirrhosis, the average annual cost of inpatient care per patient was $27,248 (95% CI: $24,247-$30,250) and for HCC $32,996 (95% CI: $26,658-$39,333). For cirrhosis, the average annual length of hospital stay for 1999-2001 (9.7 days) was 18% lower than the average for 1993-1998 (11.8 days); the average annual inpatient cost for 1999-2001 ($22,828) was 23% lower than the average for 1993-1998 ($29,459).

Conclusions:

The estimated annual cost of inpatient care per hospitalized cirrhosis or HCC patient who did not undergo transplantation was more than twice the average annual cost for all hospital admissions (~$12,000) and about eight times the per capita annual medical care expenditure (~$4,176). Cirrhosis and HCC cause substantial economic burden in the United States, underscoring the need to prevent these outcomes though hepatitis B, hepatitis C and alcohol abuse prevention.

 

 


Poster 1142

THE FIRST DETAILED ANALYSIS OF PREDICTORS OF RESPONSE IN HBEAG-NEGATIVE CHRONIC HEPATITIS B: DATA FROM A MULTICENTER, RANDOMIZED, PARTIALLY DOUBLE-BLIND STUDY OF PEGINTERFERON ALFA-2A (40KD) (PEGASYS(r)) ALONE OR IN COMBINATION WITH LAMIVUDINE VS LAMIVUDINE ALONE

Ferruccio Bonino, Scientific Direction of IRCCS Ospedale Maggiore, Milan, Italy; George Lau, Queen Mary Hospital, Hong Kong, Hong Kong Special Administrative Region of China; Patrick Marcellin, Hôpital Beaujon, Clichy, France; Stephanos Hadziyannis, H Dynant Hospital, Athens, Greece; George Kitis, George Papanikolau General Hospital, Thessaloniki, Greece; Rui Jin, Beijing You An Hospital, Beijing, China; Guang-Bi Yao, Shanghai Jing'An Central Hospital, Shanghai, China; Teerha Piratvisuth, Songklanakarin Hospital, Songkla, Thailand; Georgios Germanidis, Papageorgiou General District Hospital of Thessaloniki, Thessaloniki, Greece; Cihan Yurdaydin, University of Ankara, Ankara, Turkey; Moises Diago, Hospital General Universitario de Valencia, Valencia, Spain; Selim Gurel, Medical School of Uludag University, Bursa, Turkey; Ming-Yang Lai, National Taiwan University Hospital, Taipei, Taiwan Republic of China; Philip McCloud, Roche, Dee Why, Australia; Maurizia R. Brunetto, Azienda Ospedaliera Pisana, Pisa, Italy; Patrizia Farci, Universita di Cagliari, Cagliari, Italy.

 

 

Background:

In HBeAg-positive chronic hepatitis B (CHB) baseline ALT and HBV DNA, HBV genotype and degree of necroinflammation were shown to influence treatment response. Predictors of response in HBeAg-negative CHB are not well defined.

 

Aim:

To study the predictors of response in 537 HBeAg-negative CHB patients.

 

Methods:

Patients were treated for 48 weeks with peginterferon alfa-2a (40KD) (PEGASYS(r)) 180 µg once weekly (qw) + placebo once daily (qd) or PEGASYS(r) 180 µg (qw) + lamivudine 100 mg (qd) or lamivudine 100 mg (qd). Individual and combined co-primary endpoints (ALT normalization and HBV DNA <20,000 copies/ml) were assessed after 24 weeks of treatment-free follow-up (week 72). Logistic regression analyses were performed using the following datasets: all study patients (dataset A); PEGASYS(r) monotherapy vs lamivudine monotherapy (dataset PvL); PEGASYS(r) + lamivudine vs PEGASYS(r) monotherapy (dataset PLvP); and individual treatment arms. Variables included in the models were: treatment group, gender, race, age, body weight, baseline ALT (log10), baseline HBV DNA (log10), HBV genotype (A, B, C and D) and screening ALT strata (<=2 x ULN, 2-5 x ULN and 5-10 x ULN).

 

Results:

Overall the most significant (p<=0.01) predictors of response at week 72 were: PEGASYS(r) treatment, high baseline ALT and low baseline HBV DNA (for HBV DNA, ALT and combined endpoints); younger age (for HBV DNA and combined endpoints); and female gender (for ALT and combined endpoints).

No significant interaction between treatment and genotype was detected in dataset PvL. Across all genotypes, the chance of achieving both ALT normalization and HBV DNA <20,000 copies/ml at week 72 was 90% higher in patients treated with PEGASYS(r) than in those treated with lamivudine (Odds Ratio, OR, 1.9 [95% CI 1.2-3.2]).

A significant interaction between treatment and genotype was detected overall and in dataset PLvP (PEGASYS(r) + lamivudine vs PEGASYS(r)). In dataset PLvP, a univariate analysis for the combined response stratified by genotype showed: for genotype B an OR of 0.4 [95% CI: 0.1-1.0], p=0.040; for genotype C an OR of 1.2 [95% CI: 0.6-2.3], p=0.665; and for genotype D an OR of 3.0 [95% CI: 1.2-7.4], p=0.017.

 

Conclusions:

Baseline elevated ALT, low HBV DNA and PEGASYS(r) treatment were independently associated with both biochemical and virologic response endpoints in HBeAg-negative CHB. Patients infected with genotype D appear to benefit from PEGASYS(r) + lamivudine combination therapy. Because of the limited number of patients with genotype D in our study, prospective studies should be undertaken to address whether combination therapy might be beneficial.

 

 


Poster 1128

CORRELATES OF RESPONSE IN PATIENTS WITH CHRONIC HBV INFECTION RECEIVING LIBIVIRUMAB AND EXBIVIRUMAB (HEPEX-B(tm))

John Andrews, XTL Biopharmaceuticals, Durham, NC.

 

 

Background:

HepeX-B(tm) (libivirumab and exbivirumab) was demonstrated to cause a dose-related reduction in circulating concentrations of HBsAg and HBV DNA.

 

Aims:

The relationship between baseline antigen concentration and dose on the pharmacokinetics of anti-HBs antibody and the changes in HBV DNA concentration is described.

 

Methods:

Each patient in a dose-escalating study of HepeX-B(tm) in patients with chronic HBV infection received four weekly infusions of HepeX-B at doses of 10 to 80 mg. Serum concentrations of anti-HBs, HBsAg, and HBV DNA were determined prior to and immediately following each infusion.

 

Results:

Administration of HepeX-B(tm) resulted in a marked decrease in circulating concentrations of HBsAg and HBV DNA that was related to the circulating concentrations of anti-HBs antibody. Change from baseline HBV DNA levels showed a linear relationship to baseline levels <106 copies/mL. A 100% change from baseline was observed for subjects with pre-treatment HBsAg concentrations less than 4 mcg/mL Circulating levels of the anti-HBs antibody were inversely related to baseline serum HBsAg levels. This effect was related to dose of HepeX-B(tm).

 

Conclusions:

Doses of HepeX-B(tm) of 10 and 20 mg or greater appear sufficient to neutralize circulating HBsAg antigen in patients with pre-treatment levels of HBsAg < 4 mcg/mL. Doses of 40 and 80 mg, however, might be required to establish antibody excess in patients with higher viral load. HBsAg concentrations return to pretreatment concentrations by the end of 1 week, suggesting that infusions at intervals <1 week may be required to maintain suppression of HBsAg in patients with chronic HBV infection. In patients with ongoing viral replication the interval between infusions may be more important than the dose administered for maintenance of antibody-excess.

 

 


Poster1129

A PHASE II, RANDOMIZED TRIAL EVALUATING THE SAFETY, PHARMACOKINETICS AND ANTIVIRAL ACTIVITY OF CLEVUDINE FOR 12 WEEKS IN PATIENTS WITH CHRONIC HEPATITIS B

Patrick Marcellin, Hosp. Beaujon, Clichy, France; Nancy Leung, Prince of Wales Hosp., Chinese Univ. of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China; Hie-Won L. Hann, Thomas Jefferson Univ. Hosp, Philadelphia, PA; George K.K. Lau, Queen Mary Hospital, University of Hong Kong, Hong kong, Hong Kong Special Administrative Region of China; Christian Trepo, Hotel Dieu, Lyon, France; Stephen Sacks, Viridae, Vancouver, BC, Canada; Herve Mommeja-Marin, Cary Moxham, Andrea Snow, Franck Rousseau, Gilead Sciences, Inc., Durham, NC; Seng Gee Lim, National Univ. Hosp, Singapore, Singapore.

 

 

Background:

Clevudine (CLV, L-FMAU) is a potent inhibitor of Hepatitis B virus (HBV) replication in vitro. In woodchucks and in a Phase I/II clinical study, CLV produced a potent and sustained viral suppression following a 4-week dosing period.

 

Methods:

A multicenter, international, randomized, double-blind study comparing 10, 30 and 50 mg CLV once daily (QD) for 12 weeks. Patients were followed post-treatment for at least 24 weeks. Eligible patients had chronic HBV infection with Baseline serum HBV DNA levels (VL) = 3x106 copies/mL (c/mL) as measured by the Chiron Quantiplex(tm) assay, and were nucleoside treatment-naïve without HIV, HDV or HCV co-infection. VL was assayed using Digene Hybrid Capture II (lower limit of detection of 4700 c/mL) and genotypic analysis of Baseline, Week 12 and Week 26 samples was performed using di-deoxy sequencing.

 

Results:

Thirty-one patients were enrolled (10, 11 and 10 in the 10, 30 and 50 mg cohorts, respectively) of whom 55% were male, 84% Asian, and 81% HBeAg positive. At Baseline, median VL was 8.7, 7.9, and 8.7 log10 c/mL and median alanine aminotransferase (ALT) level was 53, 63, and 80 IU/L in the 10, 30 and 50 mg CLV cohorts, respectively. After 12 weeks of dosing, the median log10 VL change from Baseline was -3.2, -3.7 and -4.2 log10 c/mL in the 10, 30, and 50 mg cohorts, respectively (P=0.012 for trend). One out of 10, 5/11 and 2/10 patients had VL below the assay LOD at Week 12 in the 10, 30, and 50 mg cohorts, respectively. Only 2 patients seroconverted to anti-HBe (both in the 30mg cohort). CLV was generally well tolerated without dose related adverse events, and no severe or serious adverse events. One patient reported a transient Grade 3 increase in ALT and another a transient Grade 3 creatine phosphokinase increase, both while on study drug. The pharmacokinetics of CLV were dose proportional with a mean plasma half-life of 70 hours. Pharmacodynamic modeling shows that 97% of the maximal treatment effect was reached with a dose of 30 mg QD.

Conclusion:

These preliminary results confirm the potent antiviral activity of once daily clevudine and further demonstrate the tolerability of the drug over 12 weeks of dosing. Based on these results, the optimal dose of clevudine appears to be >10 mg QD.

 

 


Poster 1130

A 12-WEEK CLEVUDINE THERAPY SHOWED DURABLE ANTIVIRAL ACTIVITY AND NORMALIZATION OF ALANINE TRANSMINASE LEVELS FOR 6 MONTHS AFTER DISCONTINUATION OF TREATMENT IN PATIENTS WITH CHRONIC HEPATITIS B

Hyo-Suk Lee, Seoul National University Hospital, Seoul, Republic of Korea; Young-Hwa Chung, Asan Medical Center, Seoul, Republic of Korea; Kwan Sik Lee, Yongdong Severance Hospital, Seoul, Republic of Korea; Kwan Soo Byun, Korea University Medical Center Guro Hospital, Seoul, Republic of Korea; Seung Woon Paik, Samsung Medical Center, Seoul, Republic of Korea; Joon-Yeol Han, The Catholic University of Korea, St. Mary's Hospital, Seoul, Republic of Korea; Kwon Yoo, Ewha Woman's University Mokdong Hospital, Seoul, Republic of Korea; Soo Geum Hwang, Bukwang Pharm.Co., Ltd., Seoul, Republic of Korea; Byung Chul Yoo, Samsung Medical Center, Seoul, Republic of Korea; Jin Heon Lee, Hallym University Hospital, Seoul, Republic of Korea.

 

 

Introduction:

Clevudine is a pyrimidine analogue that is a potent inhibitor of HBV replication in vitro. In woodchucks, clevudine caused potent and durable post-treatment viral suppression. In a preliminary clinical trial of 4-week clevudine treatment, potent antiviral activity was demonstrated along with the marked post-treatment antiviral effect.

 

Aims:

The primary objectives of the study were to evaluate the tolerability, safety and antiviral activity of clevudine in a 12-week, double-blind, randomized, multicenter, phase II clinical trial and to assess the durable antiviral response at week 24 off therapy.

 

Methods:

The safety and efficacy of clevudine (30mg/day or 50mg/day orally) were compared with those of placebo, and then each group was observed for 24 weeks off therapy (a total period of 36 weeks). The study was conducted at a total of 8 sites in South Korea. Eligible patients were female or male with HBeAg-positive chronic hepatitis B with HBV DNA levels greater than or equal to 3 million copies/mL. Among a total of 99 patients(33, 32 and 34 in the placebo, 30mg and 50mg cohorts, respectively) enrolled, 88 patients (25, 31 and 32 in the placebo, 30mg and 50mg cohorts, respectively) have completed 36-week study period, the data of whom were analyzed.

Results: Median HBV DNA level at baseline was 8.4, 8.4 and 8.2 log10 copies/mL, and the median change in HBV DNA levels from baseline at week 12 was -0.12, -4.47 and -4.45 log10 copies/mL in the placebo, 30mg and 50mg cohorts, respectively. Post-treatment antiviral activities were sustained with 3.32 and 2.99 log10 reduction at week 12 off therapy and 2.28 and 1.40 log10 reduction at week 24 off therapy in 30mg and 50mg cohorts, respectively. In the placebo, 30mg and 50mg cohorts, 0 and 63 and 52% had HBV DNA levels below the lower limit of detection assay (4.7 thousand copies/mL) at the end of 12-week treatment, and 4, 16 and 9% at week 36, respectively. Normalization of alanine transminase levels was observed in 7, 53 and 55% at week 12 and 12, 71 and 63% at week 36 in the placebo, 30mg and 50mg cohorts, respectively. Clevudine was well tolerated and most adverse events were comparable in all study groups.

 

Conclusion:

A 12-week clevudine therapy showed potent antiviral activity against HBV with durable viral suppression and normalization of aminotransferase levels at week 24 off therapy and was well tolerated.

 

 


Poster 1131

PROFOUND ON-TREATMENT VIRAL SUPPRESSION WITH PEGINTERFERON ALFA-2A (40KD) (PEGASYS(r)) PLUS LAMIVUDINE COMBINATION THERAPY LIMITS THE DEVELOPMENT OF YMDD MUTATIONS, BUT DOES NOT IMPROVE SUSTAINED RESPONSE RATES OVER PEGINTERFERON ALFA-2A (40KD) ALONE

Georgios Germanidis, Papageorgiou General District Hospital of Thessaloniki, Thessaloniki, Greece; Patrick Marcellin, Hôpital Beaujon, Clichy, France; George Lau, Queen Mary Hospital, Hong Kong, Hong Kong Special Administrative Region of China; Ferrucio Bonino, Scientific Direction of IRCCS Ospedale Maggiore, Milan, Italy; Patrizia Farci, Universita di Cagliari, Cagliari, Italy; Stephanos Hadziyannis, H Dynant Hospital, Athens, Greece; Rui Jin, Beijing You An Hospital, Beijing, China; Guang-Bi Yao, Shanghai Jing'An Central Hospital, Shanghai, China; Teerha Piratvisuth, Songklanakarin Hospital, Songkla, Thailand; Cihan Yurdaydin, University of Ankara, Ankara, Turkey; Moises Diago, Hospital General Universitario de Valencia, Valencia, Spain; Selim Gurel, Medical School of Uludag University, Bursa, Turkey; Ming-Yang Lai, National Taiwan University Hospital, Taipei, Taiwan Republic of China; Matei Popescu, Roche, Basel, Switzerland; Nigel Pluck, Roche, Welwyn, United Kingdom.

 

 

Background:

The long-term use of lamivudine for the treatment of chronic hepatitis B (CHB) is limited by the development of YMDD mutations and subsequent lamivudine resistance. Previous studies have indicated that more potent on-treatment viral suppression may assist in reducing the development of antiviral drug resistance.

 

Aim:

To evaluate the pattern of viral suppression and YMDD mutations in patients with HBeAg-negative CHB enrolled in a randomized, partially double-blind, multinational study of peginterferon alfa-2a (40KD) (PEGASYS(r)) ± lamivudine vs lamivudine alone.

 

Methods:

Patients with HBeAg-negative CHB received one of the following: peginterferon alfa-2a (40KD) (PEGASYS(r)) 180 µg once weekly (qw) + placebo once daily (qd) (n=177); peginterferon alfa-2a (40KD) (PEGASYS(r)) 180 µg qw + lamivudine 100 mg qd (n=179); or lamivudine 100 mg qd (n=181). Patients were treated for 48 weeks with 24 weeks treatment-free follow-up. HBV DNA was regularly assessed using the COBAS AMPLICOR HBV MONITOR(r). Detection of changes to the YMDD motif was performed on all available serum samples from patients receiving combination therapy or lamivudine monotherapy at the end of treatment (week 48) using the INNO-LiPA HBV DR assay.

 

Results:

By the end of treatment, suppression of HBV DNA was greatest with the combination therapy; HBV DNA suppression was similar with peginterferon alfa-2a monotherapy and lamivudine monotherapy (see table). Development of YMDD mutations by the end of treatment was significantly lower in the combination therapy group (<1%) compared with the lamivudine monotherapy group (18%; P<0.001 Fisher's Exact test). After 24 weeks of follow-up, the proportion of patients with HBV DNA <20,000 copies/ml was similar between peginterferon alfa-2a monotherapy and the combination therapy; both were significantly superior to lamivudine.

 

Conclusions:

Results from this study indicate that a more profound on-treatment HBV DNA suppression, as seen with peginterferon alfa-2a (40KD) (PEGASYS(r)) plus lamivudine, contributes towards a lower incidence of lamivudine resistance than with lamivudine alone. However, the potent on-treatment viral suppression achieved with the combination therapy does not translate into improved off-therapy HBV DNA response rates compared with peginterferon alfa-2a alone in patients with HBeAg-negative CHB.

 

 

PEGASYS®
+ placebo

PEGASYS®
+ lamivudine

lamivudine

Mean HBV DNA reduction at
end of treatment (log10 copies/ml)
[95% confidence intervals]

- 4.1
[-3.8 to -4.5]

- 5.0
[-4.7 to -5.3]

- 4.2
[-3.9 to -4.5]

YMDD mutation at end of treatment

N/A

1/173 (<1%)*

32/179 (18%)

HBV DNA <20,000 copies/ml
at end of follow-up
(24 weeks after end of treatment)

76/177 (43%)§

79/179 (44%)

53/181 (29%)

* P<0.001 for comparison with lamivudine
§ P=0.007 for comparison with lamivudine
‡ P=0.003 for comparison with lamivudine

 

 


Poster 1132

DIFFERENT PROFILES OF RESPONSE TO ADEFOVIR DIPIVOXIL AND FACTORS THAT MAY INFLUENCE RESPONSE IN PATIENTS WITH CHRONIC HEPATITIS B

Sandra Durantel, Bettina Werle, David Durantel, Christian Pichoud, INSERM U271, Lyon, France; Graeme Currie, Shelly Xiong, Carol L. Brosgart, Gilead Sciences, Foster City, CA; Christian Trépo, Fabien Zoulim, INSERM U271, Lyon, France.

 

 

Background/Aim:

Adefovir dipivoxil (ADV), an oral prodrug of adefovir, has demonstrated activity against wild-type and lamivudine-resistant hepatitis B virus (HBV). After 48 weeks of therapy a median 3.5-4.7 log10 decrease in viral load is observed. Furthermore, ADV therapy is associated with delayed and infrequent selection of drug resistant viruses. Our aim was to characterize the different profiles of response in patients who receive ADV in relation to the in vitro susceptibility of viral strains to ADV.

