Saturday Poster Sessions, October 28, 2006
HCV: Pathogenesis
#271. Immunization with Hepatitis C Virus-Like Particles Induces Partial Protection against Hepatitis C Virus Infection in Chimpanzees.
G. A. Elmowalid; M. Qiao ; S. Jeong ; B. Borg; T.
Baumert; R. Sapp; K. Murthy; Z. Hu; J. Liang.
Introduction:
Recombinant hepatitis C virus-like particles (HCV-LP)
containing HCV structural proteins (core, E1, and E2) produced in insect cells
resemble the putative HCV virions and are capable of inducing strong and broad
humoral and cellular immune responses in mice and baboons.
Aim:
Here we present evidence on the immunogenicity and induction
of protective immunity by HCV-LP in chimpanzees.
Methods:
Two groups of two chimpanzees each were immunized with HCV-LP
or HCV-LP + adjuvant ASO1B (a lipid-based adjuvant from GSK).
Results:
After four immunizations over an eight-month period, all
animals developed strong HCV-specific cellular immune response including
IFN-γ+CD4+ and IFN-γ+CD8+ T-cell and proliferative lymphocyte
responses against core, E1 and E2. The immunogenicity of HCV-LP was not
enhanced by the adjuvant. The chimpanzees in both groups were challenged with
100 CID 50 of HCV CG1B inoculum. Upon challenge with HCV, one chimpanzee
developed transient viremia with low HCV RNA titers (~104 copies/ml) in the
third and fourth weeks post-challenge. The three other chimpanzees became
infected with higher levels of viremia (up to 10 5 copies/ml) but their viral
levels became unquantifiable ( 600 copies/ml) ten to twelve weeks
post-challenge. After HCV challenge, all four chimpanzees demonstrated a
significant increase in both peripheral IFN-γ+ and IL-2+ T-cell and
proliferative responses as well as the presence of intrahepatic T-cell response
against the HCV structural proteins. The T-cell responses against various nonstructural
proteins also became detectable within 3 weeks after infection. These T-cell
responses coincided with the fall in HCV RNA level. Previously, three other
naďve chimpanzees have been challenged with the same HCV inoculum at lower
CID50 (3-10). One cleared the infection and two developed persistent infection
with viremia in the range of 105-6 copies/ml.
Conclusion:
Our results suggest that HCV-LP immunization induces strong HCV-specific cellular immune responses and confers partial protection against HCV challenge in the chimpanzee model.
#277. Mouse Model for HCV Pathogenesis.
R. Witek; J. R. Bess; J. Dong; J. S. Elyar; C. Liu.
Introduction:
Hepatitis C virus (HCV) infection is a major health threat in
the world. An estimated 170 million people are infected with the virus.
Although it has been postulated that the host immune system plays a role in
pathogenesis, the interaction of the virus and the host immune system remains
obscure. One major obstacle to HCV investigations is the lack of a robust small
animal model. Considering the vast amount of knowledge on mouse immune system,
availability of a reliable murine model for HCV undoubtedly has a great
advantage.
Aim:
The current research tests the feasibility of developing an
animal model to study HCV immunopathogenesis.
Methods:
The experimental design utilized repopulation of mouse
(Balb/cJ) livers with an HCV positive CG1b cell line (hepatocytes derived from
Balb/cJ mouse) following partial hepatectomy (PHx) and monocrotaline (MCT)
pre-treatment. MCT blocks endogenous hepatocyte proliferation, thus providing a
proliferative advantage to the transplanted cells. Since the mice used in the
experiment are immune competent, the effects of the immune system on HCV
positive hepatocytes and their effects on liver pathology was systemically
investigated by performing histological stainings and molecular analysis by
RT-PCR.
Results:
In the first part of the experiment, HCV positive CG1b cell
line was established by stably transfecting immortalized male BNL murine
hepatocyte cell line with HCV-ribozyme based expression plasmid pEHr/Neo
containing full-length HCV RNA genome. The RT-PCR analysis of the BNL-CG1b cell
line reveals high expression of HCV RNA, and Western Blot shows expression of
NS5a proteins. In the second part of the experiment, transplantation of
BNL-CG1b cells led to liver repopulation in BALB/cJ mice verified by SRY
analysis at 30 days post transplantation. H&E examination of liver tissue
revealed increasing levels of inflammation and fibrosis progressing with time
post transplantation (30, 60, and 90 days). However, SRY was not detected at 60
and 90 days.
