Transcript HBV Pol-Gene Mutant

Efficacy of Hepatitis B Vaccine Against
Antiviral Drug-Resistant Hepatitis B
Virus Mutants in the Chimpanzee Model
Abstract
Hepatitis B virus (HBV) mutants resistant to
treatment with nucleoside or nucleotide
analogs and those with the ability to escape
from HBV-neutralizing antibody have the
potential to infect HBV-vaccinated individuals
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Abstract
To address this potential serious public health
challenge, we tested the efficacy of immunity
induced by a commercial hepatitis B vaccine
against a tissue culture-derived, clonal HBV
polymerase mutant in HBV seronegative
chimpanzees
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Abstract
The polymerase gene mutant contained a
combination of three mutations (rtV173L,
rtL180M, rtM204V), two of which resulted in
changes to the overlapping viral envelope of
the hepatitis B surface antigen (sE164D,
sI195M)
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Abstract
Prior to the HBV mutant challenge of vaccinated
chimpanzees, we established virologic, serologic,
and pathologic characteristics of infections
resulting from intravenous inoculation of the
HBV polymerase gene mutant and the sG145R
vaccine-escape surface gene mutant
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Abstract
Cloning and sequencing experiments
determined that the three mutations in the
polymerase gene mutant remained stable and
that the single mutation in the surface gene
mutant reverted to the wild-type sequence
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Abstract
Immunological evidence of HBV replication
was observed in the vaccinated chimpanzees
after challenge with the polymerase gene
mutant as well as after rechallenge with serumderived wild-type HBV (5,000 chimpanzee
infectious doses administered intravenously),
despite robust humoral and cellular anti-HBV
immune responses after hepatitis B vaccination
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Abstract ——Conclusion
Our data showing successful experimental
infection by HBV mutants despite the presence
of high anti-HBs levels considered protective
in the vaccinated host are consistent with
clinical reports on breakthrough infection in
anti-HBs-positive patients infected with HBV
mutants.
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Abstract ——Conclusion
In the absence of a protective humoral
immunity, adaptive cellular immune responses
elicited by infection may limit HBV
replication and persistence
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Background

Hepatitis B is a global health problem that related to chronic
active hepatitis, liver cirrhosis, and primary liver cancer

Vaccination against HBV prevents new infections and
efforts toward the control of chronic disease have involved
the therapeutic inhibition of viral replication using analogs
of nucleotides or nucleosides

Lamivudine was the first drug in this class to be licensed for
the treatment of chronic hepatitis B and remains in
widespread use
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Mechanism of analogs of nucleotides or
nucleosides
Infective HBV particles
Lamivudine
Adefovir
Entecavir
Infective HBV particles
Partial Doublestranded DNA
DNA
polymerase
RT
(-)-DNA
cccDNA
A(n)
mRNA
wrapped
Pre-genome mRNA
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
HBsAg
capsid
Background
Pol gene (HBV Polymerase gene)
Its reverse transcriptase district contains seven
functional sequence (A-G)，The main target area of
nucleoside or nucleotide analogs widely used in
clinical is located in the reverse transcriptase (RT)
domain B and C, therefore resistant variantion is
located in the domain B and C
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Background
Antiviral drug-resistant mutations selected
during treatment with lamivudine in the HBV
polymerase gene (Pol) clustered within its B
domain (rtV173L, rtL180M) and in the C
domain in the conserved YMDD motif
(rtM204V)
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

It has been reported that these mutant
viruses are transmissible and may have the
potential to cause breakthrough infections
among recipients of hepatitis B vaccine

The development of neutralizing antibody
escape in those individuals may result from
the changed amino acid composition of the
HBV envelope protein, as the envelope and
polymerase open reading frames overlap in
the circular HBV genome
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
ORF
S: coding for
HBV envelope protein
[neutralization of the “a”
determinant of the hepatitis B
surface antigen (HBsAg)
protein]
P: coding for
DNA polymerase
Hepatitis B virus genome
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

alterations in the neutralization of the “a” determinant
of the hepatitis B surface antigen (HBsAg) protein
have been reported in patients with mutations in the
Pol-gene receiving therapy with lamivudine

A markedly reduced binding of anti-HBs antibodies
to HBsAg with mutations corresponding to Pol-
protein changes (rtV173L, rtL180M, rtM204V) has
been demonstrated in vitro
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

These findings raise the possibility of the
selection of lamivudine-resistant HBV mutants
with antigenically modified HBsAg proteins
that could act as vaccine escape mutants and
infect vaccinated individuals in whom antiHBs exerts a further positive selection pressure
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