 

Methods:

In an international phase III randomized, placebo-controlled study of ADV in HBeAg positive patients, the response to ADV at 10 mg/day can be analyzed by the four quartiles that comprise the median reduction in serum HBV DNA The top 25% patients (quartile 1, Q1) showed a higher than 4.91 log10 reduction in serum HBV DNA at week 48. In Q2, patients demonstrated a 3.52 to 4.90 log10 reduction at week 48. In Q3, a 2.22 to 3.51 log10 reduction was observed at week 48. The bottom 25% of patients (Q4) showed less than 2.22 log10 reduction in serum HBV DNA levels at week 48. The influence of baseline characteristics (HBV DNA and ALT levels, body weight, BMI , liver fibrosis score, age, race, gender), concomitant medications, and drug compliance on response was investigated by comparison of summary statistics across the quartiles. The replication capacity and drug susceptibility of HBV genomes of selected clinical isolates that were representative of the treatment response quartiles were analyzed using a novel phenotypic assay based on PCR amplification, cloning, transfection of Huh7 and HepG2 cells. For all quartiles, a polyclonal analysis was conducted using pre-treatment viral isolates. For 2 patients belonging to Q4, monoclonal and polyclonal studies were performed at day 0 and week 48 of treatment.

 

Results:

Analysis of clinical parameters showed that the lowest quartile of response (Q4) appear to have worse compliance, a higher BMI and less fibrosis. Higher ALT at baseline is related to improved response. No specific mutation was associated with poorer response to ADV. Phenotypic analysis of these viral strains in vitro in Huh7 and HepG2 cells showed that HBV genomes remained susceptible to ADV regardless of treatment response observed in patients. The analysis of week 48 viral isolates from patients Q4, showed no modification in the IC50 and IC90 of ADV compared to wild-type clones. By contrast, the N236T polymerase mutant has a 4-6 fold reduced susceptibility to ADV in the same experimental conditions.

Conclusion:

Suboptimal response ( Q4) and acquired resistance to ADV are most likely to be due to two different phenomena. Sub-optimal response to ADV may be due to a host pharmacological effect or to patient compliance rather than to a reduced susceptibility to ADV.

 

 


Poster 1133

CORRELATION BETWEEN BASELINE GENOTYPE AND ANTIVIRAL RESPONSE TO EMTRICITABINE 200 MG QD AMONG HBEAG NEGATIVE PATIENTS WITH CHRONIC HEPATITIS B INFECTION

Andi Snow, Gilead Sciences, Durham, NC; Richard Guan, Mount Elizabeth Medical Centre, Singapore, Singapore; Ionnis Diamantis, Regional University General Hospital of Herakleio, Crete, Herakleio, Crete, Greece; Jeff Sorbel, Elsa Mondou, Franck Rousseau, Gilead Sciences, Durham, NC.

 

 

Background/Aim:

To evaluate the interaction of genotypic variations at Baseline (BL) and antiviral response among HBeAg negative (HBeAg-) patients with chronic hepatitis B virus (CHB) receiving emtricitabine (Emtriva, FTC) 200 mg QD.

 

Methods:

Patients were enrolled in one of two double-blind, randomized 48-week studies. Serum HBV DNA levels were assessed by Digene Hybrid Capture II; lower limit of detection (LOD) of 4700 copies/mL [cp/mL]. Sequence analysis of the HBV POL and HBsAg reading frames was performed by di-deoxy sequencing.

 

Results:

Two hundred patients were randomized to receive FTC 200 mg QD for 48 weeks [B102; 33 (21 naïve, 12 experienced from FTCB 101), B301; 167 naïve]. One hundred ninety one patients were evaluable for virologic and biochemical responses and for the emergence of drug resistance mutations based on BL genotype. Thirty-six percent (36%, 68/191) were HBeAg- and phylogenetic analysis at BL revealed that 4, 28, 7 and 29 harbored HBV of genotype A, B, C and D, respectively. At 1 year, improvement in liver histology, i.e., reduction of at least 2 points in the Knodell necroinflammatory score and lack of fibrosis progression was observed in 75% (A), 68% (B), 60% (C) and 75% (D) of HBeAg- patients (missing=excluded). Median log10 decreases in HBV VL at 1 year were -2.79, -2.14, -2.60 and -2.85 cp/mL and proportion of patients with undetectable VL was observed in 50%, 84.6%, 85.7% and 92.9% of patients with genotype A, B, C and D virus, respectively. Median changes in ALT values were -134, -35, -38 and -53.5 IU/L at 1 year across genotypes A-D, respectively. Resistance surveillance for patients with detectable viremia at 1 year revealed that 1/4 and 2/26 of the patients with genotype A and B, respectively harbored HBV DNA with mutations associated with resistance (rt204I/V+/-rt180M+/-rt173L). There were no statistically significant differences between genotypes in any of the markers of response evaluated (p>0.05).

 

Conclusions:

Antiviral response among HBeAg negative patients with CHB receiving FTC 200 mg QD for 1 year was not significantly different based on HBV genotype at Baseline. Overall, FTC 200 mg QD produced potent viral suppression, demonstrated a low incidence of resistance mutations and ultimately resulted in improvement in liver histology in approximately 71% of these HBeAg- patients.

 

 


Poster 1134

ANALYSIS OF HBV POLYMERASE POLYMORPHISMS AND VIROLOGICAL RESPONSE TO ADEFOVIR DIPIVOXIL (ADV) IN CHRONIC HEPATITIS B (CHB) PATIENTS

Xiaoping Qi, Sandy Chang, Ching-Fai Pang, Sarah Arterburn, Graeme Currie, Chris E. Westland, Carol L. Brosgart, Michael Miller, Shelly Xiong, Gilead Sciences, Inc., Foster City, CA.

 

 

Background:

HBV polymerase is the antiviral target of adefovir and lamivudine. The reverse transcriptase (RT) domain of HBV polymerase consists of 344 amino acids. Natural variants at polymorphic sites of the HBV RT can be found in the majority of patients. Two natural polymorphisms rt91L and rt256C have been found to be correlated with lamivudine treatment failure (Ciancio, Hepatology, 2004;39:64). It is unknown if any natural polymorphism of HBV RT is associated with reduced virological response to ADV therapy.

 

Aims:

To determine the potential association of HBV RT polymorphisms with reduced virological response to ADV therapy.

 

Methods:

This analysis included all intent-to-treat CHB patients in the ADV 10 mg dose groups of two ADV phase 3 clinical studies (n=171 in the HBeAg+ study and n=123 in the HBeAg- study). The two studies were analyzed separately for cross-validation. HBV RT sites with > 1% of sequence variations are considered as polymorphic sites. The baseline HBV RT sequences were determined by sequencing. Serum HBV DNA level was determined by Roche Amplicor Monitor assay (LLQ = 400 c/mL). Each amino acid that occurred in =2% of patients at a polymorphic site was analyzed. The serum HBV DNA change from baseline to week 48 was compared between patients with and without the polymorphic amino acid at baseline by using a Wilcoxon rank sum test. The Bonferroni correction was applied to identify an appropriate adjusted p-value cutoff for statistical significance.

 

Results:

Fifty-nine and 78 of the 344 residues in the HBV RT at baseline were determined to be polymorphic sites in the HBeAg+ and HBeAg- patients, respectively. Within the HBeAg+ analysis, there were no polymorphisms associated with altered serum HBV DNA responses that showed statistical significance when corrected for multiple comparisons (number of polymorphic sites) (p<0.0008). Within the HBeAg- study, patients with rt332S (n=15) showed significantly smaller HBV DNA declines (a median decline of 2.48 log10, p<.0006) as compared to patients without rt332S (n=102, median decline of 3.84 log10). However, rt332S occurred frequently in the HBeAg+ patients (n=63 with; n=89 without). The HBeAg+ patients showed nearly identical median HBV DNA declines with and without rt332S (3.44 log10 vs. 3.56 log10, p=0.9851), suggesting that the rt332S result observed in the HBeAg- patients may have been due to chance.

 

Conclusions:

No HBV RT polymorphisms were identified to be associated with a significant reduced virological response to adefovir dipivoxil therapy in chronic hepatitis B patients in both adefovir phase 3 pivotal studies.

 

 


Poster 1135

LONG TERM EFFICACY AND SAFETY OF ADEFOVIR DIPIVOXIL (ADV) 10 MG IN HBEAG+ CHRONIC HEPATITIS B (CHB) PATIENTS: INCREASING SEROLOGIC, VIROLOGIC AND BIOCHEMICAL RESPONSE OVER TIME

P Marcellin, Hopital Beujon, Clichy, France; T T. Chang, National Cheng Kung University Hospital, Tainan, Taiwan Republic of China; S Lim, National University Hospital, Singapore, Singapore; W Sievert, Monash Medical Centre, Victoria, Australia; M Tong, Huntington Medical Center Research Institute, Huntington, CA; Sarah Arterburn, Shelly Xiong, Carol L. Brosgart, Graeme Currie, Gilead Sciences, Inc., Foster City, CA.

 

 

Background:

Approximately 400 million individuals worldwide have CHB, a leading cause of cirrhosis and HCC. ADV has potent activity against wild-type and lamivudine-resistant HBV.

 

Aim:

Treatment with ADV 10 mg over 48 wks demonstrated significant histological, virological, serological and biochemical improvement compared to placebo (PLB) in CHB.

 

Methods:

Eligibility: HBsAg+ = 6 months, HBeAg+, serum HBV DNA =106 c/mL (Roche Amplicor(tm) Monitor PCR, LLQ 1,000 c/mL) and ALT 1.2-10 x ULN. Prior interferon therapy allowed, if > 6 months prior to enrollment. Patients (pts) were randomized to receive ADV 10 mg or 30 mg, or PLB. The study continued to evaluate the long term safety and efficacy of ADV 10 mg in pts who received ADV 10 mg in the first 48 wks. Results are reported up to 144 wks of therapy. Most pts had = 1 dose PLB in year 2 due to a misallocation of dosing error. As the length of on study follow-up varies, K-M* estimates were used to conservatively evaluate proportions of pts achieving HBV DNA undetectability, ALT normalization, HBeAg loss and seroconversion.

Results: 309 pts received = 1 dose ADV 10 mg; 296, 231, and 84 pts were followed through 48, 96, and 144 wks; 74% male, 57% Asian and 38% Caucasian; median age 34 years. Baseline (BL) median serum HBV DNA 8.11 log10 c/mL; median ALT was 85 IU/L (2 x ULN).

 

Baseline

Week 48

Week 96

Week 144

% Serum HBV DNA Undetectable by PCR (<1000 copies/mL)*

28%

45%

56%

% HBeAg seroconversion*

12%

29%

43%

% HBeAg loss*

21%

42%

51%

% ALT normalization*

58%

71%

81%

 

Note: Pts with confirmed HBeAg seroconversion or HBeAg loss were followed off-treatment in an observational study

*Kaplan-Meier analysis

 

The frequency and nature of adverse events (AEs), serious AEs and laboratory abnormalities was similar to PLB over 48 wks. Over 96 and 144 wks safety was consistent with that seen in the first 48 wks. Through 144 wks, no pt had a confirmed serum creatinine increase from BL of = 0.5 mg/dL or a serum phosphorus < 1.5 mg/dL. No pt developed resistance by 48 wks; 2 (3.1%) developed resistance (1 N236T, 1 A181V) through 144 wks.

 

Conclusions:

Treatment with ADV 10 mg over 144 wks resulted in increasing HBeAg loss and seroconversion rates. Continued reductions in serum HBV DNA and ALT levels were demonstrated with increasing proportions of patients achieving undetectable serum HBV DNA and ALT normalization. Resistance was delayed and infrequent. ADV 10mg was well-tolerated with a safety profile consistent with that seen in PLB. No nephrotoxicity was observed. This study continues to evaluate the long term safety and efficacy of ADV 10 mg.

 

 


Poster 1136

ENTECAVIR IS SUPERIOR TO LAMIVUDINE AT REDUCING HBV DNA IN PATIENTS WITH CHRONIC HEPATITIS B REGARDLESS OF BASELINE ALANINE AMINOTRANSFERASE LEVELS

M Rosmawati, University Malaya Medical Center, Kuala Lumpur, Malaysia; E Schiff, University of Miami, Miami, FL; R Parana, Federal University of Bahia, Salvador, Brazil; W Sievert, MMC Clayton, Victoria, Australia; J Zhu, A Cross, D DeHertogh, D Apelian, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT; the BEHoLD Study Group.

 

 

Background:

Entecavir (ETV) is a potent and selective inhibitor of hepatitis B virus (HBV) polymerase. Chronic hepatitis B patients with low serum alanine aminotransferase (ALT) levels typically show reduced or absent responses to interferon and lamivudine (LVD). Data from a Phase II dose-ranging trial in nucleoside-naive patients suggested that this is not the case for ETV.

 

Aim:

This analysis assesses the influence of baseline ALT levels on the clinical efficacy of ETV 0.5 mg QD compared to LVD 100 mg QD in a Phase III trial in 709 nucleoside-naive patients with chronic hepatitis B (ETV-022).

 

Methods:

Patients were HBeAg(+) with baseline HBV DNA = 3 MEq/ml by bDNA assay and ALT = 1.3 x ULN. Efficacy endpoints included proportions of patients with HBV DNA < 0.7 MEq/ml by bDNA, proportions with HBV DNA < 400 copies/mL by PCR, and HBV DNA reduction by PCR. Efficacy results for both treatment groups were analyzed within subgroups with baseline ALT < 2.6 x ULN or = 2.6 x ULN.

 

Results:

Baseline disease characteristics were comparable in the ETV and LVD groups. Mean baseline viral loads were ETV: 9.61 log10 c/mL; LVD: 9.69 log10 c/mL. The baseline ALT < 2.6 x ULN and = 2.6 x ULN subgroups comprised 186 and 168 ETV patients, respectively, and 190 and 164 LVD patients, respectively. Virologic efficacy results at week 48 by treatment and baseline ALT subgroup are shown in the table. ETV was highly effective at reducing HBV DNA regardless of baseline ALT level, and was superior to LVD by all virologic assessments. In contrast, among LVD-treated patients, HBV DNA reduction and the proportion of patients with undetectable HBV DNA levels by bDNA or PCR assays was attenuated in the lower baseline ALT subgroup.

 

 

Baseline ALT

ETV

LVD

p-value

*Log reduction from baseline in HBV DNA by PCR

< 2.6 x ULN
≥ 2.6 x ULN

-6.79
-7.18

-4.85
-6.15

< 0.0001
< 0.0001

% of patients with:
HBV DNA < 0.7 MEq/mL
by bDNA

< 2.6 x ULN
≥ 2.6 x ULN

90%
92%

58%
74%

< 0.0001
< 0.0001

HBV DNA < 400 copies/mL by PCR

< 2.6 x ULN
≥ 2.6 x ULN

59%
81%

28%
49%

< 0.0001
< 0.0001

* n = 179 (<2.6xULN), 161 (> 2.6xULN) for ETV;
n = 173 (<2.6xULN), 150 (> 2.6xULN) for LVD

 

Conclusions:

These data confirm Phase II observations showing that in patients with chronic hepatitis B, entecavir 0.5 mg is highly effective and is superior to LVD at reducing HBV DNA regardless of baseline ALT levels.

 

 


Poster 1137

ALT FLARES AND SUSTAINED ALT RESPONSE IN PATIENTS WITH HBEAG-NEGATIVE CHRONIC HEPATITIS B TREATED WITH PEGINTERFERON ALFA-2A (40KD) (PEGASYS(r)), PEGINTERFERON ALFA-2A (40KD) PLUS LAMIVUDINE OR LAMIVUDINE ALONE

Teerha Piratvisuth, Songklanakarin Hospital, Songkla, Thailand; Patrick Marcellin, Hôpital Beaujon, Clichy, France; George Lau, Queen Mary Hospital, Hong Kong, Hong Kong Special Administrative Region of China; Ferrucio Bonino, Scientific Direction of IRCCS Ospedale Maggiore, Milan, Italy; Patrizia Farci, Universita di Cagliari, Cagliari, Italy; Stephanos Hadziyannis, H Dynant Hospital, Athens, Greece; Rui Jin, Beijing You An Hospital, Beijing, China; Zhi-Meng Lu, Ruijin Hospital, Shanghai, China; Georgios Germanidis, Papageorgiou General District Hospital of Thessaloniki, Thessaloniki, Greece; Cihan Yurdaydin, University of Ankara, Ankara, Turkey; Moises Diago, Hospital General Universitario de Valencia, Valencia, Spain; Selim Gurel, Medical School of Uludag University, Bursa, Turkey; Ming-Yang Lai, National Taiwan University Hospital, National Taiwan University Hospital, Taiwan Republic of China; Peter Button, Roche, Dee Why, Australia; Nigel Pluck, Roche, Welwyn, United Kingdom.

 

 

Background:

Prominent ALT flares occurring during, or shortly after, interferon-based treatment of HBeAg-positive chronic hepatitis B (CHB) have been associated with HBeAg seroconversion and increased viral suppression. The relationship between ALT flares and response in patients with HBeAg-negative CHB has not been defined.

 

Aim:

 To investigate the relationship between ALT flares and response in a randomized, partially double-blind, multinational study of peginterferon alfa-2a (40KD) (PEGASYS(r)) with and without lamivudine vs lamivudine alone in 537 patients with HBeAg-negative CHB.

 

Methods:

Patients with HBeAg-negative CHB received one of the following: peginterferon alfa-2a (40KD) (PEGASYS(r)), 180 µg once weekly (qw) + placebo once daily (qd); peginterferon alfa-2a (40KD) (PEGASYS(r)), 180 µg qw + lamivudine 100 mg qd; or lamivudine 100 mg qd. Patients were treated for 48 weeks with 24 weeks treatment-free follow-up. Co-primary endpoints were (1) ALT normalization, and (2) HBV DNA <20,000 copies/ml, assessed after 24 weeks follow-up (week 72). For this analysis, marked ALT flares were a peak ALT >10 x the upper limit of normal (ULN) and moderate ALT flares were a peak ALT between 5 and 10 x ULN.

 

Results:

The incidence of marked on-therapy ALT flares was significantly higher with peginterferon alfa-2a monotherapy than with combination therapy or lamivudine monotherapy (P=0.007 and P=0.038, respectively). During follow-up, marked flares were significantly more frequent with lamivudine monotherapy or combination therapy than peginterferon alfa-2a monotherapy (P=0.033 and P=0.021, respectively). A similar trend was observed with moderate ALT flares during follow-up (see table). Overall, there was a significant association between marked on-therapy ALT flares and ALT normalization at week 72 (P=0.011), but not between moderate flares and ALT normalization at week 72.

 

Conclusions:

Marked on-therapy ALT flares occurred more frequently in patients who achieved a sustained response. This suggests that a marked flare during therapy with peginterferon alfa-2a (40KD) (PEGASYS(r)), lamivudine or the two agents combined may be beneficial in patients with HBeAg-negative CHB. It is noteworthy that, although this relationship has previously been observed in HBeAg-positive CHB, these data represent the first report of such a relationship in patients with HBeAg-negative CHB.

 

 

PEGASYS®+ placebo

PEGASYS® + lamivudine

lamivudine

Patients with sustained ALT response*

105/177 (59%)

107/179 (60%)

80/181 (44%)

During Therapy

Marked ALT flare (>10 x ULN)

22/177 (12%)§

8/179 (4%)

11/181 (6%)

Moderate ALT flare (5-10 x ULN)

44/177 (25%)

53/179 (30%)

21/181 (12%)

ALT response in pts WITH a marked flare

16/22 (73%)

6/8 (75%)

8/11 (73%)

ALT response in pts WITH a moderate flare

24/44 (55%)

27/53 (57%)

9/21 (43%)

ALT response in pts WITHOUT a
marked or moderate flare

65/111 (59%)

74/118 (63%)

63/149 (42%)

During Follow-up

Marked ALT flare (>10 x ULN)

13/177 (7%)

27/179 (15%)

26/181 (14%)

Moderate ALT flare (5-10 x ULN)

22/177 (12%)

28/179 (16%)

33/181 (18%)

ALT response in pts WITH a marked flare

3/13 (23%)

12/27 (44%)

5/26 (19%)

ALT response in pts WITH a moderate flare

6/22 (27%)

9/28 (32%)

9/33 (27%)

ALT response in pts WITHOUT a
marked or moderate flare

96/142 (68%)

86/124 (69%)

66/122 (54%)

* ALT normalization 24 weeks after end of treatment (week 72)
‡ P=0.004 in comparison with lamivudine
§ P=0.007 in comparison with PEGASYS® + lamivudine; P=0.038 in comparison with lamivudine.
¶ P=0.021 in comparison with PEGASYS® + lamivudine; P=0.033 in comparison with lamivudine.