Conclusion:
The results presented in the current study support the
feasibility of creating a small animal model for HCV pathogenicity utilizing
BNL-CG1b cell transplantation to MCT/PHx treated animals. The inflammation,
lymphocytic infiltration and liver fibrosis observed in the experimental mice
conform to liver pathology observed in patients with chronic HCV. It is
possible that transplanted BNL-CG1b cells are invoking CD8+ CTL response (day
30) that led to the initial clearance of HCV producing cells (day 60).
The presented animal model offers an innovative approach to
investigate HCV immunology and will be used for future studies to elucidate
mechanisms of HCV liver immunopathogenesis.
#282. Persistence of hepatitis C virus (HCV) in chimpanzees is associated with a loss of intrahepatic T cell function during the late acute phase.
H. Watanabe; M. E. Major.
Background:
Infection with HCV frequently leads to chronic hepatitis and
cirrhosis and is associated with hepatocellular carcinoma. The liver is the
primary site of viral replication therefore we undertook a detailed
intrahepatic study of the dynamics of T-cells, apoptosis, and gene expression
during the acute phase of HCV infection in chimpanzees.
Methods:
We examined sequential liver biopsies from chimpanzees that
developed persistent infection or spontaneously cleared the virus and
correlated this data with viral kinetics and clinical signs of hepatitis.
Studies used formalin-fixed biopsies and RNA extracted from frozen liver
tissue. T cell infiltration was assessed in liver sections using antibodies
specific for CD4 or CD8 and H&E staining. Apoptosis was assessed using a
TUNEL assay and M30 antigen staining. Real-time PCR was used to assess mRNA
levels for specific response genes.
Results:
Several features were common to all animals regardless of
disease outcome. Using histological analyses we observed increased intrahepatic
T-cell infiltration (5-10-fold above baseline) in both groups of animals with
CD8+ T-cells representing the major population throughout infection. In some
cases the onset of T-cell infiltration was early (2 weeks post infection) but
in all animals the appearance of immune T cells was associated with liver
apoptosis and mild ALT elevations. In all animals apoptosis (5-20% of liver
cells) occurred prior to the ALT peak with no direct correlation between
maximal apoptosis and peak ALT. Major differences associated with outcome were
observed during the late acute phase. Liver biopsies from cleared animals
showed an increased frequency of apoptosis, relative to persistently infected
animals, which correlated with increased intrahepatic CD8+ T cell frequency
(8-10-fold above baseline) in this group. Up to 20% of the infiltrating T cells
observed during the late acute phase in the cleared animals stained positive
for perforin expression whereas only 1-2% of liver infiltrating T cells in the
persistently infected groups at this same time point were perforin positive.
Conclusions:
These data support the hypothesis that although both groups
of animals mount immune responses during the acute phase these are not
maintained in frequency or efficacy in animals that develop persistent
infections. There is ongoing intrahepatic immune control of replication in the
animals that clear virus but there is a reduction in both the numbers of T
cells infiltrating the liver during the late acute phase in animals that develop
persistent infections and significantly fewer of these cells are functional in
clearing the virus by inducing apoptosis.
#292. Altered Hepatic Metabolic Function in Patients with Chronic Hepatitis C is Associated with Obesity, Insulin Resistance, or Hepatic Steatosis, Independent of Cirrhosis: Results from the HALT-C Trial.
G. T. Everson; C. C. Kulig; M. L. Shiffman; R. K.
Sterling; T. R. Morgan; J. C. Hoefs; T. M. Curto; J. E. Everhart; D. Wagner.
Introduction:
Hepatic dysfunction in patients with chronic hepatitis C
(CHC) could be related to metabolic effects of obesity, insulin resistance
(IR), or hepatic steatosis, or underlying cirrhosis. In our study, we used
multiple quantitative tests (QLFTs) and controlled for cirrhosis, to define
associations of obesity, IR, and hepatic steatosis with altered hepatic
function.
Patients and Methods:
All patients (N=285) were enrolled in the Hepatitis C
Antiviral Long-Term Treatment to Prevent Cirrhosis (HALT-C) Trial. Patients had
either bridging fibrosis (Ishak fibrosis score 3 or 4) or compensated cirrhosis
(Ishak fibrosis score 5 or 6, 40%), 92% were infected with genotype 1, and all
had failed prior treatment with interferon or interferon/ribavirin. Test
compounds were given intravenously (lidocaine, 0.5mg/kg, galactose, 30g,
[24-13C]cholate, 20mg, technetium sulfur colloid, 5mCi) and orally (antipyrine,
500mg, caffeine 300mg, [1-13C]methionine, 200 mg, [2,2,4,4-2H]cholate, 40mg).