The most common mutation in the S-gene product is
glycine to arginine at position 145 (sG145R)

It has been suggested that the immunity induced by
existing vaccines may not be protective against various
HBV mutants with altered surface proteins because of
the conformational nature of the “a” determinant

Furthermore, the existence of such HBV isolates and
the potential of these mutants to infect hepatitis Bvaccinated individuals may lead to occult HBV
infection and, consequently, have serious public health
implications
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Materials and Methods
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Tissue Culture-Derived Mutant and WildType HBV Inocula

Point mutations were introduced by site-directed
mutagenesis into a 1.5-times genome-length wt-HBV
belonging to genotype A, subtype adw 2

The sG145R mutation was produced in the S-gene with
overlapping rtW153Q mutation in the Pol-gene（HBsAg
Classical immune evasion strain ）

A combination of three HBV Pol-gene mutations, rtV173L,
rtL180M, and rtM204V, was produced that correspond to
sE164D and sI195M mutations in the S-gene (rtL180M does
not result in an amino acid change in the S-gene)
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Chimpanzee Infectivity Experiments
Eleven colony-born chimpanzees (six females and five males,
ranging in age from3 to 11 years and weighing between 11 to
37 kg), determined to be seronegative for markers of HBV
infection, were used for infectivity, vaccination, and challenge
studies
All chimpanzees inoculated with the wild-type and mutant
HBV inocula were followed up for 180 to 200 days after
inoculation
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Chimpanzee HBV Immunization and
Challenge Experiments

Two chimpanzees (CH10364 and CH10369) were given a
pediatric dose (10 μg/0.5mL) of a licensed recombinant
hepatitis B vaccine at 0, 28, and 56 days

One control chimpanzee(CH10301) was vaccinated with
hepatitis A vaccine

All three vaccinated chimpanzees were challenged with the
clonal HBV Pol-gene mutant containing 9.8* 109 copies of
HBV DNA 42 days after the third vaccine dose
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Chimpanzee HBV Immunization and
Challenge Experiments

the control chimpanzee was challenged 35 days after the
hepatitis A vaccination

The two hepatitis B-vaccinated chimpanzees were further
cross-challenged with 5,000 chimpanzee infectious doses (CID)
of human serum-derived wt-HBV inoculum 216 days after the
Pol-gene mutant challenge

A naive chimpanzee, CH10270, served as a control for the wtHBV challenge and was also inoculated with 5,000 CIDs of
the wt-HBV inoculum
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Chimpanzee HBV Immunization and
Challenge Experiments

Serial serum specimens were collected from all the
chimpanzees twice weekly and whole blood samples for
peripheral blood mononuclear cells (PBMCs) isolation were
collected every 2 weeks

Serum alanine aminotransferase (ALT) levels were measured
in fresh serum specimens. Individual cutoff values for ALT
levels were established for each animal using at least 10
preinoculation measurements
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Serological Markers of HBV Infection

All serum samples were tested for :
HBsAg
antibody to HBsAg (anti-HBs)
total antibody to hepatitis B core antigen (anti-HBc)
IgM anti-HBc
hepatitis B e antigen (HBeAg)
total anti-HBe

The levels of anti-HBs in samples collected during vaccination and
challenge periods were also quantitatively measured
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
T-Cell Responses

PBMCs from the chimpanzees were enriched and tested in an interferonΥELISpot assay for reactivity to HBV surface and polymerase antigens

Millipore were precoated with antibodies to human interferon-Υ and 2*105
chimpanzee PBMCs in medium added to each well

Duplicate wells received pools of overlapping synthetic peptides that
spanned either amino acids 67-237 of the HBsAg or amino acids 1-344 of the
reverse transcriptase domain of the polymerase protein

Plates were incubated and developed for spot formation using a second
antibody to interferon-Υ conjugated to enzyme followed by substrate

Spot-forming cells (SFCs) in the microtiter wells were quantified using a CTL
analyzer