 

 


Poster 1138

CLEVUDINE THERAPY FOR 24 WEEKS FURTHER REDUCED SERUM HEPATITIS B VIRUS DNA LEVELS AND INCREASED ALT NORMALIZATION RATES WITHOUT EMERGENCE OF VIRAL BREAKTHROUGH THAN 12-WEEK CLEVUDINE THERAPY

Kwan Sik Lee, Yongdong Severance Hospital, Seoul, Republic of Korea; Kwan Soo Byun, Korea University Medical Center Guro Hospital, Seoul, Republic of Korea; Young-Hwa Chung, Asan Medical Center, Seoul, Republic of Korea; Seung Woon Paik, Samsung Medical Center, Seoul, Republic of Korea; Joon-Yeol Han, The Catholic University of Korea, St. Mary's Hospital, Seoul, Republic of Korea; Kwon Yoo, Ewha Woman's University Mokdong Hospital, Seoul, Republic of Korea; Soo Geum Hwang, Bukwang Pharm.Co.,Ltd., Seoul, Republic of Korea; Byung Chul Yoo, Samsung Medical Center, Seoul, Republic of Korea; Hyo-Suk Lee, Seoul National University Hospital, Seoul, Republic of Korea.

 

 

Background:

Clevudine(CLV, L-FMAU) is a pyrimidine analogue that is a potent inhibitor of HBV replication in vitro.

 

Aim:

In woodchucks and in a phase I/II clinical study (L-FMAU-102), CLV produced a potent and durable post-treatment viral suppression. In a phase II clinical study (L-FMAU-201), CLV showed potent antiviral activities during 12-week dosing period and prolonged antiviral activities after discontinuation of treatment, which was associated with sustained normalization of ALT. AIMS: The primary objectives of this study were to evaluate the safety and antiviral activity of CLV during longer treatment period of 24 weeks.

 

Methods:

The safety and efficacy of CLV (30mg/day, orally) for 24 weeks was evaluated. The study was conducted at a total of 7 sites in South Korea. Eligible patients were chronic hepatitis B patients previously included in the placebo group of L-FMAU-201 study, whose HBeAgs were not seroconverted to anti-HBe. A total of 21 patients were enrolled and among them, 17 patients have completed 24-week treatment period, the data of which were analyzed for this interim report.

 

Results:

Median HBV DNA level at baseline was 7.53 log10 copies/mL, and the median changes in HBV DNA levels from baseline were -4.05, 4.64 log10 copies/mL at week 12 and week 24, respectively. 59 % of patients had HBV DNA levels below the lower limit of detection of Supersensitive Digene II assay (LOD, 4.7 × 103 copies/mL) at week 12, and 82 % at the end of 24-week treatment. 24 % of patients had HBV DNA levels below LOD in Amplicor PCR assay (400 copies/mL) at week 12, and 59 % at the end of 24-week treatment. Viral breakthrough was not observed during the entire dosing period. Normalization of alanine transaminase level was observed in 47 % and 76% at week 12 and 24, respectively. HBeAg loss occurred in 12 % and 24% at week 12 and 24, respectively. No serious or severe adverse events relevant to CLV were reported during the study and no adverse events led to treatment discontinuation.

 

Conclusion:

CLV treatment for 24 weeks showed more potent antiviral activity against HBV without the emergence of viral breakthrough and higher rates of HBeAg loss as well as ALT normalization rates than 12-week treatment of CLV with an excellent safety profile.

 

 


Poster 1139

IN VITRO CHARACTERISATION OF THE ANTI-HBV ACTIVITY AND CROSS-RESISTANCE PROFILE OF 2',3'-DIDEOXY-3'-FLUOROGUANOSINE

Anne-Carole Jacquard, Marie-Noelle Brunelle, Christian Pichoud, Christian Trépo, Fabien Zoulim, INSERM U271, Lyon, France.

 

 

Background/Aim:

The fluorinated guanosine analogue 2',3'-dideoxy-3'-fluoroguanosine (FLG, MIV-210) has been shown to have an efficient antiviral effect on human hepatitis B virus (HBV) in vitro in HepG2 2.2.15 cell line and in vivo in the duck model of HBV infection. The aim of the study was to evaluate the anti-HBV activity of FLG and determine its detailed mechanism of action on wild-type (WT), lamivudine resistant mutant (L180M+M204V, ie LAM-R), adefovir resistant mutant (N236T, ie ADV-R) or both LAM-R and ADV-R mutant (L180M+M204V+N236T) HBV and DHBV.

 

Methods:

We determined whether FLG-triphosphate (TP) can inhibit priming and/or elongation of the viral replication and whether FLG-TP could inhibit the incorporation of the next nucleotide by chain termination using a cell free assay for the expression of enzymatically active WT and drug resistant mutant polymerases. Its inhibitory activity was compared to that of lamivudine-TP and adefovir-DP. The evaluation of anti-HBV activity of FLG was also performed after transfection of Huh7 cells with either WT, LAM-R, ADV-R or LAM-R+ADV-R HBV genomes cloned in a vector allowing the expression of the pregenomic RNA. FLG was administered daily from day 4 to day 9, and intracellular viral DNA was analyzed by Southern Blot hybridization. In parallel, cells were treated with lamivudine or adefovir.

 

Results:

In the cell free assay, FLG-TP showed a more potent inhibition of the DHBV polymerase activity (IC50 = 4.9 uM) compared with lamivudine-TP (IC50 = 8.0 uM), and a similar activity compared to ADV-DP or tenofovir-DP (IC50 = 4.6 and 4.9 uM). FLG-TP inhibited the elongation of viral minus strand DNA by terminating DNA chain whereas it inhibited the priming reaction of the reverse transcription with only 40% inhibition at 100 uM. Furthermore, FLG-TP inhibited equally the elongation of viral minus strand DNA by WT (IC50 = 4.9 uM), LAM-R (IC50 = 4.8 uM), ADV-R (IC50 = 5.5 uM) polymerase mutants. In Huh7 cell culture experiments, FLG had lower IC50 (9 uM) on HBV replication compared with ADV (15 uM) but higher than LAM (0.5 uM). Its inhibitory activity on the LAM-R, ADV-R, and LAM-R + ADV-R mutants was comparable to that of WT HBV (IC50 = 8, 13 and 15 uM). By contrast LAM and ADV were not active against the LAM-R+ADV-R mutant.

 

Conclusions:

Our data provide new insight in the mechanism of inhibition of FLG on HBV replication and demonstrate its inhibitory activity on drug resistant mutant reverse transcriptases in vitro in both cell free and cellular assays. Noteworthy, FLG inhibited the replication of LAM-R, ADV-R, and multiple drug resistant (LAM-R + ADV-R) mutants. Its in vitro cross-resistance profile warrants further clinical evaluation to rescue resistance to lamivudine and/or adefovir, and to prevent the emergence of resistance in combination trials.

 

 


Poster 1140

BINDING AND INHIBITION OF HEPATITIS B VIRUS C GENE PROMOTER BY ADENO-ASSOCIATED VIRUS REP78 PROTEIN IN VITRO

Hong You, Zhongyu Yan, Min Cong, Ping Wang, Baoen Wang, Jidong Jia, Beijing Friendship Hospital, Capital University of Medical Sciences, Beijing,, China; Yong Liu, Paul L. Hermonat, University of Arkansas for Medical Sciences, Little Rock, AR.

 

 

Background:

Adeno-associated virus (AAV) Rep78 is known to inhibit a variety of important viral and cellular genes, including human papillomavirus type 16 p97 and cellular H-ras, c-fos, c-jun, and c-myc, specifically by binding to their transcriptional promoters. Thus, potentially AAV Rep78 might be used therapeutically as an anti-viral/anti-cancer “drug.”

 

Aim:

The study was designed to investigate if AAV Rep78 might also regulate hepatitis B virus gene expression.

 

Methods:

Woodchuck hepatitis virus (WHV) was removed from the plasmid pWHV and recircularized. Liver derived HepG2 cells were cotransfected with a Rep78 expression plasmid (6 ugs) plus recircularized WHV (3 ugs). Ten ug of total cellular DNA, isolated at day 5, was digested with DpnI, size separated, Southern blotted and probed with 32P-WHV DNA. Binding of the Rep78 protein to the HBV c promoter binding was also analyzed by electrophoretic mobility shift assay (EMSA). Finally in vitro transcription in nuclear extracts was utilized to study Rep78 inhibition of the HBV-c promoter.

 

Results:

WHV replication was found to be significantly reduced around 70% by the presence of an AAV Rep78 expression plasmid. EMSA showed Rep78 protein did bind to HBV-c promoter strongly in a dose-dependent manner. This binding could be retarded by specific anti-Rep78 antibody. In addition, HBV-c gene transcription was significantly inhibited about 50% by Rep78 by in vitro transcription.

 

Conclusions:

AAV Rep78 could inhibit the WHV replication and transcription of HBV-c gene through its binding with HBV-c promoter. Furthermore, these data are fully consistent with Rep78 inhibition of other important genes as being by direct binding of promoter sequences and down-regulation of transcription.

 

 


Poster 1141

SAFETY, TOLERANCE, PHARMACOKINETICS AND PHARMACODYNAMICS OF REMOFOVIR, A LIVER-TARGETING PRODRUG OF PMEA IN HBV PATIENTS FOLLOWING DAILY DOSING FOR 28 DAYS

Chin-chung Lin, Valeant Pharmaceuticals International, Costa Mesa, CA; You-Chen Chao, Tri-Service General Hospital, Taipei, Taiwan Republic of China; Ming-Yang Lai, National Taiwan University Hospital, Taipei, Taiwan Republic of China; Ting-Tsung Chang, National Cheng Kung University Hospital, Tainan, Taiwan Republic of China; Wan-Long Chuang, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan Republic of China; Sien-Sing Yang, Cathy General Hospital, Taipeh, Taiwan Republic of China; Rene Braeckman, Valeant Pharmaceuticals International, Costa Mesa, CA; Ding-Shinn Chen, National Taiwan University Hospital, Taipei, Taiwan Republic of China.

 

 

Background:

Adeofovir dipivoxil, an esterase-activated prodrug of PMEA has a recommended dose of 10 mg QD, which is sub-optimized due to dose-limiting nephrotoxicity. Remofovir is a CYP3A4-activated prodrug of PMEA with excellent liver-target properties in rats and better safety than adeofovir dipivoxil in one-month monkey toxicity study. Thus, remofovir may optimize its clinical efficacy with lower nephrotoxicity potential than adeofovir dipivoxil.

 

Aim:

To evaluate safety, tolerance, pharmacokinetics and pharmacodynamics of remofovir in HBV patients following daily dosing for 28 days.

 

Methods:

Forty-five adult Asian patients with compensated HBV infection completed this study. All patients had a HBV DNA viral load of =3,000,000 copies/mL at screening. Eight to ten patients were randomized to each dose group of remofovir (5, 10, 20 and 30 mg daily) or placebo and were dosed for 28 days. Safety assessment included adverse events, physical examination, vital signs and safety laboratory tests. Serum samples were analyzed for remofovir and PMEA by validated LC-MS/MS methods. Predose serum HBV DNA levels in all patients on Days 1, 8, 15, 21 and 28 after the first dose and Weeks 4, 8 and 12 after the last dose were determined using the COBAS Amplicor assay (LOQ: 200 copies/mL).

 

Results:

Following oral doses of remofovir up to 30 mg/day for 28 days in HBV patients, no unexpected adverse events were reported. The majority of events were mild in severity and similar between the placebo and remofovir groups. The most frequently reported event among all study patients was upper respiratory infection (30%). No dose related trends regarding safety were identified and no events resulted in a patient being withdrawn prematurely from treatment. Remofovir was rapidly absorbed and converted to PMEA with a Tmax of 1 hr or less for both remofovir and PMEA. On Days 1 and 28, Cmax and AUC for remofovir and PMEA increased with dose. T 1/2 for PMEA (43-53 hr) was longer than that for remofovir (6.8-11 hr). At all doses, serum HBV DNA levels decreased with time. After 4 weeks of remofovir treatment, the median log HBV decline from baseline (Day 1 predose) was 1.64 at 5 mg dose, 2.48 at 10 mg dose, 2.72 at 20 mg dose and 2.66 at 30 mg dose.

 

Conclusions:

(1) Remofovir was rapidly absorbed in HBV patients, and rapidly converted into PMEA. (2) Remofovir was well tolerated in patients up to doses of 30 mg/day for 28 days. (3) Cmax and AUC increased with dose for both remofovir and PMEA. (4) After 4 weeks of treatment, daily doses of 10, 20 and 30 mg of remofovir yielded clinical significant median declines in log HBV.

 

 


Poster 1143

LONG-TERM SAFETY AND EFFICACY OF ADEFOVIR DIPIVOXIL (ADV) IN THE TREATMENT OF CHRONIC HEPATITIS B IN PATIENTS PRE-AND POST-LIVER TRANSPLANTATION (OLT) WITH LAMIVUDINE-RESISTANT (LAM-R) HEPATITIS B VIRUS (HBV)

Eugene Schiff, Center for Liver Disease, Miami, FL; Ching-Lung Lai, Queen Mary Hospital, Hong Kong, China; Peter Neuhaus, Charite Campus Virchow, Berlin, Germany; Hans Tillmann, Hannover Medical School, Hannover, Germany; Didier Samuel, Paul Brousse Hospital, Villejuif, France; Jean-Pierre Villeneuve, CHUM-Campus Saint-Luc, Montreal, PQ, Canada; Stefanos Hadziyannis, Henry Dunant Hospital, Athens, Greece; Shelly Xiong, Sarah Arterburn, Carol L. Brosgart, Graeme Currie, Gilead Sciences, Inc., Foster City, CA.

 

 

Background:

Adefovir (ADV) has potent in vivo and in vitro activity against wild-type and LAM-R HBV. In pre- and post-OLT patients (pts) with LAM-R HBV, 48 weeks (wks) of ADV resulted in serum HBV DNA reductions of 4.1 and 4.3 log10 c/mL and ALT normalization in 68% and 79% of pts, respectively. No ADV resistance was seen in the first 48 weeks.

 

Aim:

To evaluate long-term safety, efficacy and resistance of ADV 10 mg in pre- and post-OLT pts with LAM-R HBV.

 

Methods:

241 post-OLT (A) and 226 pre-OLT, decompensated liver disease (B) pts failing LAM enrolled. ADV dose adjusted for renal impairment (CLcr < 50 mL/min).

 

Results:

Baseline (BL): median HBV DNA 8.2 and 7.4 log10 c/mL, and median ALT 2.0 and 1.8 x ULN in A and B, respectively; 83% (A) and 88% (B) male; median age 53 (A) and 52 (B) years; 78 % (A) and 70% (B) Caucasian. Median ADV duration 99 wks (max: 203 wks) in A and 52 wks (max: 153 wks) in B. 4 (2%) pts had re-OLT (A); 61 (27%) underwent OLT (B). HBV DNA reductions in the first 48 wks were maintained and/or improved throughout 144 wks. Increasing proportions of pts normalized ALT over time. Based upon assessable Child-Pugh-Turcotte (CPT) scores, 95% (A) and 100% (B) had a stable or improved CPT at wk 96. Survival by wk 96 was 88% (A) and 78% (B) (Kaplan-Meier estimates). Resistance up to 144 wks was seen in 2 (1.8%) pts between wks 48 and 96. Both pts had discontinued LAM prior to emergence of resistance. Addition of LAM to ADV resulted in re-suppression of HBV DNA.

 

Cohort

A*

B*

 

Wk 48

Wk 96

Wk 144

Wk 48

Wk 96

Wk 144

HBV DNA median change from baseline (log10 copies/mL)

-4.3
(n=98)

-4.7 (n=40)

-4.6
(n=20)

-4.1
(n=63)

-4.4
(n=18)

NA

ALT median change from baseline (IU/L)

-35
(n=138)

-33
(n=84)

-30
(n=33)

-46 (n=102)

-60
(n= 23)

NA


* 68% (A) and 57% (B) withdrew due to study closure with commercial availability

 

A low rate of related serious adverse events (AEs) were reported (6% A, 6% B), AEs leading to drug discontinuation (11% A, 15% B) were infrequent. A total of 67 deaths were reported (27 A, 40 B); 22% and 38% of these occurred within 30 days of starting ADV. 15 pts died prior to initiating ADV. Confirmed changes in serum creatinine = 0.5 mg/dL above BL by 144 wks were observed in 21% pts; the majority had renal dysfunction and/or multiple risk factors for renal dysfunction at BL. Only 2% of pts discontinued ADV for renal adverse events.

 

Conclusion:

ADV treatment over 144 wks resulted in significant reductions in serum HBV DNA, ALT normalization and other liver functions. The survival experience in the patients, pre- and post-OLT, together with the improvement in HBV DNA and other efficacy parameters is evidence of a clinically meaningful benefit. Long-term treatment was not associated with treatment limiting toxicity.

 

 


Poster 1157

ANTIGEN-PRESENTING DENDRITIC CELLS LOADED WITH HEPATITIS B SURFACE ANTIGEN IS CAPABLE OF INDUCING AND MAINTAINING ANTI-HBS IN IMMUNOSUPPRESSED MOUSE: STRATEGY FOR DEVELOPING PROPHYLACTIC VACCINE AGAINST HEPATITIS B VIRUS IN IMMUNOSUPPRESSED INDIVIDUALS

Shinya Furukawa, Sk. Md. Fazle Akbar, Aki Hasebe, Norio Horiike, Morikazu Onji, Ehime University School fo Medicine, Shitukawa, Japan.

 

 

Background:

Administration of prophylactic vaccines containing hepatitis B surface antigen (HBsAg) induces antibody to HBsAg (anti-HBs) and protect more than 90% of normal individuals against HBV infection. However, HBsAg-based vaccines usually fail to induce anti-HBs in a considerable numbers of immunosuppressed individuals.

 

Aims:

The aim of this study was to develop a powerful cell-based vaccine for immunosuppressed subjects by loading HBsAg on dendritic cell (DC), the most potent antigen-presenting cells.

 

Methods:

Based on the data of preliminary studies for production of immunosuppressed mouse, we injected normal C57BL/6 mouse with tacrolimus (FK-506), an immunosuppressive agent (2-8 mg/kg body wt, intraperitoneal), daily for 2 weeks. The immunosuppressive status of FK-506-injected mouse was assessed from the levels of mRNAs for interleukin-2 and interferon-gamma in spleen cell cultures. DCs were isolated from the single cell suspensions of mouse spleen by density centrifugation, adherence on plastic surface and depletion of lymphocytes and macrophages. Spleen DCs were cultured with graded doses of HBsAg (10-100 microgram) for 24-48 hours in RPMI 1640 plus 10% fetal calf serum. After washing for 5 times, 1-2 million HBsAg-pulsed DCs were injected to immunosuppressed C57BL/6, twice at an interval of 2 weeks.

 

Results:

The expression of mRNAs for interferon-gamma and interleukin-2 was lower in spleen cell cultures of immunosuppressed C57BL/6 (2 weeks after starting of injection of FK-506) compared to that of normal C57BL/6 mouse. Immunosuppressed C57BL/6 mice did not develop anti-HBs in the sera after injecting twice with HBsAg in adjuvant (2.5 microgram). However, 1.0 microgram of HBsAg in adjuvant induced anti-HBs in all normal C57BL/6 mouse. Interestingly, administration of HBsAg-pulsed DCs for two times induced production of anti-HBs in immunosuppressed C57BL/6 within 2-4 weeks after second injection. In 3 of 5 immunosuppressed C57BL/6 mice, the levels of anti-HBs were more than 1000 mIU/ml at 8 weeks after second injection of HBsAg-pulsed DCs, although these immunosuppressed mice received FK-506 on a daily basis for this entire duration.

 

Conclusions:

The concept of this study might be used for induction and maintenance of anti-HBs in immunosuppressed subjects, especially in children borne from human immune deficiency virus-infected mothers and persons receiving immunosuppressive drugs for autoimmune disorders or for protecting transplanted organs.