Caffeine elimination (Cf kelim), antipyrine elimination (AP kelim) and
clearance (AP Cl), and MEGX formation quantified microsomal function.
Methionine breath test (MBT) quantified mitochondrial function. Galactose
elimination capacity (GEC) assessed cytosolic metabolism and blood flow.
Clearance of orally administered cholate (CA Cloral), cholate shunt (CA Shunt),
and perfused hepatic mass (PHM) measured by SPECT liver-spleen scan assessed
portal inflow and shunt. Body mass index (BMI), insulin resistance (homeostasis
model assessment (HOMA) = {[Glucose, mmol/L] x [Insulin, µU/ml]} / 22.5), and
hepatic steatosis (graded 0, 1, 2, 3 by panel of HALT-C pathologists) were the
independent variables.
Results:
Relationships between QLFTs and quartiles of BMI or HOMA, or
grades of steatosis, were analyzed by ANOVA with a test for trend. BMI
quartiles were associated inversely with MBT (P=0.03) and GEC (P<0.0001).
HOMA quartiles were associated inversely with AP kelim (P<0.0001), AP Cl
(P=0.03), MBT (P=0.0007), GEC (P=0.0001), PHM (P=0.0002), CA Cloral (P=0.002),
and directly with CA Shunt (P=0.006). Hepatic steatosis was associated
inversely with both AP Cl (P=0.05) and MBT (P=0.0006). After controlling for
cirrhosis, the remaining significant relationships were BMI with GEC
(P<0.0001), HOMA with GEC (P=0.04) and AP kelim (P=0.0004), and hepatic
steatosis with AP Cl (P=0.02) and MBT (P=0.02).
Conclusion:
Altered hepatic metabolic function in patients with CHC is
associated with obesity and insulin resistance, independent of cirrhosis. Hepatic
steatosis, but not obesity or insulin resistance, is associated with impaired
hepatic mitochondrial function.
#295. The Role of Hepatitis C Genotype 3 Core Protein Domain 3 in Intrahepatic Steatosis.
R. Jhaveri; J. G. McHutchison; K. Patel; A. Diehl.
Background:
Steatosis is a common histological finding and a poor
prognostic indicator in patients with Hepatitis C virus (HCV) infection. The
etiology of steatosis is multifactorial, but appears to be closely correlated
with unknown viral factors in HCV genotype 3 infected patients. We previously
identified novel amino acid polymorphisms at residues 182/186 within domain 3
of HCV Core protein that correlate with intrahepatic steatosis in a well
characterized group of HCV genotype 3a infected patients. The combination of
leucine-isoleucine (LI) and phenylalanine-valine (FV) at these positions
correlated with steatosis while phenylalanine-isoleucine (FI) correlated with
the absence of steatosis. In this project, we expressed these patient derived clones
and corresponding mutants to examine if lipid accumulation occurred in cultured
liver cell lines.
Methods:
We transfected human and rat liver cell lines with steatosis
and non-steatosis associated HCV genotype 3a Core clones. Transfected cells
were then stained using a combined immunofluorescence and oil red o protocol.
Cells were analyzed using MetaMorph software that quantified the amount of oil
red o in cells expressing HCV Core protein.
Results:
Expression of all the HCV Core isolates led to increased
intracellular lipid compared to controls in 5H cells at transfection efficiency
of 5%. Expression of a steatosis-associated clone (FV) led to significantly
more intracellular lipid in transfected cells when compared with a
non-steatosis clone (FI) (11.4%±6.7% vs. 7.8%±3.3%; p=0.02). Expression of a
mutant designed to reverse the steatosis phenotype (FV to FI) resulted in a 27%
decrease in the amount of intracellular lipid compared to the parent clone
(11.4%±6.7% vs. 8.3%±4.8%; p=0.03). Mutation of another steatosis-associated
clone (LI to FI) resulted in a 37% decrease in intracellular lipid compared to
its parent clone (p=0.01).
Conclusions:
We have verified the importance of specific amino acids
within domain 3 of HCV Core protein genotype 3a in altering host lipid
metabolism and/or trafficking. Future work will attempt to identify the
mechanisms involved.
