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The Liver Meeting |
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October 29 - November 2 |
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.
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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.
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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.
|
|
|
|
|
|
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 |
LVD
100 mg N=355 (treated) |
p-value |
|
*Histologic Improvement (%) |
72% |
62% |
0.0085 |
|
†HBV
DNA |
−
6.98 |
−
5.46 |
<0.0001 |
|
HBV
bDNA |
91% |
65% |
<0.0001 |
|
HBV DNA |
69% |
38% |
<0.0001 |
|
ALT
Normalization (<1.25 x ULN) (%) |
78% |
70% |
0.0136 |
|
*n = 314 with evaluable histology in each treatment group |
|||
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 |
LVD |
p-value |
|
*†Histologic
Improvement |
70% |
61% |
0.0143 |
|
†Ishak
Fibrosis Score Improvement |
36% |
38% |
NS |
|
‡HBV
DNA |
−5.20 |
−4.66 |
<0.0001 |
|
HBV
DNA |
91% |
73% |
<0.0001 |
|
Composite
Endpoint Response |
84% |
78% |
0.0401 |
|
*Primary
Endpoint |
|||
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 |
ADV+OL-ADV+ADV |
ADV+OL-ADV+PLA |
|
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 |
Mild/Moderate
Liver Disease (N=281) |
Cirrhosis/HCC |
|
Undetectable |
393
(43.8%) |
83
(29.5%): |
93
(27.2%) |
|
Low |
226
(25.2%) |
71
(25.3%) |
60
(17.5%): |
|
High |
278
(31.0%) |
127
(49.2%) |
189
(55.3%) |
*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.
|
|
|
|
|
|
|
|
%
US-Born |
84 |
55 |
0.01 |
65 |
0.09 |
|
%
HBeAg+ve |
57 |
30 |
0.04 |
10 |
0.0003 |
|
Mean
HBV DNA± SD |
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, % |
2 |
0 |
NS |
75 |
<0.0001 |
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.
|
|
|
|
|
|
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) |
Controls
(n=61) |
RR (95%
CI) |
|
Undetectable |
5
(13.5%) |
32
(51.6%) |
Reference |
|
Low
Titer |
7
(18.9%) |
11
(18.0%) |
4.1
(1.1-15.5) |
|
High
Titer |
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 |
|
|
|
|
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:
- We have developed a
sensitive method for the quantification of (-) and (+)strand HBV DNA in
the liver.
- 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.
- 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:
- The majority (79%)
of HBeAg(-) patients harbor BCP mutations and one-third of them (31%) have
mutations other than T/A.
- 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.
- 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® |
PEGASYS® |
lamivudine |
|
Mean
HBV DNA reduction at |
- 4.1 |
- 5.0 |
- 4.2 |
|
YMDD
mutation at end of treatment |
N/A |
1/173
(<1%)* |
32/179
(18%) |
|
HBV DNA
<20,000 copies/ml |
76/177
(43%)§ |
79/179
(44%)‡ |
53/181
(29%) |
|
*
P<0.001 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 |
-6.79 |
-4.85 |
<
0.0001 |
|
% of
patients with: |
<
2.6 x ULN |
90% |
58% |
<
0.0001 |
|
HBV DNA
< 400 copies/mL by PCR |
<
2.6 x ULN |
59% |
28% |
<
0.0001 |
|
* n =
179 (<2.6xULN), 161 (> 2.6xULN) for ETV; |
||||
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 |
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 |
96/142
(68%) |
86/124
(69%) |
66/122
(54%) |
|
* ALT
normalization 24 weeks after end of treatment (week 72) |
|||
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 |
-4.7
(n=40) |
-4.6 |
-4.1 |
-4.4 |
NA |
|
ALT
median change from baseline (IU/L) |
-35 |
-33 |
-30 |
-46
(n=102) |
-60 |
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 |
Child-Pugh |
Child-Pugh |
Hepatic Impaired |
Healthy |
|
Cmax (ng/mL) |
8.99 (32.7) |
7.25 (39.6) |
8.52 (34.3) |
8.22 (29.9) |
|
AUC(INF) (ng·h/mL) |
29.33 (24.3) |
28.17 (38.4) |
29.03 (26.9) |
31.28 (21.6) |
|
Tmax (h) |
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) |
87.07 (41.0) |
109.94 (75.6) |
92.79 (48.3) |
92.10 (14.5) |
|
%UR (%) |
44.52a (25.9) |
47.15 (13.3) |
45.22b (22.8) |
52.77 (15.2) |
|
CLR (mL/min) |
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 |
LVD |
p-value |
|
*†Histologic Improvement |
55% |
28% |
<0.0001 |
|
*Composite |
55% |
4% |
<0.0001 |
|
†Ishak Fibrosis Score Improvement |
34% |
16% |
0.0019 |
|
‡HBV
DNA Mean change from baseline (PCR) |
-5.14 |
-0.48 |
<0.0001 |
|
HBV
bDNA |
66% |
6% |
<0.0001 |
|
HBV DNA |
21% |
1% |
<0.0001 |
|
ALT
Normalization |
75% |
23% |
<0.0001 |
|
*Co-primary endpoints |
|||
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 |
LVD 100
mg |
ETV 1.0
mg |
LVD 100
mg |
|
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 |
95 (27) |
114
(33) |
24 (17) |
44 (31) |
|
ALT
Flares |
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) |
|||
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.