 

 


Poster 1144

HBSAG SEROCONVERSION IN CHRONIC HBV PATIENTS TREATED WITH PEGYLATED INTERFERON ALPHA-2B ALONE OR IN COMBINATION WITH LAMIVUDINE. THE ROLE OF HBV GENOTYPE

Harry L. A. Janssen, Hajo J. Flink, Monika van Zonneveld, Hubert G. M. Niesters, Robert A. de Man, Solko W. Schalm, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands; for the HBV 99-01 Study Group.

 

 

Background:

HBsAg seroconversion is the hallmark of a complete response during antiviral therapy in chronic hepatitis B (CHB). It is accepted as the most definite evidence of sustained disease remission. HBsAg seroconversion is almost absent in patients treated with lamivudine and is 1.6% after one year of adefovir.

 

Aim:

Since pegylated interferon alpha-2b (PEG-IFN) is known to induce a response (HBeAg loss) by immune modulation rather than a direct antiviral effect we investigated the frequency of HBsAg seroconversion during treatment with PEG-IFN.

 

Methods:

In a global, double-blind, randomized controlled trial 266 HBeAg-positive CHB patients were treated for 52 weeks with PEG-IFN 100 µg/week in combination with either lamivudine 100 mg/day or placebo. PEG-IFN dose was halved after 32 weeks of treatment. Follow-up lasted for 26 weeks post-treatment.

 

Results:

Ninety-five (36%) of the 266 patients exhibited HBeAg loss before the end of follow-up. HBeAg loss was 47% for genotype A (n=90), 44% for genotype B (n=23), 28% for genotype C (n=39) and 25% for genotype D (n=103) (A vs D and B vs C: P< 0.001). By multivariate analysis genotype was an important independent predictor for HBeAg loss. During the study 18(7%) patients showed loss of HBsAg and 16 (6%) HBsAg seroconversion. Adding lamivudine did not enhance HBeAg loss, HBsAg loss or HBsAg seroconversion. HBsAg seroconversion occurred in 3 patients during treatment and in 13 patients after treatment. All patients with HBsAg seroconversion had normal ALT and HBV DNA < 10e3 copies/ml by PCR at the end of follow-up. HBsAg seroconversion rate also differed according to HBV genotype: 13% (n=11) for genotype A, 9% (n=2) for genotype B, 0% for genotype C and 2% (n=2) for genotype D (A vs D: P=0.04). Among responders with HBeAg loss the HBsAg seroconversion rate was 28%, 20%, 0%, 8% for genotype A, B, C and D, respectively.

 

Conclusion:

In conclusion, one year of PEG-IFN in HBeAg-positive CHB patients leads to an HBsAg seroconversion rate of 6%. Adding lamivudine did not increase HBsAg seroconversion. Almost one third of the responders with genotype A exhibited HBsAg seroconversion. Our study indicates that future intervention studies for HBeAg-positive CHB may need stratification according to HBV genotype and that PEG-IFN is most beneficial to achieve an HBeAg- and HBsAg response in patients with genotype A and B.

 

 


Poster 1145

DEVELOPMENT OF A NON-REPLICATIVE, SINGLE-SHOT DNA-BASED VACCINE EXPRESSING ALL HEPATITIS B VIRAL ANTIGENS

Christian Freddy Grimm, Stefanie Gaiser, Hubert Erich Blum, Michael Geissler, University Hospital Freiburg, Freiburg, Germany.

 

 

Background:

The activity of a broad based immune response to HBV antigens has been shown to be one of the most important factors contributing to virus elimination from infected hepatocytes.

 

Aim:

Our aim was, therefore, to develop a DNA-based vaccine expressing HBV gene products but lacking HBV replication. Therefore, a CMV-promoter based plasmid producing a HBV pregenome lacking the first 43 nucleotides resulting in a mutated epsilon-signal and subsequently a defective HBV pregenome encapsidation (pCH3143) was employed.

 

Methods:

pCH3143 was transfected in G8 murine myoblast and Huh-7 human hepatoma cells. Expression of HBcAg, HBsAgs, HBeAg, polymerase, and x-antigen was determined by Western-blot, immunofluorescence and ELISA techniques. HBV replication and RNA analysis was assessed by Southern blot and Northern blot, respectively. Balb/c and C57BL/6 mice were immunized once with 100 mg pCH3143 and, subsequently, antibody as well as CTL responses against all viral gene products were determined by ELISA and cytotoxicity assays. Protective immunity was determined by re-challenge of pCH3143-immunized Balb/c mice with syngeneic tumor cells expressing HBcAg, HBeAg, the small and large HBsAgs, and polymerase.

 

Results:

After transfection of both G8 and Huh-7 cells high level expression of HBeAg, polymerase, and all HBsAgs (small, pre-S2, pre-S1) could be detected. By contrast, expression of x-antigen was weak. No HBV replication was detectable, whereas high level replication was observed in cells transfected with a non-mutated control plasmid. No encapsidation of pregenomic RNA in cores could be observed and the corresponding RNA was detected in 10-20 fold increase amounts compared to wild-type HBV RNAs produced from the HBV wild-type construct. Immunization of Balb/c and C57BL/6 mice resulted in strong anti-HBs and anti-HBc antibody responses. Only low-titer antibodies against polymerase and no anti-HBx could be detected. Strong CTL responses could be observed against all viral antigens in a hierarchical manner in both mouse strains (HBcAg>HBsAg>polymerase>HBxAg). Corresponding to the in vitro CTL activity, pCH3143 vaccinated animals were protected against a s.c. challenge with syngeneic tumors expressing HBcAg, HBsAg, and polymerase. Mice immunized with Mock-DNA were not protected. Similarly, there was a rapid growth of x-antigen expressing tumors in pCH3143 immunized animals.

 

Conclusion:

This is the first demonstration of a single shot vaccine expressing all HBV antigens, lacking HBV replication, and inducing strong humoral and cellular immune responses against all viral proteins except x-antigen. This vaccine may be of value for the treatment of chronic viral hepatitis, for vaccine non-responders, and the prophylaxis of HCC.

 

 


Poster 1146

EMERGENCE OF ENTECAVIR RESISTANT HEPATITIS B VIRUS AFTER ONE YEAR OF THERAPY IN PHASE II & III STUDIES IS ONLY OBSERVED IN LAMIVUDINE REFRACTORY PATIENTS

RJ Colonno, RE Rose, SM Levine, K Pokornowski, M Plym, CF Yu, CJ Baldick, S Zhang, AW Walsh, L Discotto, J Fang, DJ Tenney, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT.

 

 

Background:

Entecavir (ETV) is a potent and selective antiviral with efficacy in the treatment of patients with chronic hepatitis B virus (HBV), including those infected with lamivudine (LVD) refractory HBV.

 

Aim:

Previous analyses of HBV isolates from 2 patients with virologic rebound in phase II ETV trials revealed that additional substitutions at positions rtT184, rtS202 and rtM250 in pre-existing LVD-resistant (LVDR) HBV reverse transcriptase (RT) were required for expression of ETV resistance. These three substitutions did not result in reduced ETV susceptibility in the absence of LVD-resistance changes.

 

Methods:

Clinical samples from studies ETV-022 and -027 (nucleoside naïve) and ETV-014 and -026 (lamivudine refractory) patients were monitored for emergence of ETV resistance. Genotypic analysis compared HBV RT sequences amplified from patient serum DNA during therapy with sequences at study entry and wildtype HBV. The phenotypes of substitutions that emerged on therapy were determined using antiviral assays in HepG2 cells measuring HBV DNA yields after transfection of recombinant plasmids containing patient RT sequences.

 

Results:

Genotypic analysis was performed on 604 patient samples (including all patients exhibiting virologic rebound on therapy) with compensated liver disease completing at least 1 year of ETV therapy. None of 432 (219 HBeAg+, 213 HBeAg-) nucleoside treatment naive patients developed recognized ETV genotypic resistance during one year of 0.5 mg daily ETV. Furthermore, phenotypic analysis of emergent changes to date has shown no signature ETV-resistance mutations in the absence of pre-existing LVDR mutations. A range of substitutions at previously identified residues rt184, rt202 and rt250 associated with clinically relevant ETV resistance were noted in 10 of 172 (5.8%) patients with prior LVDR HBV after 1 year of therapy with 1.0 mg daily ETV. In all cases, pre-existing LVD-signature resistance mutations were required to achieve an ETV resistant phenotype; however, not all ETV related genotypic changes resulted in phenotypic resistance in vitro or in virologic rebounds clinically. Extended resistance analysis of patients in ETV clinical trails is ongoing to determine the incidence of resistance upon prolonged therapy.

Conclusion:

There was no evidence of ETV resistance emergence in nucleoside naïve patients and a low incidence (5.8%) of ETV resistance in LVD refractory patients following 48 wk of ETV treatment. Phenotypic resistance to ETV required the presence of pre-existing LVD resistance mutations. Coupled with superior clinical efficacy to LVD and a comparable safety profile, ETV represents a highly effective antiviral for the primary treatment of chronic HBV infection.

 

 


Poster 1147

PREDICTION OF BREAKTHROUGH HEPATITIS DUE TO LAMIVUDINE-RESISTANT HEPATITIS B VIRUS BY A SENSITIVE SEMI-QUANTITATIVE ASSAY FOR THE MUTANT VIRUS

Kojiro Mori, Masahito Minami, Toshihiko Kirishima, Koji Kunimoto, Kohichiro Yasui, Yoshito Itoh, Takeshi Okanoue, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan.

 

 

Background:

The emergence of lamivudine-resistant YMDD mutants resulting in breakthrough hepatitis is a serious problem for treatment of chronic hepatitis B (HBV).

 

Aim:

Early prediction of the emergence of a lamivudine-resistant virus may help to interrupt the proliferation of the mutant virus and provide insights into better therapeutic strategies.

 

Method:

We previously reported a highly sensitive assay utilizing peptide nucleic acid (PNA) and restriction fragment length polymorphism (RFLP) for the detection of YMDD mutants. Using this assay, YMDD mutants were detected early during lamivudine therapy, even in patients who had not been treated with lamivudine (J Hepatol 2002; 37: 259). To predict breakthrough hepatitis at an early stage of lamivudine therapy and to analyze the dynamics of YMDD mutants in patients who experienced HBV DNA breakthrough, we developed a sensitive semi-quantitative assay using competitive polymerase chain reaction (PCR) and analyzed serial sera during lamivudine treatment.

Methods: Subjects were 55 consecutive Japanese patients with chronic hepatitis B who underwent daily 100mg lamivudine therapy. First, emergence of YMDD mutants was detected by a PNA mediated PCR clamping method with a detection limit for the YMDD mutant of 101 co-existing with as many as 104 fold excess of wild type viruses. In subjects where YMDD mutants were detected, we performed semi-quantitative assay. This assay detects 102.5 - 107.5 copies of mutant virus per one milliliter of serum.

 

Results:

YMDD mutants were detected in 26 (47%) of the 55 patients during periods of lamivudine administration by PNA mediated PCR clamping. Eight patients developed breakthrough hepatitis; 7 stopped taking medication before viral breakthrough. YMDD mutants appeared temporally and disappeared later despite continuance of lamivudine therapy in 11 patients. Thus, the existence of a small quantity of mutant viruses does not always lead to prediction of breakthrough hepatitis. We then tried to quantify these mutant viruses by a semi-quantitative assay. In all 8 patients who developed breakthrough hepatitis, quantities of YMDD mutants ranged from 102.5 - 103.5 copies/ml in the 2 to 3 months before clinical breakthrough. In contrast, in 11 patients without viral breakthrough YMDD mutants were always less than 102.5 copies/ml. Thus a mutant virus quantity of over 102.5 copies precisely predicted emergence of breakthrough hepatitis among the 19 patients tested.

 

Conclusions:

(1) Small populations of YMDD mutants can be detected early, but often disappear during lamivudine therapy.

(2) Our sensitive quantitative assay is useful for early detection of YMDD mutants and a threshold quantity of 102.5 copies/ml is suggested for prediction of viral breakthrough.

 

 


Poster 1148

LONG-TERM OUTCOME OF PATIENTS WITH HBeAG-NEGATIVE CHRONIC HEPATITIS B (CHBe-) UNDER NUCLEOS(T)IDE ANALOGUES MAINTENANCE THERAPY STARTING WITH LAMIVUDINE (LAM)

George V. Papatheodoridis, Hippokration General Hospital, Athens, Greece; Evangelini Dimou, Henry Dunant Hospital, Athens, Greece; Konstantinos Dimakopoulos, George Papanikoloau General Hospital, Thessaloniki, Greece; Spilios Manolakopoulos, Polyclinic General Hospital, Athens, Greece; Irene Rapti, Henry Dunant Hospital, Athens, Greece; Dimitrios Tzourmakliotis, Polyclinic General Hospital, Athens, Greece; George Kitis, George Papanikolaou General Hospital, Thessaloniki, Greece; Emanuel K. Manesis, Hippokration General Hospital, Athens, Greece; Stephanos J. Hadziyannis, Henry Dunant Hospital, Athens, Greece.

 

 

Background:

Data on the long-term outcome of CHBe- patients under maintenance antiviral therapy are lacking.

 

Aims:

We evaluated the outcome of 201 patients with CHBe- who started LAM prior to 12/2001 (Group A) and compared it with the outcome of two cohorts of CHBe- patients [interferon-alfa (IFN) treated: 209 (Group B)-sustained responders (SRs):57, non-SRs:152; untreated: 195 (Group C) - J Hepatol 2001;34:306]. There was no significant difference among the 3 groups at baseline.

 

Methods:

Group A patients were followed-up for a median of 43 (12-94) months with clinical examinations and LFTs every =3 months and serum HBV-DNA (Monitor, Roche; sens. 400 cp/ml) every =6 months and on biochemical breakthroughs (BTH). The probability of virologic remission was 73%, 52%, 40% and 34% and of biochemical remission 84%, 64%, 50% and 36% at 12, 24, 36 and 48 months after LAM onset, respectively. Another nucleot(s)ide analogue was administered in 79/109 (72%) patients with virologic BTH or no response at a median 15 (1-91) months or in 76/97 (78%) patients with biochemical BTH or no response at a median of 8 (0-91) months.

 

Results:

In Group A, 4 patients died (decompensation: 2, HCC: 1, liver unrelated cause: 1) and 1 was transplanted (for HCC), while 8 additional patients developed complications (decompensation: 7- reversed in 4 after addition of adefovir; HCC: 1) but were still alive. All 12 patients with liver related major events had advanced fibrosis (Ishak score 5-6) at baseline and all but one had previously experienced virologic-biochemical BTH (n=10) or no response (n=1). Another nucleot(s)ide analogue was administered in 5/9 with decompensation: at a median of 4 (0-13) months after (n=4) or at 2 months before decompensation (n=1). At the end of follow-up, OLT-free and complication-free survival were significantly better in Group A than C (P<0.04) or B-non-SRs (P<0.04), while they did not differ between Group A and B-SRs. The probability of HCC was significantly lower in Group A than B-non-SRs (P=0.02) and relatively lower in Group A than C (P=0.09). Cox regression analysis showed that, besides younger age (P<0.001) and absence of advanced fibrosis at baseline (P<0.0001), Group (A>C; A>B; A>B-non-SRs, P<0.02) was significantly associated with complication-free survival at the end of follow-up, but not at the end of LAM monotherapy.

 

Conclusions:

In CHBe-, long-term antiviral therapy starting with LAM significantly improves survival and reduces the risk of complications, compared with IFN or non-SR to IFN or no treatment. In CHBe- with advanced fibrosis, very close follow-up for LAM resistance and prompt onset of additional antiviral therapy is required in an effort to reduce the rate of complications or the ab initio use of agent(s) with low resistance rates should be considered.

 

 


Poster 1149

HEPATIC IMPAIRMENT DOES NOT ALTER SINGLE-DOSE PHARMACOKINETICS AND SAFETY OF ENTECAVIR

Marc Bifano, Jing-He Yan, Jingdong Xie, Duxi Zhang, Jessica Freund, George Hanna, Frank LaCreta, Dennis Grasela, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ.

 

 

Background:

Entecavir (ETV) is a potent and selective anti-hepatitis B agent which is primarily cleared via renal excretory pathways.

 

Aim:

This Phase I study compared the pharmacokinetics (PK), safety, and tolerability of ETV in subjects with hepatic impairment to those in healthy controls.

 

Methods:

ETV-032 was an open-label, non-randomized study in which healthy controls were matched (1:1) to hepatically impaired subjects for age (±5 years), weight (±15%), and sex. Sixteen (16) subjects with Grade B or C hepatic impairment (by Child-Pugh classification) and 16 healthy subjects with normal hepatic function were enrolled. All subjects received a single oral dose of ETV 1.0 mg. Blood samples were collected up to 336 hours post-dose and urine samples were collected up to 96 hours post-dose for PK analyses. ETV concentrations were determined using validated LC/MS/MS methods. PK analyses for ETV were conducted using non-compartmental methods.

 

Results:

Summary statistics for PK parameters are listed below.

 

Pharmacokinetic
Parameter

Child-Pugh
Grade B
(n=12)

Child-Pugh
Grade C
(n=4)

Hepatic Impaired
(Grade B Plus C)
(n=16)

Healthy
(n=16)

Cmax (ng/mL)
Geometric Mean (CV%)

8.99 (32.7)

7.25 (39.6)

8.52 (34.3)

8.22 (29.9)

AUC(INF) (ng·h/mL)
Geometric Mean (CV%)

29.33 (24.3)

28.17 (38.4)

29.03 (26.9)

31.28 (21.6)

Tmax (h)
Median (min, max)

0.75 (0.50, 1.50)

0.88 (0.50, 1.50)

0.75 (0.50, 1.50)

0.75 (0.50, 2.00)

T-HALF (h)
Mean (SD)

87.07 (41.0)

109.94 (75.6)

92.79 (48.3)

92.10 (14.5)

%UR (%)
Mean (SD)

44.52a (25.9)

47.15 (13.3)

45.22b (22.8)

52.77 (15.2)

CLR (mL/min)
Mean (SD)

309.31a (143.5)

383.10 (200.6)

328.99b (156.4)

374.23 (133.2)


a n=11;
b n=15

 

Statistical analysis showed that both Cmax and AUC(INF) were unaffected by hepatic impairment. The point estimates (90% Confidence Intervals) for the hepatic impairment to healthy Cmax ratio and AUC(INF) ratio were 1.036 (0.942, 1.139) and 0.928 (0.813, 1.059), respectively, and were both well within the pre-specified no effect interval (0.67-1.50). Other ETV PK parameters in subjects with hepatic impairment were comparable to those of healthy subjects. ETV administered as a single 1.0 mg oral dose was safe and well tolerated in healthy subjects and in subjects with moderate to severe hepatic impairment.

 

Conclusions:

ETV exposure was not significantly altered in subjects with moderate to severe hepatic impairment when compared to healthy subjects. ETV may be administered to patients with hepatic impairment without dose adjustment.

 

 


Poster 1150

SUCCESSFUL TREATMENT WITH PEGYLATED INTERFERON IN HBV NON-RESPONDERS TO STANDARD INTERFERON AND LAMIVUDINE

Hajo J. Flink, Bettina E. Hansen, Monika van Zonneveld, Solko W. Schalm, Harry L. A. Janssen, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands; for the HBV 99-01 study group.

 

 

Background:

Treatment with antiviral therapy is effective in 20-30% of HBeAg-positive chronic hepatitis B (CHB). Non-response to treatment may result in progression of liver disease and increased risk of hepatocellular carcinoma.

 

Aims:

As part of a global randomized controlled trial we investigated the efficacy (i.e. loss of HBeAg at end of follow-up) of pegylated interferon (PEG-IFN) in CHB who failed to respond to previous courses of standard interferon or lamivudine.

 

Methods:

We analyzed 59 patients non-responders to previous standard IFN and 39 non-responders to previous lamivudine therapy. All patients received a 52 week course of 100µg PEG-IFN weekly combined with, either 100mg lamivudine or placebo daily. After therapy patients were followed for 24 weeks.