Sample image of HCV Core expression and Oil Red
staining.
#308. The Hepatitis C Virus Core Protein of Genotypes 1B and 3A Down-Regulate Insulin Receptor Substrate 1 via Genotype-Specific Mechanisms.
V. Pazienza; S. Clément; P. Pugnale; S. Conzelmann; M.
Foti; A. Mangia; F. Negro.
Background/Aims:
Both molecular and clinical evidence support a link between
the hepatitis C virus (HCV) infection and insulin resistance. We examined the
in vitro interaction between the HCV core protein of genotypes 1b and 3a with
the insulin signalling pathway.
Methods:
We measured the levels of insulin receptor substrate 1 (IRS-1),
IRS-2 and other factors involved in the insulin signal transduction in human
hepatoma cells (Huh-7) transiently expressing the HCV core protein of genotypes
3a or 1b by different molecular biology and immunofluorescence techniques.
Results:
IRS-1 (but not IRS-2) protein level was significantly reduced
in Huh-7 expressing the core protein of both genotype 3a (P= 0.0067) and 1b (P=
0.04) as compared to cells transfected with the empty pIRES2-EGFP vector. IRS-1
degradation was associated with an increased phosphorylation at Ser636/639.
However, whereas the core protein of genotype 3a promoted IRS-1 degradation by
upregulating the suppressor of cytokine signal 7 (SOCS-7) and the
downregulation of peroxisome proliferator-activated receptor γ (PPARγ),
the downregulation of IRS-1 by the core protein of genotype 1b proceeded
through the activation of the mammalian target of rapamycin (mTOR). These
findings were confirmed by using specific inhibitors (siRNAs for SOCS-7,
rapamycin for mTOR) or agonists (rosiglitazone for PPARγ).
Conclusions:
Despite the little sequence divergence of the HCV core
proteins of genotypes 3a and 1b, the two proteins seem to interfere with the in
vitro insulin signaling using genotype-specific mechanisms. This, coupled with
mounting clinical evidence, suggests an evolutionary advantage for HCV to
maintain an insulin resistant state.
#310. Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C.
J. M. Pestka; M. B. Zeisel; P. Schürmann; B. Bartosch;
F. Cosset; A. H. Patel; H. Meisel; J. Baumert; S. Viazov; K. Rispeter; H. E.
Blum; M. Roggendorf; T. F. Baumert.
Background and aim:
In contrast to a detailed understanding of antiviral cellular
immune responses, the impact of neutralizing antibodies for resolution of acute
hepatitis C is poorly defined. The analysis of neutralizing responses has been
hampered by the fact that patient cohorts as well as HCV strains are usually
heterogeneous and that clinical data from acute-phase and long-term follow-up
after infection are not easily available.
Methods:
Using an infectious retroviral HCV pseudo-particle model
system, we studied a cohort of women accidentally exposed to the same HCV
strain of known sequence.
Results:
In this single-source outbreak of hepatitis C virus infection
in East Germany 1978-1979, viral clearance was associated with a rapid
induction of neutralizing antibodies in the early phase of infection.
Neutralizing antibodies decreased or disappeared following recovery from HCV
infection. In contrast, chronic HCV infection was characterized by absent or
low-titer neutralizing antibodies in the early phase of infection and
persistence of infection despite the induction of cross-neutralizing antibodies
in the late phase of infection.
Conclusions:
These data indicate that rapid induction of neutralizing
antibodies during the early phase of infection may play an important role for
control of viral infection and contribute to HCV clearance. This finding may
have important implications for understanding of the pathogenesis of HCV
infection and the development of novel preventive and therapeutic antiviral
strategies.
#313. Insulin Resistance and Liver Fibrosis in Virus C Chronic Hepatitis and in Nonalcoholic Fatty Liver Disease.
G. Svegliati-Baroni; E. Bugianesi; E. Peruzzi; F.
Ridolfi; F. Tarsetti; F. Ancarani; E. Petrelli; E. Brunelli; M. Lo Cascio; M.
Rizzetto; G. Marchesini; A. Benedetti.
Background:
Insulin resistance, the hallmark of nonalcoholic fatty liver
disease (NAFLD), is also frequently found in patients with chronic HCV
hepatitis (CHC), and has been associated with histological liver damage
(steatosis and fibrosis).
Aims:
To examine the relationship between histological findings and
biochemical parameters of insulin resistance in CHC and NAFLD patients.