Responses were considered positive when an average of 10 SF Cover
background was detected in the duplicate wells
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Results
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Wild-Type HBV
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Sequencing studies showed that all clones
from the inoculum and samples collected
from the two infected chimpanzees at the
beginning, during, and at the end of viremia
maintained the wild-type sequence
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
HBV S-Gene Mutant (sG145R)
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Sequencing studies of 24 clones from the
first HBV DNA-positive serum sample
postinoculation (day 98) showed the
preservation of the inoculum sequence in
only 33% of the clones, whereas 67% had
the wild-type sequence
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
HBV Pol-Gene Mutant
(rtV173L, rtL180M,rtM204V)
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Sequencing studies showed that all clones
from the two inocula and samples collected
at the beginning, during, and at the end of
viremia in the three infected chimpanzees
showed the preservation of the rt173L,
rt180M, and rt204V mutations
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Protective Efficacy of HBV Vaccine
The ability of a licensed hepatitis B vaccine to
provide protection against challenge with the
HBV Pol-gene mutant was evaluated in two
chimpanzees (CH10364 and CH10369) that
received the hepatitis B vaccine and a control
chimpanzee (CH10301) that received the
hepatitis A vaccine
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Both hepatitis B vaccine recipients seroconverted to antiHBs 1 week after the first dose of the vaccine, and antiHBs levels were boosted to steady-state levels of >75
mIU/mL after the administration of the second and third
vaccine doses
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Neither humoral nor cellular immunity to HBV
antigens was detected in the control
chimpanzee CH10301 vaccinated with HAV
vaccine
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
All three chimpanzees were challenged with
the triple mutant to determine if coding
changes in the corresponding S-gene (sE164D
and sI195M) compromised protective
immunity elicited by hepatitis B vaccination
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Anti-HBc, which is a marker of exposure or
infection, was detected on day 7 in CH10364
and days 7, 10, and 17 in CH10369
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009




The hepatitis A-immunized control chimpanzee
CH10301 was infected because HBV DNA was
detected in serum from days 42 to 91, not exceeding
1 log IU/mL
The chimpanzee seroconverted to total anti-HBc from
day 87 and anti-HBs from day 84 post infection.
Despite replication of the Pol-gene mutant virus in
this chimpanzee, peripheral blood T-cell responses
against the surface or polymerase proteins were not
detected
No ALT elevation was observed during the follow-up
period
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

The presence of a humoral immune response to the
HBV core antigen in both ENGERIX-B-vaccinated
chimpanzees and of circulating T-cells against the
HBV polymerase antigen in one chimpanzee
(CH10364) indicated that sterilizing immunity against
the HBV Pol-gene mutant was lacking

Therefore, chimpanzees CH10364 and CH10369
were challenged with human-derived wt-HBV to
determine if they were protected against challenge
with a virus encoding intact (that is, unmutated)
HBsAg
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Challenge with the wt-HBV inoculum (HLD1,
5,000 CIDs) was carried out when anti-HBs
levels were 63,610 mIU/mL and 18,868
mIU/mL in CH10364 and CH10369,
respectively, and after loss of HBV mutantstimulated T-cell responses in both
chimpanzees
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
High frequencies of circulating T-cells against
HBsAg suggested an anamnestic response
primed by vaccination and perhaps the prior
infection with the HBV Pol-gene mutant.
Responses to the polymerase protein were
delayed by comparison
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

Polymerase-specific T-cell lines expanded from the
blood of both chimpanzees expressed CD4+ and not
CD8+ (data not shown).

Anti-HBs titers increased 6-fold in chimpanzee
CH10369 and 10-fold in chimpanzee CH10364

However, HBV DNA, HBsAg, IgM anti-core, and
HBeAg were not detected in any of the chimpanzees
during the follow-up period after the challenge
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

A markedly different profile of wt-HBV infection was
observed in a naive unvaccinated chimpanzee
CH10270

The infection was characterized by the presence of
HBV DNA, which reached the peak levels of 4.1 log
IU/mL on day 66, and of HBsAg, HBeAg, and IgM
anti-HBc. The chimpanzee subsequently developed
acute hepatitis with elevated serum ALT levels from
days 66 to 129, with the peak of 2,500 IU/L (cutoff
73 IU/L) observed on day 91.
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

This chimpanzee subsequently seroconverted
to total anti-HBc, anti-HBe, and anti-HBs

Circulating T-cells specific for the surface and
polymerase antigens were not detected in
chimpanzee CH10270 by the ELISpot assay
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Discussion
Breakthrough infections etiologically linked to
S-gene HBV mutants have been reported to
occur among HBV-immunized populations,
despite the presence of seroprotective levels of
anti-HBs in the infected individuals
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Discussion
In this experimental study, they evaluated the
protective efficacy of a commercial hepatitis B
vaccine against the Polg-ene HBV mutant
(rtV173L, rtL180M, rtM204V) encoding the
surface protein mutations (sE164D, sI195M)
that was previously shown to have
significantly reduced anti-HBs binding
properties
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

The inoculation of naive chimpanzees with clonal HBV
mutants allowed studying the virologic, immunologic,
and pathologic findings exclusively related to the
mutant infection

The subsequent challenge experiment provided an
opportunity to determine whether HBV mutants are
infectious to vaccinated chimpanzees