 

Median treatment duration was 24 weeks (range 3 - 52) for previous standard IFN (median dose 18 MU/week), and 52 weeks (range 2 - 411) for previous treatment with lamivudine. Median interval until retreatment was 119 weeks (range 26 - 576) and 43 weeks (range 28 - 366) for prior IFN and lamivudine, respectively. Eleven patients had a YMDD-mutant at start of Peg-IFN therapy. Seventeen (29%) non-responders to previous IFN and 9 (23%) non-responders to previous lamivudine responded (loss of HBeAg at end of follow-up) to treatment with Peg-IFN.

 

Results:

Treatment with the combination of Peg-IFN and lamivudine was not superior to Peg-IFN alone. Non-responders to prior IFN therapy with baseline ALT above 4 x ULN responded better to Peg-IFN than those with ALT levels below 4 x ULN (44% vs. 16 % respectively, p = 0.019). A similar trend was found for non-responders to previous lamivudine with ALT above vs. below 4 x ULN (response 37% vs. 13%, respectively; p = 0.089).

 

Conclusion:

In conclusion, Peg-IFN is effective in almost one-third of patients who failed previous treatment with standard IFN or lamivudine. High ALT levels at baseline of Peg-IFN therapy was the best predictor for increased response in prior non-responders to either standard IFN or lamivudine therapy.

 

 


Poster 1151

BREAKING IMMUNE TOLERANCE IN CHRONIC HEPATITIS B BY DENDRITIC CELL VACCINATION IN HBV TRIMERA MICE

Raja Reddy Vuyyuru, Fareed Khan Rahman, Silke Schmitt, Sabine Herzog-Hauff, Soheila Tavakoli, Sandra Weyer, University Hospital, Mainz, Germany; Josef Koeck, Michael Geissler, University Hospital, Freiburg, Germany; Dennis Strand, Peter Galle, Wulf O. Bocher, University Hospital, Mainz, Germany.

 

 

Background:

Whereas spontaneous resolution from hepatitis B is associated with strong antiviral T cell responses, only weak immune responses are found in patients with chronic infection.

 

Aims:

Thus, induction of strong HBV-specific T cell reactivities might represent a new therapeutic strategy. Dendritic cells (DC) are highly specialised antigen presenting cells (APC) and might therefore serve as an effective therapeutic vaccine.

 

Methods:

DC were generated from MACS -seperated monocytes of patients and healthy cnotrols by incubation in IL-4 and GM-CSF. Apoptosis of HepG2 cells transfected with a 1.3 overlength HBV plasmid was induced by UV irradiation. Balb/c mice were irradiated, transplanted with nod.scid mouse bone marrow and reconstituted with human PBMC from donors with chronic HBV infection (HBsAg seropositive) or controls. Vaccination of such trimera was performed i.p. with DC pulsed with apoptotic HBV-transfected or non-transfected HepG2 cells, rHBcAg or HBc18-27 peptide; vaccination with rHBc antigen and EBV peptide280-288 served as controls. HBV specific human T cell frequencies were analysed from peritoneal lavage by IFNy ELISpot with autologous APC pulsed with HBV core (HBc) and surface (HBs) antigens or one core and five envelope peptides 10 days after vaccination.

 

Results:

Cross presentation of HBV core antigen by DC from chronic HBV patients and controls was similarly effective, as demonstrated by stimulation of an HLA A2-restricted HBc18-27-specific CTL clone by IFNy Elispot. Patient PBMC revealed no or very weak Th cell and CTL responses against core or envelope epitopes, indicating their antiviral immune tolerance. However, when DC pulsed with apoptotic HBV-transfected HepG2 cells were transferred together with autologous PBMC from chronic HBV carriers into trimera mice, strong and multispecific HBc and HBs specific Th cell and CTL responses were detected.

 

Conclusions:

Our studies demonstrate that HBV transfected apoptotic body pulsed DC might represent a tool for therapeutic vaccination of chronic HBV patients. Moreover, core specific CTL, that are barely detectable ex vivo in chronic HBV patients, can rapidly be recovered under our experimental conditions, arguing against deletion of such cells in patient PBMC.

 

 


Poster 1152

ENTECAVIR IS SUPERIOR TO CONTINUED LAMIVUDINE FOR THE TREATMENT OF LAMIVUDINE-REFRACTORY, HBeAG(+) CHRONIC HEPATITIS B: RESULTS OF PHASE III STUDY ETV-026

M Sherman, Toronto General Hospital, Toronto, ON, Canada; C Yurdaydin, Ankara University Medical School, Ankara, Turkey; J Sollano, University of Santo Tomas, Manila, Philippines; M Silva, Hospital Universitario Austral, Pilar, Argentina; Z Goodman, Armed Forces Institute of Pathology, Washington DC, WA; L Chen, A Cross, D DeHertogh, R Hindes, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT; the BEHoLD Study Group.

 

 

Background:

Entecavir (ETV) is a potent and selective inhibitor of hepatitis B virus (HBV) polymerase.

 

Aims:

This Phase III trial compared ETV 1.0 mg QD to LVD 100 mg QD for 48 weeks in LVD-refractory, HBeAg(+) chronic hepatitis B patients.

 

Methods:

ETV-026 is a multinational, randomized, double-blind, Phase III trial (N=286 treated) in which LVD-refractory patients either switched to ETV or continued LVD. Patients were HBeAg(+) with HBV DNA > 3 MEq/mL (bDNA assay) and ALT levels 1.3-10 x ULN. LVD-refractory was defined as persistent viremia while on LVD with or without documented YMDD mutation (present in 85% of pts). Co-primary endpoints, assessed independently with a Bonferroni adjustment, were the proportion of patients achieving (1) Histologic Improvement: a > 2 point decrease in Knodell necroinflammatory score and no worsening of fibrosis (worsening: > 1-point increase in Knodell fibrosis score), and (2) a Composite Endpoint: HBV DNA < 0.7 MEq/mL and ALT normalization. Results: Mean baseline viral loads by PCR were ETV: 9.48 log10 c/mL; LVD: 9.24 log10 c/mL. Mean baseline ALTs were ETV: 124 U/L; LVD: 132 U/L.

 

Results:

Results of primary and major secondary endpoints are shown below.

 

Study Endpoints at Week 48 (non-completer = failure)

Endpoint

ETV
1.0 mg
N=141 (treated)

LVD
100 mg
N=145 (treated)

p-value

*†Histologic Improvement

55%

28%

<0.0001

*Composite
Endpoint (HBV DNA
< 0.7 MEq/mL and ALT normalization)

55%

4%

<0.0001

Ishak Fibrosis Score Improvement

34%

16%

0.0019

‡HBV DNA Mean change from baseline (PCR)
(log10 c/mL)

-5.14

-0.48

<0.0001

HBV bDNA
< 0.7 MEq/mL

66%

6%

<0.0001

HBV DNA
< 400 c/mL (PCR)

21%

1%

<0.0001

ALT Normalization
(<1.25 x ULN)

75%

23%

<0.0001

*Co-primary endpoints
n=124 for ETV group and n=116 for LVD group (pts with evaluable histology)
‡n=133 for ETV and n=128 for LVD with baseline and Week 48 results

 

HBeAg loss occurred in 10% and 3% of ETV and LVD patients, respectively (p=0.0278). A greater proportion of LVD than ETV patients were observed to have virologic non-response (HBV DNA > 0.7 MEq/mL) at Week 48, and met protocol criteria to discontinue treatment. Mutations associated with ETV resistance were infrequently observed. Safety was comparable between groups: 10% and 8% of patients had serious adverse events in the ETV and LVD groups, respectively.

 

Conclusion:

In patients with LVD-refractory chronic hepatitis B, switching to ETV provided superior histologic, virologic, and biochemical response compared to continuing LVD, with comparable safety.

 

 


Poster 1153

ENTECAVIR IS WELL-TOLERATED FOR TREATMENT OF CHRONIC HEPATITIS B: PHASE III SAFETY ANALYSIS IN NUCLEOSIDE-NAïVE AND LAMIVUDINE-REFRACTORY PATIENTS

J Sollano, University of Santo Tomas, Manila, Philippines; E Schiff, University of Miami, Miami, FL; F Carrilho, University of São Paulo School of Medicine, São Paulo, Brazil; M Raptopoulou-Gigi, Aristotle University of Thessaloniki, Thessaloniki, Greece; E Cooney, R Hindes, A Cross, D DeHertogh, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT; the BEHoLD Study Group.

 

 

Background:

Entecavir (ETV) is a potent and selective inhibitor of hepatitis B virus (HBV) polymerase that was well-tolerated in Phase II clinical trials.

 

Aims:

Two randomized, double-blind, double-dummy Phase III clinical trials evaluated ETV at two different doses in nucleoside-naïve (ETV-022) and lamivudine (LVD)-refractory (ETV-026) HBeAg(+) chronic hepatitis B patients. This analysis compared the safety of ETV treatment to LVD treatment for approximately one year in these trials.

 

Methods:

In ETV-022, patients received LVD 100 mg daily (n=355) or ETV 0.5 mg daily (n=354); in ETV-026, they received LVD 100 mg daily (n=145) or ETV 1.0 mg daily (n=141). Safety evaluations included adverse events (AEs), common AEs, serious AEs (SAEs), discontinuations due to AEs, abnormal liver function tests, ALT flares, malignancies, and deaths.

 

Results:

The mean duration of treatment was 57 and 52 weeks for LVD (ETV-022 and ETV-026, respectively), and 62 and 63 weeks for ETV 0.5 mg and 1.0 mg, respectively. The majority of patients in each treatment group reported at least one AE while on treatment, and the AE profiles were comparable in LVD and ETV groups, with the exception of ALT flares and discontinuations due to AEs which were observed more frequently in LVD patients. The most frequently reported AEs, which were generally of mild-to-moderate severity, were headache, upper respiratory tract infection, cough, nasopharyngitis, fatigue, and upper abdominal pain. The frequencies of other categories of AEs are given below.

 

Adverse Events-On Treatment

 

Nucleoside-naïve (ETV-022)

LVD-refractory (ETV-026)

 

ETV 0.5 mg
N =354

LVD 100 mg
N=355

ETV 1.0 mg
N=141

LVD 100 mg
N=145

Any AE (%)

301 (85)

293 (83)

120 (85)

117 (81)

SAEs (%)

26 (7)

26 (7)

14 (10)

11 (8)

Discontinuation due to AE (%)

1 (<1)

9 (3)

2 (1)

10 (7)

Grade 3/4
ALT elevations

95 (27)

114 (33)

24 (17)

44 (31)

ALT Flares
(>2 x bl and > 10 x ULN)

12 (3)

20 (6)

1 (<1)

16 (11)

*Malignant neoplasms

3 (<1)

2 (<1)

2 (1)

1 (<1)

*Deaths

0 (0)

4 (1)

1(<1)

2 (1)

* On study: on-treatment or during 24-week follow-up

 

Conclusions:

Phase III trial safety results confirm earlier observations from Phase II trials and show that ETV at doses of 0.5 mg or 1.0 mg daily is well-tolerated in nucleoside-naïve and LVD-refractory patients. The frequencies of AEs reported during ETV treatment at both doses are comparable to the frequencies reported during LVD treatment.

 

 


Poster 1154

EFFECT OF SWITCHING TO TENOFOVIR WITH EMTRICITABINE OR LAMIVUDINE IN PATIENTS WITH CHRONIC HEPATITIS B FAILING TO RESPOND TO AN ADEFOVIR-CONTAINING REGIMEN

Jennifer L. Lucas, Damaris Ciprian Carriero, Alison Juriel, David Jaffe, Douglas T. Dieterich, Mount Sinai Medical Center, New York, NY.

 

 

Background:

Lamivudine (3TC) and Adefovir (ADV) are the only licensed drugs for the treatment of chronic Hepatitis B (HBV). However, long-term use of 3TC is limited by resistance. ADV has a very low rate of resistance, but there have been recent reports describing a resistance mutation (N 236 T). Tenofovir (TDF) and Emtricitabine (FTC), both FDA-approved for HIV treatment, also show potent activity against HBV.

 

Aim:

To evaluate the effect of switching to TDF + (FTC or 3TC) in HIV seronegative patients (pts) with chronic HBV failing to achieve undetectable HBV DNA levels on ADV.

 

Methods:

Seven HIV seronegative patients with detectable HBV DNA levels on ADF were switched to TDF + (FTC or 3TC). Variables collected retrospectively and prospectively included demographics, HBV serology, HBV DNA, AST/ALT, serum creatinine, and length of ADF, TDF + (FTC or 3TC) therapy.

 

Results:

Six patients were HB e Ag positive, one was HB e Ag negative, baseline (bl) AST/ALT were elevated in 3/7 (43%), and median bl HBV DNA was 265,000 IU/ml (8,740 to > 185 x 106). Mean age was 33 yrs (23 to 45), 5/7 (71%) were male and 4/7 (57%) were Asian. HBV treatment at bl was ADV + 3TC (4) and ADV alone (3). Median length of therapy with ADV was 11 months (4 to 19). Six patients were switched to TDF + FTC and one pt was treated with TDF + 3TC. Outcome of TDF + (FTC or 3TC) therapy to date indicate that 1/3 patients normalized both AST and ALT; 3/7 (43%) achieved undetectable HBV DNA levels (< 100 IU/ml on ultraquantitative PCR); and 4/7 (57%) sustained a = 2 log 10 drop in viral load. No patients have undergone HBeAg to HbeAb seroconversion. All patients remain on TDF + (FTC or 3TC). Median length of therapy is 5 months (3 to 6) and there is no evidence of renal toxicity to date.

 

Conclusions:

These results indicate that after a median of 5 months on TDF + (FTC or 3TC), 3/7 (43%) achieved an undetectable HBV DNA level and 4/7 (57%) sustained drop of = 2 log 10 in serum HBV DNA. TDF was well-tolerated with no adverse events noted. We believe that these results indicate that TDF + (FTC or 3TC) may be efficacious in the setting of suboptimal response to ADV in HBV monoinfection. We will continue to follow this cohort to evaluate long-term HBV suppression.

 

 


Poster 1155

RANDOMIZED, DOUBLE-BLIND STUDY COMPARING ADEFOVIR DIPIVOXIL (ADV) PLUS EMTRICITABINE (FTC) COMBINATION THERAPY VERSUS ADV ALONE IN HBEAG (+) CHRONIC HEPATITIS B: EFFICACY AND MECHANISMS OF TREATMENT RESPONSE

George K. K. Lau, Queen Mary Hospital, Hong Kong SAR, China; Helen Cooksley, University College London, London, United Kingdom; Ruy M. Ribiero, Kimberly A. Powers, Los Alamos National Laboratory, Los Alamos, NM; Scott Bowden, Victorian Infectious Diseases Reference Laboratory, Victoria, Australia; Herve Mommeja-Marin, Jeff Sorbel, Elsa Mondou, Franck Rousseau, Gilead Sciences, Inc., Durham, NC; Sharon Lewin, The University of Melbourne, Victoria, Australia; Alan S. Perelson, Los Alamos National Laboratory, Los Alamos, NM; Stephen Locarnini, Victorian Infectious Diseases Reference Laboratory, Victoria, Australia; Nikolai V. Naoumov, University College London, London, United Kingdom.

 

Background:

Improvement of treatment response in chronic hepatitis B requires new antiviral regimens and better understanding of viral and host factors associated with sustained control of HBV replication.

 

Aims:

i) To compare the efficacy of a new combination therapy with ADV plus FTC versus ADV alone; ii) To examine early HBV kinetics and virus-specific T-cell reactivity to gain understanding of the mechanisms of successful HBV control.

 

Methods:

Thirty treatment naïve, HBeAg (+) patients (HBV genotype B - 9; genotype C - 21) with ALT> 1.3xULN were randomized to receive ADV 10 mg + FTC 200 mg qd (Group A, n=14) or ADV 10 mg + placebo qd (Group B, n=16) for 48 weeks. Blood samples were collected at Day 0, 1, 3, 5, 7, 9, 11, 14; Week 3, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48. HBV DNA was quantitated by Cobas amplicor and by molecular beacons assay (range 3x102 to 109 copies/mL). Mathematical modeling was employed to determine the patterns of viral kinetics (Beacon). HBV-specific CD4 and CD8+ T-cells were enumerated by IFN? ELISpot assays and tetramer staining for CD8+ T-cells.

 

Results:

The combination (ADV+FTC) therapy showed greater antiviral activity compared to ADV alone: median log10 reduction of viremia at treatment week 28 (TW28) was 5.04 vs 3.2 (p=0.04); at TW48 5.3 vs. -3.4 (p=0.13), respectively. HBeAg seroconversion occurred in 3 patients - 2 in Group A and 1 in Group B. Modeling of early HBV kinetics (baseline to TW12) revealed a 2-phase decay in most patients with the 1st phase t1/2 from 0.5 day to 3.3 days (mean: 1.4 ± 0.7 day) and the 2nd phase t1/2 from 3.6 to 112 days (mean: 24.4 ± 22.6 days). The infected cell loss rate (d) was significantly higher in the combination arm (d =0.07 day-1) vs monotherapy ( (d =0.04 day-1 p=0.04). Early HBV kinetics identified two subsets - patients who cleared the virus (<300 copies/mL) by TW12 (fast responders) and those who did not (slow responders). Fast responders had a larger d (0.07 day-1) than slow responders (d =0.03 day-1, p=0.006). Patients on combination treatment were more likely to be fast responders than those on monotherapy (11/14 vs 5/16,p=0.01). A fast response was associated with enhanced T-cell reactivity (for T-cell response to HBcAg p=0.05; and to HBsAg p=0.03). All 3 cases with HBeAg seroconversion were fast responders. No mutations or safety concerns were observed.

 

Conclusions:

i) ADV plus FTC combination therapy shows faster and greater HBV suppression in comparison with ADV alone; ii) this faster suppression is reflected in the second phase viral kinetic parameters; and iii) early HBV suppression during therapy is associated with enhanced antiviral immunity.

 

 


Poster 1156

HBV-DNA AND INFECTED HEPATOCYTES DYNAMICS IN HBeG-NEGATIVE CHRONIC HEPATITIS B PATIENTS TREATED WITH PEGINTERFERON ALFA-2A (40KD) (PEGASYS(r)), LAMIVUDINE, OR PEGASYS(r) PLUS LAMIVUDINE COMBINATION THERAPY USING A NEW BIO-MATHEMATICAL MODEL

Piero Colombatto, Pisa University Hospital, Pisa, Italy; Ranieri Bizzarri, Scuola Normale Superiore of Pisa, Pisa, Italy; Luigi Civitano, Filippo Oliveri, Pisa University Hospital, Pisa, Italy; Ferruccio Bonino, IRCCS Ospedale Maggiore, Milano, Italy; G. Germanidis, Papageorgiou General Hospital, Thessalonika, Greece; Patrizia Farci, University of Cagliari, Cagliari, Italy; G. Kitis, G. Papanikolau Hospital, Thessalonika, Greece; Stefanos Hadziannis, Henry Dunant Hospital, Athens, Greece; Somesh Choudhury, Roche, Nutely, CT; Ronald Gieschke, F. Zahm, Roche, Basel, Switzerland; Maurizia Brunetto, Pisa University Hospital, Pisa, Italy.

 

 

Background:

Standard biphasic models are suitable to describe the HBV-DNA decline in the sera of a significant number of HBeAg-positive chronic hepatitis B (CHB) patients (patients) during the first weeks of antiviral therapy, whereas modeling data are lacking in HBeAg-negative patients.

 

Aims/Methods:

To analyze viremia kinetics in these patients, a new multiphasic model describing virus and infected-hepatocyte dynamics during the whole therapy was applied in 72 patients of PEGASYS(r) Phase III trial who received 48 weeks of either lamivudine (Group A; n=25); PEGASYS(r) 180 µg qw plus lamivudine (Group B; n=23) or PEGASYS(r) 180 µg qw plus placebo (Group C; n=24). Decay constants for the different phases of viremia decline and infected hepatocytes declines were computed using HBV-DNA and ALT measures at days 0, 0.3, 1, 2, 4, 5, 7, 14, 21, 28, 35, 42, 56, 84 days and every 6 weeks thereafter.