Methods:
We assessed the degree of basal insulin resistance (by the
homeostasis model assessment, HOMA-R) and post-load insulin sensitivity (by the
oral glucose insulin sensitivity index, OGIS) in 90 patients with CHC (23
genotype 3) and in 90 pair-matched patients with NAFLD. Basal and post-load
insulin resistance were defined as HOMA-R ≥ 2.7 and OGIS ≤ 9.8
ml/kg*min, respectively corresponding to the upper and lower quartile of a control
population. Steatosis was scored according to Brunt in both groups, fibrosis
according to Brunt in NAFLD and to Ishak in CHC.
Results:
Severe steatosis (grade 3) was associated with HOMA-R (OR
4.42; CI 1.16-16.85; P=0.029) in NAFLD and with HOMA-R (OR 14.87; CI
1.17-89.70; P=0.038) and OGIS (OR 7.43; CI 1.25-44.07; P0.027) in genotype
non-3 CHC, but not in genotype 3. After adjustment for age, gender and BMI, in
NAFLD severe fibrosis ( stage 3-4) was predicted by elevated aminotransferases,
fasting hyperglycemia, basal and post-load insulin resistance and steatosis at
univariate analysis, but only by OGIS ≤ 9.8 (0.56; 0.35 – 0.91; P =
0.019) at multivariate analysis. In CHC, OGIS ≤ 9.8 (OR 9.43; CI
1.43-62.13; P=0.020) was the sole independent predictor of severe (stage 4-6)
fibrosis. When split according to genotype, post-load insulin resistance was
associated with severe fibrosis only in genotype non-3 patients.
Conclusions:
Post-load insulin resistance (OGIS ≤ 9.8 ml/kg*min) is
an independent predictor of severe fibrosis in NAFLD and genotype non-3 CHC and
represents a useful tool to select patients who may benefit of
insulin-sensitizing therapy.
#317. Inhibition of Hepatitis C viral (HCV) replication by in-vivo dimerization of STAT1.
X. Li; H. Zhu; M. Butera; D. R. Nelson; C. Liu.
Background:
It is known that type I interferons (IFN) induce
intracellular antiviral state via the JAK-STAT signaling pathway. Dimerization
of STAT1, STAT2, and STAT3 are key steps in this pathway. However, the precise
role for each individual STAT in antiviral defense is not completely defined.
Aim:
The goal of this study is to determine the role of STAT1
homodimers in the establishment of intracellular antiviral activity.
Methods:
To create an inducible STAT1 dimerization system, STAT 1 open
reading frame is fused with estrogen receptor (ER) domain, resulting in
STAT1-ER fusion expression construct. The fusion protein dimerization is
inducible by estrogen analog (4-HT). The construct was then transfected into
HCV replicon cell line, FL-Neo. Various doses of 4-HT were incubated with the
cells and the STAT1-ER dimerization was monitored by Western blot analysis
using an anti-STAT1 antibody. The target genes of the STAT1 dimers were
examined by cDNA microarray analysis and real-time RT-PCR assay. The effect of
STAT1 homodimers on HCV replication was determined by HCV-specific real-time
RT-PCR assay.
Results:
The STAT1-ER fusion protein formed homodimers with 4-HT stimulation.
The amount of dimers is positively correlated with the doses of 4-HT. By
transfection assay, the formation of STAT1 homodimers substantially inhibited
HCV full-length RNA in correlation with the formation of homodimers indicating
the establishment of antiviral activity in the replicon cells. The dimmers also
induced many interferon-stimulated genes (ISGs) in human hepatoma cells.
Conclusions:
We have established an inducible and cytokine-independent STAT1
activation system. This system would provide a valuable tool to understand the
molecular events triggering the intracellular anti-HCV activity in liver cells.
The system would also allow us to identify STAT1-specific target genes.
#318. Hepatitis C virus is present in CD4+ T cells of chronically infected patients impairing their IFN-g production.
A. Perrella; A. D'Antonio; C. Esposito; D. Vergani; S.
Grattacaso; C. Sbreglia; L. Atripladi; A. Di Spirito; D. Guarnaccia; O.
Perrella.
Background/Aim:
HCV, a hepatotropic RNA virus responsible for frequent
evolution to chronic hepatitis, impairs IFN- gamma production by T helper 1
lymphocytes (Tsai T et al. Hepatology
1999). HCV has been recently shown to infect B cells, monocytes and dendritic
cells. We aimed to investigate whether HCV also infects CD4+ T lymphocytes,
replicates in them, and influences IFN-gamma production.