Because the cellular immunity data indicated that the
commercial vaccine did not induce sterilizing immunity
against the Pol-gene HBV mutant, they further
challenged two immunized chimpanzees with the
serum-derived wt-HBV
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
In naive nonimmunized chimpanzees：

they observed variations in the infectivity profile
following injection with inocula carrying various
viral titers of HBV mutants

The length of the incubation period, measured by first
appearance of HBV DNA in serum, ranged from 1
week (clonal Pol-gene HBV mutant and wt-HBV) to
14 weeks (sG145R mutant) and did not correlate with
the number of HBV DNA copies in the inocula
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Also of interest were differences in the stability of
mutations between Pol-gene and S-gene mutants during
the experimental infection of chimpanzees

mutations in the Pol-gene remained stable throughout the
period of viremia in all four chimpanzees

the sG145R mutant in one infected chimpanzee readily
reverted to wild-type, which are at variance with data
from previous experimental infectivity studies, may be
attributed to the mutation site, replication fitness of the
mutants, the size of the inocula, or a combination of these
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

Vaccinated chimpanzees in this study developed a
robust T-cell immunity and had pronounced anti-HBs
serum antibody titers (75 mIU/mL) that significantly
exceeded those considered protective (10mIU/mL)

Detection of anti-HBc antibodies after challenge with
the Pol-gene HBV mutant in one serum sample from
CH10364 and three consecutive samples from
CH10369 provided immunological evidence of the
HBV replication despite high anti-HBs levels
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

Further evidence of the breakthrough infection in the
challenge experiment using the clonal Pol-gene HBV
mutant was provided by priming of T-cell responses
against the polymerase protein in at least one
vaccinated chimpanzee

This protein is not a component of the vaccine and it
is unlikely that sufficient quantities are physically
associated with the Pol-gene mutant virus particles in
the original inoculum to prime T-cell immunity in the
absence of virus replication

this latter possibility seems remote, as they did not
observe polymerase specific responses in
unvaccinated chimpanzees that received high doses of
Pol-gene mutant or wild-type particles that resulted in
infection
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

High serum titers of anti-HBs antibodies are a reliable
marker of vaccine-induced protective immunity
against HBV infection

It is conceivable that virus-specific T cells provide
protection when mutant or wild-type viruses
breakthrough sterilizing humoral immunity

Their observation that Pol-specific T-cell responses
are elicited in hepatitis B-vaccinated animals
challenged with mutant and wild-type viruses is
clearly consistent with this possibility.
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

Efficacy of anti-HBV immunity induced by the
commercial vaccine was further tested by
rechallenging the two chimpanzees, previously
vaccinated and challenged with the Pol-gene mutant
with serum-derived wt-HBV

This experiment showed a subsequent boost of antiHBs levels (6-fold to 10-fold increase) in both
chimpanzees and a surge of T-cell responses against
both surface-derived and polymerase-derived
peptides
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

This pattern of cellular and humoral immune
responses against HBV antigens strongly
implicated viral replication despite very high
levels of anti-HBs suggesting breakthrough
HBV infection after wt-HBV challenge

It is possible, however, that a dose of 5,000
CIDs used in the challenge experiment may
have been large enough to overcome the
seroprotective barrier
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009

A further challenge inoculum titration study
would be needed to evaluate the relationship
between the size of inoculum and protective
level of anti-HBV immunity induced by a
vaccine and measured by the dynamics of
cellular immunity to HBV polymerase protein
and the level of anti-HBs antibody
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
conclusion

They demonstrated that in vitro-generated HBV
clonal Pol-gene mutant, bearing the lamivudineresistance-associated mutations (rtV173L, rtL180M,
rtM204V) and the prototype vaccine-escape S-gene
mutant (sG145R), are infectious in naive
chimpanzees

The S-gene mutant readily reverted to the wild-type
sequence but the Pol-gene mutant was stable during
the course of infection
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
conclusion

Vaccination of naive chimpanzees with a commercial hepatitis
B vaccine that resulted in the induction of both humoral and
cellular immune responses did not seem to confer sterilizing
immunity against challenge with the Pol-gene mutant and
subsequent challenge with a serum-derived wt-HBV

The data showing successful experimental infection by HBV
mutants despite the presence of high anti-HBs levels
considered protective in the vaccinated host are consistent with
clinical reports on breakthrough infection in anti-HBs positive
patients infected with HBV mutants and warrant further
studies of efficacy of HBV vaccine
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009
Thanks!!
Saleem Kamili, Vitini Sozzi, Geoff Thompson. et al. HEPATOLOGY, Month 2009