 

Results:

The model was able to describe HBV-DNA decline in 24 (96%) patients of group A, 20 (87%) patients of group B and 19 (79%) patients of group C. During the 1st month in most group A and B patients an intermediate phase (decay constant Ф) was observed between the 1st rapid phase (circulating virions clearance upon block of virus production, λ) and the last phase (HBV-DNA decline by infected hepatocyte clearance δ). Only 3 (12.5%) patients of group A and 1 (5%) pt of group B had biphasic profiles. Group C patients had biphasic profile (decay constants: ώ  and δ) except 2 (10.5%) who had monophasic decrease. Means ± SD of HBV-DNA decay constants (day-1), infected cell % at baseline (I0) and its Log10 reduction at the end of therapy (Ieot) are shown:

 

Parameter

δ

I0

λ

Ф

Log10 Ieot/I0

Therapy

Average

0.077

20.0

1.91

0.34

-3.31

Lam (A)

Std

0.038

18.4

0.59

0.13

1.18

Average

0.091

20.9

2.79

0.44

-5.02

PEG+Lam (B)

Std

0.037

15.7

1.24

0.15

2.60

A vs B p value:

ns

ns

0.005

0.023

0.028

ANOVA

 

δ

I0

λ

ω

Log10 Ieot/I0

 

Average

0.105

18.6

 

0.70

-2.91

PEG (C)

Std

0.059

20.2

 

0.55

1.82

A vs B vs C p value:

ns

ns

NA

NA

0.017

 

A vs C p value:

0.065

ns

NA

NA

Ns

ANOVA

B vs C p value:

ns

ns

NA

NA

0.026

 

 

Conclusion:

In conclusion HBV dynamics during PEGASYS(r) monotherapy are significantly different from those of lamivudine monotherapy. PEGASYS(r) may increase the early-phase HBV-DNA decay when combined with lamivudine and contribute to achieve a lower number of infected cells at the end of therapy.

 

 


Poster 1177

LAMIVUDINE TREATMENT FOR ACUTE SEVERE HEPATITIS B

Hemda Schmilovitz-Weiss, Golda Campus,Rabin Medical Center, Petach-Tikva, Israel; Ziv Ben-Ari, Beilinson Campus,Rabin Medical Center, Petach-Tikva, Israel; Emanuel Sikuler, Soroka Medical Center, Be'er Sheva, Israel; Eli Zuckerman, Bnei Zion Medical Center, Haifa, Israel; Wisam Sbeit, Western Galilee Hospital, Nahariya, Israel; Zvi Ackerman, Hadassah University Hospital, Mount Scopus, Jerusalem, Israel; Rifat Safadi, Hadassah University Medical Center, Jerusalem, Israel; Yoav Lurie, Guy Rosner, Tel Aviv Sourasky Medical Center, Tel-Aviv, Israel; Ran Tur-Kaspa, Beilinson Campus,Rabin Medical Center, Petach-Tikva, Israel; Ron Reshef, Western Galilee Hospital, Nahariya, Israel.

 

 

Background/Aim:

The aim of the study was to evaluate the safety and efficacy of lamivudine for the treatment of acute severe hepatitis B virus (HBV) infection in immunocompetent adults.

 

Methods:

Fifteen patients (10 men, 5 women, mean age 34.3±7.3 years) with severe acute HBV infection were treated with lamivudine 100 mg daily for 3-6 months, starting 3-12 weeks after onset of infection. Prior to treatment, 5 patients had grade 1-4 encephalopathy; all patients had severe coagulopathy (mean INR was 4.5±6.4), and all patients had evidence of severe hepatocyte lysis (mean alanine aminotransferase 3738±1659 U/L, and mean total serum bilirubin 18±6.8 mg/dl). All patients had evidence of highly replicative HBV(mean HBV DNA 13.5x106 ± 11x106 copies/ml).

 

Results:

Two patients in whom lamivudine therapy was delayed developed fulminant hepatitis and underwent urgent liver transplantation. (One died of vascular complications 1 month later). Thirteen patients (86.6%) responded to treatment . Encephalopathy disappeared within 3 days of treatment and coagulopathy improved within 1 week. Serum HBV DNA was undetectable (by polymerase chain reaction) within 4 weeks, and serum liver enzyme levels normalized within 8 weeks. The 11 patients who were serum HBeAg-positive before treatment seroconverted, and HBeAb developed within 12 weeks in 9 of them; HBsAg was undetectable in all 11 tested patients, and protective titer of HBsAb developed within 12-16 weeks in 9 of them. Therapy was well tolerated in all cases.

 

Conclusion:

These data indicate that lamivudine induces a prompt clinical, biochemical, serological and virological response in immunocompetent patients with de novo HBV infection. Lamivudine may prevent the progression of severe acute disease to fulminant or chronic hepatitis and should be considered for use in selected patients. A large randomized prospective study is needed.

 

 


Poster#:1158

BETTER SAFETY AND QUALITY OF LIFE IN PATIENTS WITH CHRONIC HEPATITIS B THAN IN PATIENTS WITH CHRONIC HEPATITIS C WHEN TREATED WITH PEGINTERFERON ALFA-2A (40KD) (PEGASYS(r)) MONOTHERAPY

Presentation Time:

11/2/2004 10:00:00 AM

Author Block:

Patrick Marcellin, Hôpital Beaujon, Clichy, France; George Lau, Queen Mary Hospital, Hong Kong, Hong Kong Special Administrative Region of China; Teerha Piratvisuth, Songklanakarin Hospital, Songkla, Thailand; Kang Xian Luo, Nangfang Hospital, Guangzhou, China; Ferrucio Bonino, Scientific Direction of IRCCS Ospedale Maggiore, Milan, Italy; Satawat Thongasawat, Chiang Mai University, Chiang Mai, Thailand; Graham Cooksley, Royal Brisbane Hospital, Herston, Australia; Stephanos Hadziyannis, H Dynant Hospital, Athens, Greece; Anuchit Chutaputti, Pramongkutklao Hospital, Bangkok, Thailand; Zhi-Meng Lu, Ruijin Hospital, Shanghai, China; Michael Fried, University of North Carolina, Chapel Hill, NC; Wan Cheng Chow, Singapore General Hospital, Singapore, Singapore; Cihan Yurdaydin, University of Ankara, Ankara, Turkey; Thomas Berg, Charité Humboldt Universität zu Berlin, Berlin, Germany; Nigel Pluck, Roche, Welwyn, United Kingdom.

 

 

Background and Objective:

The safety profile of peginterferon alfa-2a (40KD) (PEGASYS(r)) and its effects on quality of life (QoL) have been well documented in previous studies of chronic hepatitis C (CHC) [Fried et al, N Engl J Med 2002; Rasenack et al, Pharmacoeconomics 2003]. Recently, the safety and tolerability of peginterferon alfa-2a, and its effects on QoL, have been assessed in patients with HBeAg-negative chronic hepatitis B (CHB) or HBeAg-positive CHB enrolled in two, randomized, multicentre studies of peginterferon alfa-2a with and without lamivudine versus lamivudine alone.

 

Methods:

Patients with HBeAg-negative CHB (n=177) or HBeAg-positive CHB (n=271) received peginterferon alfa-2a (40KD) (PEGASYS(r)) 180 µg once weekly + placebo once daily for 48 weeks, and were followed up for a 24-week treatment-free period. Safety was assessed at baseline and throughout treatment and follow-up. QoL was measured using the SF-36 questionnaire which was completed by patients at baseline and weeks 12, 24, 48 and 72. Data from the two CHB studies were compared with pooled peginterferon alfa-2a monotherapy data from three studies in CHC, which used the same therapeutic schedule and identical methodology for assessing safety and QoL.

 

Results:

Adverse events were qualitatively similar, but the frequency of interferon-related adverse events was generally lower with peginterferon alfa-2a in patients with CHB than previously reported for patients with CHC with the exception of pyrexia (see table). The difference between the rate of adverse events seen in patients with CHB and CHC was not affected by population differences and was the same in Asians and Caucasians. Importantly, the incidence of depression and the rates of withdrawal were much lower in patients with CHB than CHC. Similarly, patients with CHB had better QoL during treatment than patients with CHC and these differences were clinically significant (>3 points) for most of the SF-36 categories.

 

Conclusions:

Peginterferon alfa-2a (40KD) (PEGASYS(r)) monotherapy appears to be better tolerated in patients with CHB than in patients with CHC. This improved tolerability was also reflected in the QoL results and lower withdrawal rates. It is currently unclear whether the markedly lower rates of depression observed in patients with CHB were due to viral-specific factors and/or host susceptibility.

 

 

PEGASYS® in HBeAg-negative CHB (n=177)

PEGASYS® in HBeAg-positive CHB (n=271)

PEGASYS® in CHC (n=575)

Adverse event

 

 

 

Pyrexia

59 [52-67]

49 [43-55]

38 [34-42]

Fatigue

42 [34-49]

37 [31-42]

51 [50-58]

Myalgia

27 [20-33]

26 [20-31]

43 [39-47]

Headache

24 [18-31]

28 [23-34]

55 [51-59]

Decreased appetite

18 [12-23]

6 [3-9]

15 [12-19]

Arthralgia

15 [10-21]

9 [5-12]

29 [22-35] §

Alopecia

14 [8-19]

20 [15-25]

23 [20-27]

Diarrhoea

11 [6-16]

7 [4-11]

21 [16-25] ‡

Injection site reaction

6 [2-9]

11 [7-15]

15 [11-19] ‡

Depression

3 [0-6]

6 [3-8]

19 [16-22]

Withdrawals

 

 

 

For any reason

8 [4-12]

6 [3-8]

19 [16-23]

For adverse events

7 [3-11]

3 [1-5]

8 [5-11] ‡

For lab abnormality

1

2

<1‡

‡ (n=351)
§ (n=224)
Included in this table are adverse events which occurred with an incidence of >10% in any study arm in one of the CHB studies and depression.

 

 


Poster 1159

HBV VIRAL DYNAMICS DURING TREATMENT OF NAIVE PATIENTS WITH HBEAG(-) CHRONIC HEPATITIS B WITH PEGYLATED INTERFERON ALPHA 2B ALONE OR IN COMBINATION WITH LAMIVUDINE

Vana Sypsa, Athens University Medical School, Athens, Greece; Konstantinos Mimidis, Alexandroupoli Hospital, Alexandroupoli, Greece; Nicholas C. Tassopoulos, Western Attica Hospital, Athens, Greece; Dimitrios Chrysages, Loimodon Hospital, Thessaloniki, Greece; Themistoklis Vassiliadis, Hippokration Hospital, Thessaloniki, Greece; Antonios Moulakakis, Hippokration Hospital, Athens, Greece; Maria Raptopoulou, Aristotelion University, Thessaloniki, Greece; Caterina Haida, Angelos Hatzakis, Athens Univeristy Medical School, Athens, Greece.

 

 

Background/Aim:

A study was conducted to assess hepatitis B virus (HBV) viral dynamics and the antiviral effect of pegylated interferon a-2b (PEG-IFN) alone or in combination with lamivudine (LAM) in chronic HBV patients with HBeAg(-) and HBV DNA>105copies/ml.

 

Methods:

Forty-four patients were treated as follows: Group A: LAM 100 mg QD x 48 weeks (N=8), Group B: PEG-IFN 100 mcg QW x 48 weeks (N=9), Group C: PEG-IFN 200 mcg QW x 4 weeks followed by PEG-IFN 100 mcg QW x 44 weeks (N=9), Group D: PEG-IFN 100 mcg QW + LAM 100 mg QD x 48 weeks (N=9) and Group E: PEG-IFN 200 mcg QW + LAM 100 mg QD x 4 weeks followed by PEG-IFN 100 mcg QW + LAM 100 mg QD x 44 weeks (N=9). Blood samples were collected at hours 0,4,8,16,20,24,28, 36,48, then at days 4,5,7,9,11,12,14,21,28,42,56 and weeks 12,16,20,24, 28,32,36,40,44,48,72. HBV DNA was tested by a real time PCR method (1). Non-linear regression was used to fit viral load data of the first 12 weeks for each patient separately and obtain estimates of virion clearance rate, death rate of infected cells (d) and antiviral efficacy (e) (2). A mixed-effects model was used to assess the effect of covariates.

 

Results:

Viral decay was biphasic. Variations in the first phase slope were explained by differences in virion clearance rate and treatment efficacy. The median half-life of free virus was 8.5 hours (range: 4.9-36.5 hours). The median e was lower in PEG-IFN 100 mcg (59.1%, p=0.001) and PEG-IFN 200 mcg (60.0%, p=0.002) compared to LAM (97.8%). The efficacy of PEG-IFN 100 mcg + LAM and PEG-IFN 200 mcg + LAM was similar to that of LAM (96.7% and 96.8%, respectively). There was no effect of patients' weight on e or d. The median half-life of infected cells (second phase decline) was estimated 8.7 days. Patients with higher ALT levels at baseline showed a faster second phase decline (p=0.018).

 

Conclusion:

In conclusion, 1) the estimated half-life of HBV virions (8.5 hours) is considerably shorter than that reported in previous studies where estimates range from 16-55 hours, 2) the combination of PEG-IFN 100-200 mcg and lamivudine was not found to have superior efficacy or improved decay rate of infected cells compared to lamivudine monotherapy, 3) baseline ALT levels were positively associated with the death rate of infected cells.

 

 


Poster 1160

DETECTION OF PREDOMINANT YMDD MUTANTS AT THE STAGE OF VIRAL DNA NEGATIVITY FOR THE PROGNOSIS OF VIRAL DNA BREAKTHROUGH

Wangdon Yoo, Soo-Ok Kim, Sun Pyo Hong, Hyun Jae Chung, Mi Sun Jee, GeneMatrix Inc., Seoul, Republic of Korea; Jong Eun Yeon, Kwan Soo Byun, Chang Hong Lee, Korea University College of Medicine, Seoul, Republic of Korea.

 

 

Background:

YMDD mutants with or without compensatory mutations are found in chronic hepatitis B patients showing the phenotypic resistance during lamivudine therapy. But it is uncertain that the detection of YMDD mutation is absolutely associated with accompanying HBV viral DNA breakthrough.

 

Aims:

In the present study we determined if predominant presence of YMDD mutants at the stage of viral DNA negativity could be prognosis marker for occurrence of viral DNA breakthrough.

 

Methods:

We retrospectively analyzed YMDD genotypes in 740 consecutive samples collected from 116 patients throughout lamivudine treatment using a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)-based genotyping assay, termed Restriction Fragment Mass Polymorphism (RFMP). The RFMP exploits differences in molecular masses between wild type and variant bases of rtM204V/I. The capacity of the RFMP assay to show relative abundance among viral species enables us to monitor YMDD genotype dynamics in the longitudinal samples more effectively.

 

Results:

The result demonstrated that YMDD mutants can be present throughout a course of lamivudine therapy irrespective of occurrence of viral DNA breakthrough, indicating that a mere detection of YMDD mutants could not always predict the viral DNA breakthrough. When mutant predominance was defined by 5-times higher amount of mutant compared to wild type, a significantly stronger association could be found between detection of YMDD mutant predominance and the following viral DNA breakthrough event.

 

Conclusion:

In conclusion our study showed that detection of predominant YMDD mutants rather than of mutant presence has a more prognostic value for viral DNA breakthrough. A close and periodical testing should be useful to detect predominant YMDD mutants for monitoring drug resistance as it develops, enabling early intervention and prevention.

 

 

 


Poster 1161

EARLY VIRAL KINETICS DURING THERAPY WITH LAMIVUDINE PLUS ADEFOVIR DIPIVOXIL IN PATIENTS WITH LAMIVUDINE RESISTANT CHRONIC HEPATITIS B

Ulrike Mihm, Barbara Gärtner, University Hospital Homburg/Saar, Homburg /Saar, Germany; Dominik Faust, University Hospital Frankfurt, Frankfurt am Main, Germany; Christoph Sarrazin, Stefan Zeuzem, University Hospital Homburg/Saar, Homburg /Saar, Germany; Eva Herrmann, University Darmstadt, Darmstadt, Germany.

 

 

Background:

Combination therapy with lamivudine and adefovir dipivoxil was reported to be as effective as monotherapy with adefovir in terms of hepatitis B virus (HBV) DNA suppression after 52 weeks in patients with lamivudine resistant hepatitis B.

 

Aims:

However, mathematical modelling of initial viral kinetics and calculation of kinetic parameters have not been performed so far in patients with lamivudine resistant chronic hepatitis B treated with combination therapy of lamivudine and adefovir dipivoxil.

 

Methods:

In 8 patients (3 HBeAg negative, 5 HBeAg positive) with lamivudine resistant chronic hepatitis B (ALT levels = 1.2 x upper limit of normal, HBV DNA = 106 c/mL despite ongoing lamivudine for at least 6 months) adefovir dipivoxil (5-10 mg/d) was added to ongoing lamivudine (100-150 mg/d). HBV DNA was quantified by PCR from frozen serum taken daily in the first week of combination therapy, then weekly in the first 4 weeks and then monthly. Viral decay during the first 72 days of combination therapy was described by a biphasic model to determine the efficacy e of blocking viral production, the clearance of free virus c and the loss of infected cells d. Viral kinetic constants were compared to those reported by Tsiang et al. (Hepatology 1999; 29:1863-1869) for monotherapy with adefovir dipivoxil 30 mg/d in patients who had not received anti-HBV therapy within 6 months.

 

Results:

The median e was 0.98 (range 0.86-0.997). Median c was 0.71/d (range 0.57-1.1/d) and median d was 0.07/d (range 0.007-0.15/d) with a median half life of infected cells of 9.5 days (range 4.5-92.7 days). No association was found in this study between viral kinetic and baseline parameters (HBeAg, ALT, viral load, genotype, weight) or treatment response (HBV DNA, HBeAg, ALT at months 3 and 6 of combination therapy). When compared with the data of Tsiang et al. (median e 0.996, range 0.98-0.999; median c 0.6/d, range 0.4-0.9/d; median d 0.04/d, range 0.02-0.06/d), e was significantly lower (p=0.026) and d was significantly higher (p=0.008) in this study whereas c did not differ significantly between both studies.

 

Conclusion:

Although it has recently been reported that monotherapy with adefovir dipivoxil was as effective in reducing HBV DNA as adefovir dipivoxil added to ongoing lamivudine in patients with lamivudine resistant chronic hepatitis B, mathematical modelling of early viral kinetics revealed significantly higher rates for loss of infected cells in adefovir-lamivudine combination therapy compared with those in patients receiving adefovir monotherapy who had not been treated within 6 months prior to adefovir therapy.

 

 


Poster 1162

A NOVEL LINE PROBE ASSAY (INNO-LIPA HBV DR V2) FOR DETECTING DRUG RESISTANCE IN HEPATITIS B VIRUS INFECTED PATIENTS DURING LAMIVUDINE AND ADEFOVIR DIPIVOXIL THERAPY

Joke Doutreloigne, Evelien Libbrecht, Els Van Assche, Erwin Sablon, Innogenetics NV, Gent, Belgium.

 

 

Background:

Since the introduction of antiviral compounds such as lamivudine and adefovir dipivoxil in the treatment schedules of chronic hepatitis B virus-infected patients, the accumulation of a variety of mutations in the HBV polymerase gene has been observed to varying degrees depending on the specific drug. These mutations are generally considered as the cause of viral non-responsiveness and treatment failure.

 

Aims:

The detection of mutations at the earliest time point is therefore of clinical importance.

 

Methods:

A single-round PCR encompassing the HBV polymerase domains A-F was developed to allow amplification of samples from as low as 1000 copies/mL This PCR protocol was used to amplify the HBV polymerase region from HBV strains isolated from treated and untreated patients and covering all known genotypes (A-H). A set of highly specific oligonucleotide probes covering confirmed and suspected drug resistance and associated compensatory mutations were selected and applied as 17 lines on a membrane strip. The different lines covered both wild-type and mutant motifs for the lamivudine resistance and compensatory mutations L80V/I, V/G173L, L180M, and M204V/I/S, and for the adefovir dipivoxil resistance mutations A181T/V and N236T. The prototype INNO-LiPA HBV DR v2 strips reveal the amino acids at these six codon positions simultaneously for each strain.