Methods:
We enrolled 20 patients with histologically proven (Grade 10,
Stage 2) chronic hepatitis C (CHC) (Group A) and 10 healthy blood donors as
controls (Group B). We measured HCV-RNA by real-time PCR (Amplicore Roche 2.5
qualitative and quantitative system; cut off 50 IU/mL and 600 IU/mL
respectively) in PBMC and CD4+ T cells (Dynal CD4+ separation kit; purity ≥90%)
before, after 6-day stimulation with HCV (Core and NS3) and Influenza A (InfMp)
peptides (2mcg/mL each), and after a further 2-days stimulation with PMA and
ionomicin. IFN-gamma production by CD4+ T cells was assessed by an ELISpot
assay (PMA and Ionomicin stimulation).
Results:
PBMC and CD4+ T cells from CHC patients were positive for
HCV-RNA by qualitative assay before stimulation with HCV peptides, while
healthy donors were negative. After 6-day stimulation, HCV-RNA was detectable
by both qualitative and quantitative assay (752 +/- 46 IU/mL) in CD4+ T cells
exposed to HCV peptides but not in those exposed to InfMp peptides. HCV-RNA
levels in CD4 cells increased to 1856 +/- 125 IU/mL after further stimulation
with PMA ionomicin, these cells showing a reduced IFN-gamma production compared
to CD4+ T cells stimulated with InfMp viral peptide (188 +/- 82 vs 322 +/- 128
SFC - U Mann Whitney p < 0.01)
Conclusion:
These preliminary results show that in patients with CHC, the
virus is present and replicates in CD4+ T cells impairing their IFN-gamma
production. This impairment may promote chronic evolution of the infection.
#330. Treatment of Cirrhotic Patients with HCV Referred for Liver Transplantation Using an Escalating Dose Regimen of Pegylated Interferon Alpha-2a and Ribavirin.
H. Massoumi; H. Elsiesy; B. Peterson; V. Khaitova; E.
Norkus; P. Grewal; L. Liu; P. Martin; P. Lopez; N. Bach; T. Schiano.
Introduction:
The rapid progression to cirrhosis and poor tolerability of
interferon post liver transplant (LT) have necessitated the consideration of
treating HCV with PEG-IFN and ribavirin in cirrhotic pts. At the Mount Sinai
Medical Center, we have initiated a protocol using PEG in pts seen in the
transplant office. The aim of our study was to assess the safety and efficacy
of this treatment.
Method:
90 cirrhotic pts were prospectively treated from 2/03 to
4/06. Exclusion criteria included co-infection with HBV or HIV or renal
insufficiency. We used an escalating dose regimen starting with 90 mcg of
PEG-IFN alpha-2a and 400 mg of ribavirin and advanced to 180 mcg and 800-1200
mg respectively over a period of 8 weeks. Hematopoietic growth factors were
started if hemoglobin was <12 or ANC was <750. Treatment was stopped if
platelet count was <20,000. Pt demographics, comorbidities, BMIs, lab
values, MELD and Child’s scores, CT liver volumes, complications and outcomes
were recorded.
Results:
Mean age was 55.3 ± 6.9 years, 69% males, and 53% whites 23% Hispanics,
16% African Americans. 34% had a history of prior treatment with IFN. 76% had
genotype 1 or 4. Mean Child’s score was 6.7 ± 2 and mean MELD 11.2 ± 3.7. 18%
of pts needed dose reduction, 30% stopped treatment due to adverse effects, 11%
had hepatic decompensation, 7% died and 14% underwent LT. Epoetin or
darbepoetin was used in 58% and filgrastim in 31% of pts. End of treatment
response (ETR) was seen in 65% of pts. During a mean follow up period of 9.6
months, 40% of responders had virological relapse; 4/32 pts followed for >
6months after therapy had SVR.
Pts who stopped PEG had significantly smaller liver volumes
(1366 ± 279 vs 1738 ± 561) (p=0.005). Child’s score (p=0.002) and MELD (p=0.03)
were strongly predictive for discontinuation of PEG; Child’s score appearing to
be a stronger predictive factor than MELD. Rate of serious complications was
22% in Child’s class A, 53% in class B and 100% in class C(p<0.0005);
Child’s C pts had a 15 fold increase risk for hepatic decompensation or death.
Every 1 g/dl lower baseline serum albumin below 4.8 predicted a 96% higher risk
of hepatic decompensation or death. No pt had significant bleeding related to
low platelet count (mean platelet count 74 ± 52×1000; range of 18-346×1000).
Conclusion:
Using an escalating dose regimen of PEG-IFN alpha-2a and
ribavirin and aggressive use of hematopoietic growth factors, we achieved a 65%
ETR in cirrhotic pts. These results were tempered by 14% risk of hepatic
decompensation or death and significant relapse rate. Serum albumin and Child’s
score were predictive of hepatic decompensation or death.
#331. Identification of candidate genes that mediate depressive side effects of pegylated IFN-αlpha 2a in patients with chronic hepatitis C.
M. Trippler; Y. Erim; S. Bein; G. Gerken; J. F.
Schlaak.
Aims and background:
Combination therapy with pegylated interferon (IFN)-alpha
plus ribavirin has been shown to be the most effective treatment for chronic
hepatitis C (HCV). One of the most common side effects is IFN-induced severe
depression, which can impair quality of life, reduce treatment adherence and
even lead to suicide. This study assessed the primary transcriptional response
to IFN-alpha-2a plus ribavirin in HCV patients to identify possible candidate
genes that mediate the depressive side effects of IFN-α.
Patients and methods:
A total of 18 Caucasian patients with histologically proven
chronic hepatitis C were treated with standard combination therapy consisting
of pegylated IFN-α2a (Pegasys, 180µg once weekly) for 12 months (HCV
genotype 1, n=14) or 6 months (HCV genotype 2/3, n=1/3) in combination with
ribavirin (800-1200 mg daily). RNA was isolated (PAXgene, PreAnalytiX) from
peripheral blood which was collected 12h before and 12h after the first
injection of IFN-α. The transcriptional profile was analysed using human
genomic microarrays (Affymetrix HG U133A) and quantitative real-time RT-PCR.
Array data were normalized (RMA Express software) and fold changes were
calculated from before and after IFN injection. Fold change values were
subjected to significance analysis (SAM software) and class prediction analysis
(PAM software). The patients were investigated using the Mini-DIPS, a
semi-structured interview, which facilitates relevant diagnostic criteria
according to the four axis of the DSM-IV. Furthermore depression scores were
evaluated with psychometric instruments, Beck`s Depression Inventory and
Hospital Anxiety and Depression Scale.
Results:
8 patients were non-responders (NR) to therapy while 10
patients had a sustained virological response (SVR). 8/18 patients suffered
from IFN-induced depression. Using class prediction analysis, the development
of an IFN-induced depression could be predicted by 24 genes with 95% accuracy.
9 genes were significantly higher expressed in patients with IFN-induced
depression compared to patients without depression. 6 of these genes were
identified as typical interferon response genes. Interestingly, 3 of these 9
genes were previously described as being associated with recurrent major
depression.
Conclusions:
These data suggest a direct role of IFN response genes in
generating depression as side effect of antiviral therapy. Finally, the
functional analysis of the differentially regulated genes that were identified
in this study could lead to the discovery of novel drug targets to improve the
efficacy of and adherence to HCV therapy.
#332. The Nonstructural 5A (NS5A) Protein of Hepatitis C Virus Genotype 1b Does Not Contain an “Interferon Sensitivity Determining Region”.
R. Brillet; F. Penin; C. Hezode; P. Chouteau; D.
Dhumeaux; J. Pawlotsky.
Introduction:
Various explanations have been forwarded for differences in IFN-alpha-based treatment outcomes. Together with a number of host parameters and disease characteristics, virus-related factors play an important role in the likelihood of permanent viral clearance after therapy. The nonstructural protein 5A (NS5A) of HCV has been suggested to contain an “interferon sensitivity determining region“ (ISDR). If NS5A indeed contains an ISDR, the latter would be expected to impact mainly on the initial viral decline, meaning that the degree of viral decline on day 1 should be associated with qualitative or quantitative differences in NS5A functions and, thus, in the amino acid sequences that subtend these functions.
Aim:
This hypothesis was tested by studying patients receiving their first injection of standard IFN alpha, the IFN molecule with the most rapid initial antiviral effect.