 

Results:

Clinical samples obtained from patients infected all known HBV genotypes (A-H), both untreated and treated with the nucleoside analogues lamivudine and adefovir dipivoxil were analyzed with the INNO-LiPA HBV DR v2 and compared to sequence analysis of the corresponding open reading frame. Due to the nature of the reverse hybridization methodology of the LiPA, the INNO-LiPA HBV DR v2 is capable of detecting emerging HBV treatment-resistant viruses earlier than sequencing, and before an increase in viral load is observed.

 

Conclusion:

The INNO-LiPA HBV DR v2 proved to be a superior method for the identification of additional wild-type and mutant minority species. The assay detects the complex quasispecies nature of HBV and can help to unravel the dynamics of emerging HBV resistance during lamivudine and adefovir dipivoxil therapy. Similarly to its predecessor, this assay should prove to be a useful asset in monitoring and tailoring of current antiviral treatment for patients with chronic hepatitis B.

 

 


Poster 1163

CLEVUDINE RE-ADMINISTERED TO THE PATIENTS WITH PREVIOUS 12-WEEKS CLEVUDINE EXPERIENCE IS AS ACTIVE AS TO THE NAIVE PATIENTS IN TERMS OF VIROLOGICAL AND BIOCHEMICAL RESPONSES

Young-Hwa Chung, Asan Medical Center, Seoul, Republic of Korea; Kwan Sik Lee, Yeonsei University Hospital, Seoul, Republic of Korea; Kwan Soo Byun, Korea University Hospital, Seoul, Republic of Korea; Seung Woon Paik, Samsung Medical Center, Seoul, Republic of Korea; Joon-Yeol Han, The Catholic University of Korea, St. Mary's Hospital, Seoul, Republic of Korea; Kwon Yoo, Ewha Woman's University Hospital, Seoul, Republic of Korea; Soo Geum Hwang, Bukwang Pharm.Co., Ltd., Seoul, Republic of Korea; Byung Chul Yoo, Samsung Medical Center, Seoul, Republic of Korea; Hyo-Suk Lee, Seoul National University Hospital, Seoul, Republic of Korea.

 

Background:

Clevudine(CLV, L-FMAU) is a pyrimidine analogue that is a potent inhibitor of HBV replication in vitro. In woodchucks and in a phase I/II clinical study (L-FMAU-102), CLV produced a potent and durable post-treatment viral suppression. In a phase II clinical study (L-FMAU-201), CLV showed potent antiviral activities during 12-week dosing period and prolonged antiviral activities after discontinuation of treatment, which was associated with sustained normalization of ALT.

 

Aim:

The primary objectives of this study were to evaluate the safety and efficacy of CLV, when re-administered to the patients who had been treated with CLV in L-FMAU-201 study.

 

Methods:

CLV was re-administered 30mg/day, orally for 24 weeks to the patients who had been previously treated for 12 weeks, and then antiviral activities and the safety were evaluated. The study was conducted at a total of 7 sites in South Korea. Eligible patients were chronic hepatitis B patients who were treated with CLV in L-FMAU-201 study, whose HBV DNA were not negative (below 4,700 copies/mL) consecutively at the last 2 visits. A total of 50 patients were enrolled and among them, 17 patients have completed 24-week treatment period, the data of which were analyzed for this interim report.

 

Results:

Median HBV DNA level at baseline was 7.12 log10 copies/mL, and the median changes in HBV DNA levels from baseline were -3.13, -3.07 log10 copies/mL, at week 12 and week 24, respectively. In contrast, the rates patients with HBV DNA levels below the lower limit of detection of Supersensitive Digene II assay (LOD, 4.7 × 103 copies/mL) were as high as 71% and 76% at week 12 and 24, respectively. The rate of patients with HBV DNA levels below LOD in Amplicor PCR assay (400 copies/mL) at week 12 and 24 were as remarkable as 35% and 65% respectively. Normalization rates of alanine transaminase (ALT) level at week 12 and 24 were 65% and 71%, respectively. No serious or severe adverse events relevant to CLV were reported during the study and no adverse events led to treatment discontinuation.

 

Conclusion:

CLV re-administered is as active to the patients with previous 12- week CLV experience as to the naïve patients in terms of potent antiviral activity and normalization rates of ALT.

 

 


Poster 1164

EFFECT OF LAMIVUDINE THERAPY ON THE SERUM COVALENTLY CLOSED CIRCULAR DNA OF CHRONIC HEPATITIS B INFECTION

MF Yuen, DKH Wong, SSM Sum, HJ Yuan, JCH Yuen, AOO Chan, BCY Wong, CL Lai, The University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China.

 

 

Background:

In a recent study we demonstrated that serum covalently closed circular (ccc) hepatitis B virus (HBV) DNA is present in 93% of HBeAg-positive patients and that it correlates well with intrahepatic cccDNA (r=0.52, p=0.004)(Wong DKH et al Hepatology 2004 in press). Serum cccDNA is good marker for intrahepatic cccDNA and has the advantage of easy monitoring. The effect of lamivudine on ccc DNA in HBV infection is unknown.

 

Aim:

To determine the effect of one-year lamivudine treatment on serum cccDNA level.

 

Methods:

Total HBV DNA and cccDNA levels at baseline, week 24 and 52 were measured in 82 lamivudine-treated patients, 17 of whom acted as controls when they received placebo in the first year.

 

Results:

There was a significant reduction in the cccDNA levels from baseline (median 3.0 X 106 copies/ml) to week 24 (33,476 copies/ml) and week 52 (48,694 copies/ml) (p<0.001 for both) in lamivudine-treated patients. The median reduction in serum cccDNA level at week 24 and 52 were 2.21 and 2.12 logs respectively, which were significantly greater than those of patients taking placebo (0.31 log, p<0.001; 0.2 log, p<0.001 respectively). Fifteen patients (18.3%) developed YMDD mutations by week 52. The median logarithmic reduction of cccDNA at week 52 was significantly less in patients with YMDD mutations compared to patients without YMDD mutations (0.8 vs. 2.35 logs respectively, p<0.001). Compared to patients without YMDD mutations at week 52, patients with YMDD mutations had a significantly lesser median logarithmic reduction of total HBV DNA and cccDNA levels at week 24 [total HBV DNA: 4.44 vs. 3.65 respectively, p=0.02; cccDNA: 2.27 vs. 1.65 respectively, p=0.016)].

 

Conclusions:

One-year lamivudine treatment decreased the serum cccDNA level by 2 logs. The chance of emergence of YMDD mutations at week 52 was related to the magnitude of viral suppression at week 24.

 

 


Poster 1165

CONTINUED EFFICACY AND SAFETY OF ADEFOVIR DIPIVOXIL IN CHRONIC HEPATITIS B PATIENTS WITH LAMIVUDINE RESISTANT HBV: 1 YEAR RESULTS

María Buti, Rafael Esteban, H. Vall D´Hebron, Barcelona, Spain; P Escartin, H. Puerta de Hierro, Madrid, Spain; J.L. Calleja, H.Puerta de Hierro, Madrid, Spain; J. Enriquez, H. de Santa Creu i San Pau, Barcelona, Spain; F Pons, J. Crespo, H.U. Marques de Valdecilla, Santander, Spain; M.G. Bengoechea, H.Donostia, San Sebastian, Spain; M. Prieto, H. La Fe, Valencia, Spain; T. Casanova, C.S.U.de Bellvitge, Barcelona, Spain; J.G. Samaniego, H. Carlos III, Madrid, Spain; M. Miras, H. Virgen de la Arixaca, Murcia, Spain; F.P Roldan, H.La Mancha Centro, Ciudad Real, Spain; M. Pelaez, H.Torrecardenas, Almeria, Spain; E. Fraga, H.Reina Sofia, Cordoba, Spain; V Moreira, H.Ramon y Cajal, Madrid, Spain; P.G. Carro, H.La Mancha Centro, Ciudad Real, Spain; G. Alonso, H.Mostoles, Mostoles, Spain; R. Barcena, H.Ramon y Cajal, Madrid, Spain; I. Fernandez, H. 12 de Octubre, Madrid, Spain; L. Garcia Buey, R. Moreno, H. de La Princesa, Madrid, Spain; A. Olveira, H. La Paz, Madrid, Spain; A Mas, Clinic i Provincial, Barcelona, Spain; M Rueda, Gilead Sciences, INC, Madrid, Spain.

 

 

Background/Aims:

Patients with chronic hepatitis B developing lamivudine resistance are at high risk of disease progression and death. To assess the efficacy and safety of Adefovir dipivoxil (ADV) therapy under compassionate use in patients with chronic hepatitis B (CHB) and lamivudine resistance.

 

Methods:

120 patients with CHB and phenotypic lamivudine resistance defined as detectable HBV DNA and elevated ALT levels were enrolled in an observational, retrospective, multicenter study performed in Spain. All patients had CHB or cirrhosis and were treated with ADV 10 mg, orally daily for at least 48 weeks. HBV DNA levels were determined at baseline, weeks 24 and 48. Virological response (VR) was defined as undetectable HBV DNA by hybridization or < 105 copies/ml by PCR assay.

 

Results:

Of the 120 patients enrolled, 82.5% were males, mean age 48.36 years (range 16-75 years), mean ALT levels 150.56 IU/L. Patients were stratified into two cohorts: HBeAg + (N=45) and HBeAg - (N=75). The majority 92.5%, received ADV monotherapy and only 7.5% were treated with ADV and lamivudine during the first three months. At week 24 of ADV therapy, a VR was observed in 40% of cases and increased to 46% at week 48. A biochemical response was observed in 46% of cases at week 24 and in 60% at week 48. A higher proportion of HBeAg - patients achieved a VR (51% in HBeAg - group vs. 33% in HBeAg + group) but the difference was not statistically significant. HBeAg seroconversion occurred in 4.5% of cases at week 24 and increased to 12.5% at week 48. No patient lost HBsAg. Two patients died during ADV therapy from liver disease complications (HCC), both had elevated ALT levels and detectable HBV DNA. No other serious adverse events were detected except a worsening of renal function in one case requiring ADV dose-adjustment.

 

Conclusions:

ADV is an effective and well-tolerated treatment for patients with CHB and lamivudine resistance. A continuous HBV DNA suppression was observed in almost 50% of cases during one year of ADV therapy.

 

 


Poster 1166

CLINICAL PHARMACOKINETICS OF TELBIVUDINE, A POTENT ANTIVIRAL FOR HEPATITIS B, IN SUBJECTS WITH IMPAIRED HEPATIC OR RENAL FUNCTION

Xiao Jian Zhou, Maureen Myers, George Chao, Gloria Dubuc, Nathaniel A. Brown, Idenix Pharmaceuticals, Cambridge, MA.

 

 

Background:

Telbivudine (LdT), an L-nucleoside with potent anti-HBV activity, is being evaluated in large randomized clinical trials in patients with chronic hepatitis B. Telbivudine is eliminated predominantly via renal clearance as unchanged drug, with minimal hepatic elimination.

 

Aims:

Telbivudine pharmacokinetics were investigated in subjects with impaired hepatic or renal function to assess the potential need for dose adjustment in patients with these conditions.

 

Methods:

The hepatic impairment study enrolled 24 subjects with normal (n=6) or impaired hepatic function (Child-Pugh score 5-6, 7-9 or 10-15, n=6/category). Subjects received a single dose of telbivudine 600 mg. The renal impairment study enrolled 36 subjects with normal or impaired renal function, as defined by serum creatinine clearance (CLCR). Normal: CLCR >80 mL/min, n=8. Mild, moderate or severe impairment: CLCR 50-80 mL/min, n=8; 30-49 mL/min, n=8; <30 mL/min, n=6, respectively. Six subjects with end-stage renal disease (ESRD) requiring hemodialysis were also enrolled. Subjects received a reduced dose in proportion to decreased renal function.

 

Results:

Telbivudine was well tolerated in all subjects. In subjects with impaired hepatic function, telbivudine pharmacokinetics were similar to those in normal subjects with respect to peak (Cmax) and overall drug exposure (AUC). However in patients with impaired renal function, plasma exposure of telbivudine was dependent upon the degree of impairment. Subjects with moderate or severe renal impairment treated with a single dose of telbivudine 400 or 200 mg, respectively, exhibited overall plasma exposure comparable to that seen in normal subjects, or subjects with mild renal impairment, who were given telbivudine 600 mg. Similarly, ESRD subjects receiving telbivudine 200 mg 2 hours prior to a 4-hour hemodialysis (intra-dialysis dosing) had overall plasma exposure comparable to normal patients treated with a single dose of 600 mg.

 

Conclusion:

Telbivudine pharmacokinetics were not altered by hepatic dysfunction, suggesting that dose adjustment will not be necessary. However, telbivudine clearance was reduced in subjects with impaired renal function, suggesting that adjustment of daily dose will be necessary for treatment of patients with moderate or severe renal impairment, or patients with ESRD receiving hemodialysis.

 

 

 

Hepatic Study

 

Renal Study

Degree of

 

Cmax

AUC

 

Dose

Cmax

AUC

Impairment

 

(µg/mL)

(µg/mL*h)

 

(mg)

(µg/mL)

(µg/mL*h)

None

 

2.8

20.1

 

600

3.4

26.5

Mild

 

3.5

22.5

 

600

3.2

29.2

Moderate

 

2.8

22.2

 

400

3.1

33.4

Severe

 

2.6

24.5

 

200

1.6

25.3

ESRD inter-dialysis

 

 

 

 

200

2.1

41.8

ESRD intra-dialysis

 

 

 

 

200

1.2

26.6

 

 


Poster 1167

ACTIVITY OF SYSTEMICALLY ADMINISTERED STABILIZED SIRNAS IN A MOUSE MODEL OF HBV REPLICATION

David V. Morrissey, Karin Blanchard, Lucinda Shaw, Kristi Jensen, Wendy Breen, Shawn Zinnen, Brent Dickinson, James McSwiggen, Chandra Vargeese, Keith Bowman, Chris Shaffer, Barry Polisky, Jennifer A. Lockridge, Sirna Therapeutics, Inc., Boulder, CO.

 

 

Background/Aims:

To develop synthetic siRNA molecules as therapeutic agents for systemic administration in vivo, chemical modifications were introduced into siRNAs targeted to conserved sites in HBV RNA. These modifications conferred significantly prolonged stability in human serum compared to unmodified siRNAs. Cell culture studies revealed a high degree of gene silencing following treatment with the chemically modified siRNAs.

 

Methods:

To initially assess activity of the stabilized siRNAs in vivo, an HBV vector-based model was employed in which the siRNA and the HBV vector were co-delivered via high volume tail vein injection.

 

Results:

More than a 3 log10 decrease in levels of serum HBV DNA and HBsAg, as well as liver HBV RNA, were observed in the siRNA treated groups compared to the control siRNA treated and saline groups. Furthermore, the observed decrease in serum HBV DNA was 1.5 log10 greater with a stabilized siRNA compared to an unmodified siRNA, indicating the value of chemical modification in therapeutic applications of siRNA. In subsequent experiments, standard systemic intravenous dosing of stabilized siRNA, following injection of the HBV vector, resulted in an approximately 1 log10 reduction of serum HBV DNA levels.

 

Conclusion:

These experiments establish the strong impact that siRNAs can have on the extent of HBV infection, and underscore the importance of stabilization of siRNA against nuclease degradation.

 

 


Poster 1168

HEPATITIS B VIRUS (HBV) RESPONSE TO THERAPY WITH LAMIVUDINE (LAM) AND TENOFOVIR (TFV) IN HBV/HIV CO-INFECTED PATIENTS: IMPACT OF GENOTYPE AND TREATMENT REGIMEN

Mamta K. Jain, U.T. Southwestern Medical Center, Dallas, TX; Lorraine Comanor, Independent Research Consultant, Palo Alto, CA; Calvin White, U.T. Southwestern Medical Center, Dallas, TX; Patricia Kipnis, Claudia Elkin, Kimmy Leung, Bayer Healthcare, Berkeley, CA; Veronica Cantu, Todd Morgan, Philip Keiser, William M. Lee, U.T. Southwestern Medical Center, Dallas, TX.

 

 

Background/Aims:

To determine the efficacy of different LAM/TFV treatment regimens in HBV/HIV co-infected patients and to identify factors predictive of response.

 

Methods:

Thirty HBV/HIV co-infected patients with detectable HBV DNA at baseline or year 1, detailed patient information, and stored samples from baseline, year 1, and in some cases, year 2 of therapy were retrospectively studied. Patients were divided into 3 groups based on their on-going treatment regimen: group 1 (n= 10) received LAM 150 mg bid only; group 2 (n= 8), LAM + TFV 300 mg qd simultaneously, and group 3 (n= 12), a minimum of one year of LAM followed by LAM + TFV. Thirty, 25% and 100% of patients in groups 1, 2, and 3 respectively, had received previous HIV therapy, which may have included LAM. HBV viral load (VL) was determined by the VERSANT(r) HBV 3.0 (bDNA) assay* (cut-off < 2,000 copies/mL) and HBV genotype and resistance profiles by the Trugene HBV 1.0 assay *(Bayer HealthCare LLC, Tarrytown, NY). Multivariate analysis was performed to determine factors predicting response with adjustment for differences in baseline HBV VL when examining treatment regimen and genotype.

 

Results:

Response to HBV therapy (HBV DNA below cut-off by year 1 or 2) was observed in 6/10(60%), 6/8(75%) and 6/12 (50%) of groups 1, 2, and 3 respectively. Group 2 showed the greatest average log drop at year 1, 3.2 logs, compared to 1.5 and 1.1 logs, in groups 1 and 3, respectively (p=0.05). No significant difference existed in the number of published LAM resistant mutations (LRMs) at baseline across the 3 groups, or in the response rate of those with and without LRMs. HBV VL drop was independent of baseline LRMs, CD4 count, and HIV VL. Forty-one percent (7/17) of HIV treatment experienced patients responded to HBV therapy with LAM ± TFV compared to 83% (10/12) HIV treatment naïve patients (p=0.02). Most patients (77%) were infected with HBV genotype A, the remainder with genotypes G, A/G mixture, D, or F. There was no difference in the distribution of genotypes among the 3 groups. Genotype A patients responded more frequently than did non-A genotypes (74% (17/23) vs. 14% (1/7), respectively; p=0.02).

 

Conclusions:

Simultaneous administration of LAM and TFV appears to be associated with a greater log drop in HBV DNA than that observed with either LAM alone or LAM prior to LAM + TFV in treatment of HBV/HIV co-infected patients, suggesting a possible synergy between the drugs. The sequential regimen was less effective in our clinic setting than in previous controlled trials. Most of our patients have HBV genotype A and appear more likely to respond to either therapy than those with non-A genotypes. We are continuing this study, as more data is needed to confirm these findings.

* for research use only, not for diagnostic purposes

 

 


Poster 1169

CHARACTERISTICS OF HEPATITIS B VIRUS (HBV) AND HIV CO-INFECTED PATIENTS IN FRANCE: A PROSPECTIVE MULTICENTER SURVEY

D Sène, CHU Pitié-Salpêtrière, Paris, France; S Pol, CHU Necker, Paris, France; L Piroth, CHU Dijon, Dijon, France; C Goujard, CHU Kremlin Bicêtre, Kremlin Bicêtre, France; P Dellamonica, CHU Nice, Nice, France; J Moussali, CHU Pitié-Salpêtrière, Paris, France; D Rey, Hop. Civils Strasbourg, Strasbourg, France; V Loustaud-Ratti, CHU Limoges, Limoges, France; L Alric, CHU Toulouse, Toulouse, France; M Chousterman, CHIC Créteil, Créteil, France; F Borsa-Lebas, CHU Rouen, Rouen, France; O Boucher, CHU Rennes, Rennes, France; D Séréni, CHU St Louis, Paris, France; P Cacoub, CHU Pitié-Salpêtrière, Paris, France.

 

 

Background/Aim/Methods:

A questionnaire itemizing the epidemiological, virological, biochemical, histological and therapeutic characteristics of HBV infection was sent to the French Internal Medicine, Infectious Diseases and Hepatogastroenterology Departments. A total of 406 patients with a chronic HBV infection (AgHBs+) and seen during a 4 weeks interval in the same department (September-October, 2003) have been included. HIV co-infection was evidenced in 205/389 (53 %) patients

 

Results:

1-Epidemiological features: HBV-HIV co-infected patients were more frequently male (88 % versus 66 %, p<10-3), intravenous drugs users (21 % versus 2 %, p<10-3), and clinically non-symptomatic (75 % versus 65 %, p=0.007).