Results:
We studied whether the degree of viral decline on day 1 is associated with differences in amino acid sequences that subtend NS5A protein functions in 16 patients receiving their first IFN injection (11 responders, 5 nonresponders). Phylogenetic analyses of the full-length protein and of particular functional domains showed no relationship between the baseline protein sequence and the antiviral response. We analyzed nonstructural protein 5A quasispecies sequences amino acid by amino acid, and studied the physicochemical properties of the proteins. The sequences showed no differences in the number of mutations in the putative ISDR relative to the prototype HCV-J sequence between responders and nonresponders, or according to IFN antiviral efficacy. No relationship was found between antiviral efficacy at 24 hours and the baseline sequence of any region of the protein. Amino acid changes were observed in a few cases at 24 hours in both responders and nonresponders, but no consistent pattern of amino acid shifts was observed, ruling out the possibility that IFN administration selected “IFN-resistant“ variants.
Conclusion:
Our findings show that there is no “interferon sensitivity determining region” in the NS5A protein of HCV genotype 1, and that the NS5A sequence does not influence the capacity of IFN to block viral replication. They do not rule out a role of NS5A in subsequent viral clearance, through effects unrelated to IFN resistance.
#335. Improved sustained virological response (SVR) rates with higher, fixed doses of peginterferon alfa-2a (40KD) (PEGASYS®) plus ribavirin (RBV)(COPEGUS®) in patients with “difficult-to-cure” characteristics.
M. Fried; D. Jensen; M. Rodriguez-Torres; L. Nyberg;
A. Biscegli; T. Morgan; P. Pockros; A. Lin; L. Cupelli; D. Nelson.
Introduction:
Patients with HCV genotype 1 (G1) and high viral load (HVL)
have historically been considered “difficult-to-cure” with response rates
<50%. High bodyweight also negatively impacts SVR for all interferon-based
therapies, although to a lesser degree. Thus, G1 pts with HVL and above average
bodyweight represent a subgroup (≈20% of total population) for which
improved treatment strategies are needed.
Aim:
The aim of this study was to determine if intensification of
treatment using higher, fixed doses of PegIFNα-2a and RBV in patients with
these treatment-resistant characteristics provide increased benefit with
manageable risk.
Methods:
In this prospective, controlled pilot study, treatment-naďve
HCV G1 adults with HCVRNA >800,000IU/mL and bodyweight >85kg were
randomized to 48wks of either PegIFNα-2a 180 or 270µg/wk + either 1200 or
1600mg/d RBV. SVR=undetectable (<50IU/mL) HCVRNA at 24wks post-therapy.
Results (Table):
188 pts, from the United States, were randomized and
treated—follow-up is available for 143 (76%); ≈18% of pts in each arm had
bridging fibrosis/cirrhosis. Similar end-of-treatment response rates were seen
in the 3 test arms; however the SVR rate was highest (47% vs 28%) with the most
intensive regimen: PegIFNα-2a 270µg/wk + RBV 1600mg/d, compared to standard
dosing. Improved SVR was driven by a lower relapse rate (40% vs 19%). Compared
to standard dosing, this regimen was associated with an increased incidence of
some hematological laboratory abnormalities, dose modifications for labs and
AEs and premature withdrawals for AEs.
Conclusions:
In pts with treatment-resistant characteristics (G1, HVL and
bodyweight >85kg), higher fixed doses of both PegIFNα-2a (40KD)
(270µg/wk) and RBV (1600 mg/d) led to a substantial increase in SVR rate (19%
more than standard dosing) but with some negative safety trends. The benefit of
an increased SVR rate with higher fixed doses of PegIFNα-2a (40KD) and RBV
should outweigh the increased potential for adverse events/lab abnormalities.
These results provide the rationale for future studies attempting to optimize
treatment response using higher doses of PegIFNα-2a and RBV and suggest
opportunities to further improve benefit/risk in patients with unfavorable
treatment characteristics.
|
|
PegIFNα-2a
(40KD) 180 µg/wk + |
PegIFNα-2a
(40KD) 270 µg/wk + |
||
|
RBV 1200 mg/d |
RBV 1600 mg/d |
RBV 1200 mg/d |
RBV 1600 mg/d |
|
|
Mean age, yrs |
47 |
50 |
47 |
49 |
|
Virological response, % (ITT) |
48 46 |
38 57 |
53 55 |
51 55 |
|
Relapse (%) |
40 |
42 |
46 |
19 |
|
Serious adverse events (%) |
9 |
13 |
13 |
11 |
|
Lab abnormalities (n, %) |
7(15) |
7(15) |
4(9) |
11(23) |
|
Dose modifications for adverse events or
lab abnormalities (%) |
20 |
26 |
||