2-Liver Histological Features: A liver biopsy was performed in 32 % of HIV co-infected compared to 51 % of HIV-negative patients (p<10-3). The severity of liver histological damages was comparable between HIV+ and HIV- patients with a METAVIR Fibrosis score of 2.1 ± 1.3 versus 1.96 ± 1.4, Activity score 1.5 ± 0.7 versus 1.6 ± 0.9, extensive fibrosis (F3F4) in 34 % versus 35 %, and histological cirrhosis (F4) in 21 % of each group, respectively.

3-Virological Features: Patients with HBV-HIV co-infection compared to HBV monoinfection were more frequently HBeAg+ (53% vs 28%, p<10-3) without difference according to HBV detectable viral replication (60 % versus 59 %), and had less frequently pre-core mutants (13% vs 34%, p<10-3). In a multivariate analysis, HIV-coinfection was the only factor negatively associated with a pre-core mutant (OR=0.3 [IC95%=0.1-0.6], p<10-4).

4-Therapeutic features: Patients with HBV-HIV co-infection compared to HBV monoinfection were more frequently receiving anti-HBV therapy (75% vs 46 %, p<10-3), mainly due to more frequent use of lamivudine (72% vs 34%, p<10-3) and tenofovir (28% vs 1%, p<10-3).

 

Conclusion:

Nearly half of HBV chronically infected patients are co-infected by the HIV in French hospitals, mostly male and intravenous drug users. Among HBV-HIV co-infected patients tested, one third present with extensive liver fibrosis. They share frequently HbeAg+, rarely pre-core mutants, and are often receiving lamivudine or tenofovir. There is a urgent need for prospective controlled studies in HBV-HIV coinfected patients.

 

 


Poster 1170

A RANDOMIZED, PLACEBO-CONTROLLED STUDY (ETV-056) IN CHINA OF THE EFFICACY AND SAFETY OF ENTECAVIR IN CHRONIC HEPATITIS B PATIENTS WHO HAVE FAILED LAMIVUDINE

Guangbi Yao, Shanghai Jing An Central Hospital, Shanghai, China; Xiaqiu Zhou, Rui Jin Hospital Affiliated to Shanghai No 2 Medical University, Shanghai, China; Daozheng Xu, Beijing Di Tan Hospital, Beijing, China; Baoen Wang, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing, China; Hong Ren, No 2 Hospital Affiliated to Chongqing Medical University, Chongqing, China; Michael Jiang, Bristol-Myers Squibb Company, Shanghai, China; Jessica Liu, Bristol-Myers Squibb Company, Beijing, China; Dong Xu, Laurie MacDonald, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT.

 

 

Background:

The treatment of patients who have failed prior therapy with lamivudine is of particular concern in China where chronic hepatitis B is endemic.

 

Aim:

Study ETV-056 evaluated treatment with entecavir (ETV) 1.0 mg QD versus placebo for 12 weeks in Chinese patients who have failed lamivudine (LVD) treatment.

 

Methods:

ETV-056 was a double-blind, placebo-controlled study conducted at five centers in China. Eligible patients had chronic hepatitis B and documented LVD treatment failure, had discontinued LVD therapy at least 12 weeks prior to enrollment, had HBV DNA > 105 copies/mL by PCR assay and ALT levels not exceeding 10 x ULN. In the double-blind phase of the study, patients were randomized (4:1) to receive ETV 1.0 mg QD (N=116) or placebo (N=29) for 12 weeks. In the subsequent open-label phase, all patients received ETV 1.0 mg QD for up to 36 weeks. The primary efficacy endpoint was the change from baseline at Week 12 in HBV DNA by PCR assay. Results:

Results from the 12-week double-blind phase are presented. Among treated patients, the majority were male (75%), the mean age was 35 years, 90% were HBe antigen-positive, 41% had ALT > 1.25 x ULN, and 42% had LVD resistant virus detected by HBV DNA polymerase sequence analysis at baseline. The mean baseline HBV DNA level by PCR assay was 8.79 log10 copies/mL. The mean change in HBV DNA levels at Week 12 was -4.30 log10 copies/mL for the ETV group compared to -0.15 log10 copies/mL for the placebo group (p < 0.0001). Entecavir was also superior to placebo for the proportion of patients with HBV DNA levels < 0.7 MEq/mL by bDNA assay at Week 12 (74% vs. 10%, p < 0.0001). Among patients with abnormal ALT at baseline, a significantly higher percentage of patients in the ETV group had achieved ALT normalization at Week 12 than in the placebo group (71% vs 7%, p < 0.0001). The overall incidence of adverse events was similar between treatment groups: 33% of ETV subjects and 28% of placebo subjects reported adverse events. Only one patient (placebo group) discontinued study drug due to adverse events. ALT flares occurred in 2 patients (2%) in the ETV group and 3 patients (10%) in the placebo group. The 2 ALT flares in the ETV group were self-limited and both patients had achieved HBV DNA levels by bDNA assay < 0.7 MEq/mL at Week 12.

 

Conclusions:

The findings from this study demonstrate the antiviral activity and safety of entecavir in adults with chronic hepatitis B who have failed prior lamivudine therapy. It is anticipated that longer duration of therapy will result in significant clinical benefit.

 

 


Poster 1171

THE LONG-TERM OUTCOME OF HBeAG-NEGATIVE PATIENTS WITH CIRRHOSIS TREATED WITH LAMIVUDINE MONOTHERAPY: A 5-YEAR PROSPECTIVE COHORT STUDY

Pietro Lampertico, Mauro Vigano, Massimo Iavarone, Elena Manenti, Raffaella Romeo, Giovanna Lunghi, IRCCS Maggiore Hospital University of Milan, Milan, Italy; Giuseppe Colucci, Roche Molecular Systems, Geneva, Switzerland; Alberto Morabito, Department of Medicine, Surgery and Dentistry, University of Milan, Milan, Italy; Ersilio Del Ninno, Massimo Colombo, IRCCS Maggiore Hospital University of Milan, Milan, Italy.

 

 

Background/Aims:

We assessed the effect of long-term continuous 100 mg/daily lamivudine treatment in 84 consecutive HBeAg-negative cirrhotics for 48 (5-84) months. Eighty-seven percent were men, 91% had genotype D, 92% had Child-Pugh A/B cirrhosis and 36% had oesophageal varices.

 

Methods:

HBV-DNA was tested by bDNA (<700.000 eq/mL, Bayer) and Cobas Amplicor HBV Monitor (1 log10 copies/mL rebound HBV DNA as compared to on-treatment nadir confirmed by INNO-LiPA assay (Innogenetics, Belgium).

 

Results:

50 (60%) patients maintained undetectable serum HBV-DNA by bDNA assay and normal ALT throughout the study period. One lost HBsAg. 34 (40%) patients who initially cleared serum HBV-DNA, later developed clinical resistance to lamivudine. The 5-yr cumulative probability of developing genotypic and clinical resistance to lamivudine was 63% and 82%, respectively. Clinical decompensation occurred in none of responders compared to 8 lamivudine-resistant patients (0% vs 24%, p<0.001). By converse, hepatocellular carcinoma (HCC) occurred in both groups at similar rates (30% and 27%). 19 (23%) patients died, underwent liver transplantation or were listed for liver transplantation because of HCC (n=14) or end-stage liver disease (n=5). The 5-year patient survival was 74% with similar rates in responders and lamivudine-resistant cirrhotics. Six patients with lamivudine resistance and clinical decompensation were rescued by another antiviral drug.

 

Conclusions:

More than half cirrhotics responded to long-term lamivudine mono-therapy and had clinical decompensation delayed compared to non-responders. HCC rate was unaffected by response to lamivudine.

 

 


Poster 1172

LONG TERM EFFECTS OF INTERFERON ALPHA THERAPY INTRAVENOUS DRUG USERS WITH TRIPLE INFECTION BY HEPATITIS B, C AND D VIRUS

Corinne Castelnau, INSERM U481, Clichy, France; Emmanuel Gordien, Service de virologie, Bobigny, France; Nathalie Boyer, Michelle MARTINOT, INSERM U481, Clichy, France; Paul Deny, Service de Virologie, Avicenne, France; Patrick Marcellin, INSERM U481, Clichy, France.

 

 

Background/Aims:

To evaluate the long term effects of interferon alpha (IFN) therapy in patients with chronic hepatitis related to HBV, HCV and HDV coinfection with or without HIV infection.

 

Methods:

16 males intravenous drug users seen from 1990 to 1994 (9 anti-HIV positive), aged 25-40 years, with chronic active hepatitis were treated for 12 months with recombinant interferon alpha 2b (Introna, Schering Plough), at the dose of 10 MU, thrice a week. All were positive for HBsAg, IgM anti-delta and anti-HCV. All were serum HBV DNA undetectable. HCV RNA was detected by PCR in one patient. 14/16 had detectable HDV RNA. There was no difference between anti-HIV positive and negative patients with respect to duration of drug use, or duration of infection and histologic lesions (cirrhosis in 3 patients). Sustained response (SR) was defined by normal serum ALT levels, undetectable HDV RNA and absence of IgM anti-delta at the end of the 12 month post-treatment follow-up.

 

Results:

Treatment was interrupted in 5 patients because of decompensated cirrhosis (1), asthenia (3), decrease of CD4 lymphocyte count (1). 12 patients were non-responders (NR) and a sustained response was observed in 4 patients (1 anti-HIV positive). All 4 sustained responders lost HBsAg, IgM anti-delta and HDV RNA. During long term follow-up (mean 9 years) in the 4 sustained responders: HBsAg, IgM anti-delta and HDV RNA remained negative, and serum ALT levels remained normal, 2/4 developed anti-HBs. 3/4 patients had a liver biopsy after treatment. One patient, showed a significant improvement of fibrosis with disappearance of necroinflammation; 2/3 with active cirrhosis at baseline showed cirrhosis without activity; immunostaining for liver HDVAg was negative n 3/3. Nine non responders were followed during 1 to 9 years. 3/9 died of decompensated cirrhosis (anti-HIV positive).

 

Conclusion:

In intravenous drug addicts with chronic hepatitis related to triple B-C-D infection, IFN therapy induced the loss of serum HBsAg and HDV RNA with a long term sustained virological and biochemical response and clinical and histologic improvement.

 

 


Poster 1173

CLINICAL SIGNIFICANCE OF HEPATITIS B VIRUS DNA LEVELS IN HBEAG-NEGATIVE, HBV GENOTYPE D-INFECTED PATIENTS

Georgios Germanidis, Papageorgiou General Hospital, Thessaloniki, Greece; Francoise Roudot-Thoraval, Fabrice Guerre, Ahmed Miladi, Magali Bouvier-Alias, Hopital Henri Mondor, Creteil, France; Irini Makri, University of Larissa, Larissa, Greece; Spilios Manolakopoulos, Evangelismos Hospital, Athens, Greece; Emanuil Pagkalos, Papageorgiou General Hospital, Thessaloniki, Greece; Alexandros Avgerinos, Evangelismos Hospital, Athens, Greece; Georgios Dalekos, University of Larissa, Larissa, Greece; Jean-Michel Pawlotsky, Hopital Henri Mondor, Creteil, France.

 

 

Background:

Hepatitis B virus DNA can easily be quantified in the serum of HBV-infected patients by means of molecular biology techniques. However, the diagnostic and prognostic value of HBV DNA levels remains largely unknown, and no clinically relevant thresholds have been established in standardized international units that could be used to make accurate clinical or therapeutical decisions.

 

Aims:

To determine the clinical information provided by HBV DNA load measurements, and identify clinically relevant HBV DNA thresholds in HBeAg-negative, HBV genotype D-infected patients with chronic hepatitis B.

 

Methods:

HBV DNA was quantified, in international units (IU)/ml, in 241 Greek patients (154 M, 87 F, mean age 50±14) with HBeAg-negative chronic hepatitis B; 19.5% had histologically proven cirrhosis. HBV DNA levels were compared to the clinical, biochemical and histological characteristics of HBV infection. Genotype was determined with INNO-LiPA HBV Genotyping (Innogenetics). Precore and core promoter mutations were sought by INNO-LiPA HBV PreCore (Innogenetics).

 

Results:

All but 3 patients were infected with HBV genotype D. HBV DNA levels varied according to the predominant precore sequence at position 1896: G (wild-type): 5.9±2.1 log IU/ml ; A (precore mutant): 4.8±1.5 log IU/ml ; mixed population: 5.0±1.4 log IU/ml, but not as a function of the type of core promoter mutation at positions 1762-1764 (AGG, TGA or other). The HBV DNA load was significantly higher in the patients with cirrhosis (5.5±1.3 vs 4.7±1.6, p=0.006). Cirrhosis was also associated with an older age, higher AST levels and a TGA rather than AGG core promoter mutation. Among 102 patients with a Metavir scoring of liver biopsy, HBV DNA load was found to be associated with the activity grade (A0-A1: 5.2±1.4 log IU/ml, A2-A3: 5.9±1.0, p=0.0005), together with the age and serum ALT and AST levels. An HBV DNA load of 600,000 IU/ml (5.8 log IU/ml) was the best predictor of chronic active hepatitis, ie A2 or A3 (PPV=64.2%, NPV=75.5%, Sp=73.9%, Se=66.1%). When combining the activity grade and fibrosis score, the HBV DNA load was significantly associated with the severity of HBV-induced liver disease (mild hepatitis: 5.1±1.3; moderate: 5.8±1.1; severe: 5.8±1.4 log IU/ml; p=0.04), together with an older age and higher AST levels.

 

Conclusion:

In genotype D-infected, HBeAg-negative patients with chronic hepatitis B, the HBV DNA load is significantly related to the type of precore mutation, and increases with the severity of HBV-induced liver disease. Combining HBV DNA load, aminotransferase levels and the patient's age may allow to discriminate among the patients who need and those who do not need antiviral therapy. Specific algorithms should now be derived for each homogeneous subpopulation of HBV patients.

 

 


Poster 1175

DYNAMICS OF HBV-DNA AND INFECTED HEPATOCYTES DESCRIBED BY A NEW BIO-MATHEMATICAL MODEL IN CHRONIC HEPATITIS B PATIENTS TREATED WITH LAMIVUDINE AND IN PATIENTS WITH YMDD MUTANTS TREATED WITH ADEFOVIR

Ranieri Bizzarri, Scuola Normale Superiore of Pisa, Pisa, Italy; Piero Colombatto, Luigi Civitano, Diego Flichman, Pietro Ciccorossi, Rodolfo Sacco, Filippo Oliveri, Barbara Coco, Anna Maina, Pisa University Hospital, Pisa, Italy; Ferruccio Bonino Sr., IRCCS Ospedale Maggiore, Milano, Italy; Teresa Santantonio Sr., Policlinico di Bari, Bari, Italy; Maurizia Brunetto Sr., Pisa University Hospital, Pisa, Italy.

 

 

Background:

Viral kinetics studies based on mathematical models allow for the analysis of viremia decline in the sera of patients with chronic viral diseases during antiviral therapy in order to understand the general dynamics of the infection.

 

Aims:

This approach has been applied in naive patients infected with HBV wild type strains undergoing antiviral therapy but it has never been used to investigate the kinetics of the infection in patients who developed resistance to antivirals.

 

Methods:

We developed a bio-mathematical multiphase model that allows to investigate the dynamics of HBV infection during all therapy and we used it to analyze viremia decline in 9 patients infected with wild type HBV treated with Lamivudine (LAM) 100 mg/die and 23 patients infected with YMDD mutant HBV treated with LAM plus Adefovir (LAM+ADV) (19 pts) or ADV only (4 pts). ALT and HBV-DNA levels (Amplicor HBV Monitor) were measured at days 0, 1, 2, 4, 7, 14, 21, 28, 42, 56, 84 and every month thereafter during therapy for 1 year. The model allows for the calculation of the parameters of distinct decay phases of viremia and the number of infected hepatocytes.

 

Results:

During the 1st month in 8 (89%) patients treated with LAM and in 6 (32%) patients treated with LAM+ADV an intermediate phase (decay constant ?) was observed between the 1st rapid phase (circulating virions clearance upon block of virus production, λ) and the last phase (infected hepatocyte clearance δ). Means ± SD of HBV-DNA decay constants (day-1), infected cell % at baseline (I0) and its Log10 reduction at the end of therapy (Ieot) and the coefficient of residual virus production per infected cell during therapy Ψres) are shown in the table below:

 

Parameter

δ

I0

λ

Ф

Ψres

Log10 Ieot/I0

Therapy

Average

0.085

31.6

2.40

0.39

0.005

- 4.34

LAM

Std

0.048

24.9

0.46

0.16

0.003

1.72

Average

0.092

20.8

1.77

0.22

0.057

- 2.58

LAM+ADV

Std

0.044

20.2

1.13

0.04

0.052

1.43

Average

0.076

13.2

1.30

 

0.135

- 1.85

ADV

Std

0.019

5.05

1.01

 

0.113

0.97

A vs B vs C p value:

ns

ns

ns

0.095

0.002

0.061

 

A vs B+C p value:

ns

ns

ns

NA

0.008

0.071

ANOVA

 

Conclusion:

Analysis of the parameter values shows: 1) no significant differences of the hepatocyte clearance rate can be found among the therapeutic regimes, thus confirming that infected hepatocyte immune clearance is related most to the host conditions and is independent of the drug effectiveness; 2) the nature of drug strongly affects the magnitude of residual viral production per infected cell, which is much lower for LAM-treated patients compared to ADV+LAM (10 times greater) or ADV alone (20 times greater).

 

 


Poster 1176

SAFETY, IMMUNOGENICITY, AND EFFICACY RESULTS OF A PHASE II TRIAL OF THERAPEUTIC VACCINES FOR CHRONIC HBV IN THE GAMBIA

Samuel J. McConkey, Dorka Awi, Medical Research Council Laboratories, The Gambia, Banjul, Gambia; James Stephens Cavenaugh, University of Oxford, Oxford, United Kingdom; Maimuna Mendy, Medical Research Council Laboratories, The Gambia, Banjul, Gambia; Joerg Schneider, Gill Pearse, Oxxon Therapeutics, Oxford, United Kingdom; Adrian Vivian Sinton Hill, University of Oxford, Oxford, United Kingdom.

 

 

Background:

Priming with a DNA plasmid followed by heterologous “boosting” with an engineered viral vector, both encoding disease specific antigens, has elicited strong cellular immune responses in animal models.

 

Aims:

Since a strong cellular immune response is essential for control of hepatitis B virus (HBV) infection, we evaluated this approach in healthy chronic carriers of HBV in The Gambia using plasmid DNA (pSG2.HBs) and recombinant modified vaccinia virus Ankara (MVA.HBs) expressing the medium surface protein (preS2 and S) of HBV ayw strain.

 

Methods:

72 chronic HBV carriers, either HBeAg+ or HBeAg- and with no biochemical evidence of liver inflammation, were allocated into 8 treatment groups. Depending on the group, vaccinees received either 1 or 2 mg of pSG2.HBs on two occasions 3 weeks apart followed by 5 or 15 x 10e7 plaque forming units of MVA.HBs on one or two occasions with or without lamivudine. Two control groups received lamivudine alone for 14 wk and another control group received control (rabies) vaccine. Cellular immune responses were evaluated by IFN-? ELISpot with pools of overlapping 15-mer peptides spanning all of the medium surface protein and by intracellular cytokine staining. Plasma HBV viremia was evaluated by quantitative PCR.

 

Results:

The therapeutic vaccine was well tolerated with no vaccine-related serious adverse events reported. HBV-specific cellular immune responses were observed. Some vaccinees exhibited IFN-? production by natural killer, NKT, CD4 and CD8 T lymphocytes after the MVA.HBs boost but not after pSG2.HBs priming alone. These responses varied between individuals both quantitatively and qualitatively by phenotype of IFN-? producing cells. The eAg+ve groups showed lower cellular immune response than eAg-ve group. In no group was there a sustained reduction of viremia following vaccination and withdrawal of lamivudine.

 

Conclusions:

This novel prime boost immunization scheme evaluated in a large Gambian group of HBV chronic carriers showed a good safety profile, and some evidence of immunogenicity, but no associated viral load reductions. Modifications of the antigen, dose, adjuvant, and delivery systems are being considered.