Posters – Tuesday
May 23, 2006 8:00AM Hepatitis C
HCV Virology, Pathogenesis and Treatment
S. Carney; W. M. Lee; S. Ray; D. Oldach;
M. Gale
The
interferon regulatory factor 3 (IRF3) pathway plays a central role in the
clearance of virus from infected host cells. When activated, this pathway initiates
transcriptional induction of interferon β (INF β) and hundreds of
interferon stimulated genes (ISGs), which act to
disrupt viral replication and to modulate the adaptive immune response. IRF-3
activation is dependant on signaling from virally
activated pathogen associated molecular pattern receptors though the signal
transduction molecule interferon-beta promoter stimulator 1(IPS-1). The
hepatitis C (HCV) NS3/4A protease blocks IRF3 activation by cleaving and
inactivating IPS-1, thus disrupting the host cell’s antiviral defense.
The
NS3/4A coding region exhibits significant sequence variation among genotypes
and quasispecies. This is reflected in functional differences in the ability to
cleave synthetic substrates demonstrated by NS3/4A isolates from different
genotypes. Differences in amino acid structure may also give rise to phenotypic
differences with respect the degree to which NS3/4A blocks the IRF3 pathway. We
hypothesize that variation in viral ability to inhibit the IRF3 pathway may
play a role in determining viral persistence following acute infection. We
propose that patients who exhibit spontaneous clearance are infected with
quasispecies variants that are less efficient inhibitors of the IRF3 pathway,
while viral quasispecies variants that efficiently inhibit the IRF3 pathway are
able to subvert the host immune response and produce chronic infection.
We
have previously identified less fit viral variants that demonstrate impaired
ability to inhibit IRF-3 activation in vitro. We are now conducting studies of
clinical isolates of the NS3/4A protease cloned from two groups of patients who
were acutely infected with HCV, one group that that spontaneously cleared the
virus without treatment and one that developed chronic infection. The dominate
NS3/4A quasispecies variants isolated from these patients show significant
differences in amino acid sequence. They are all capable of cleaving IPS-1 when
overexpressed in cultured cells. However, they show
variation in the degree to which they block the production of INF β in
response to viral challenge. Further assays to assess if this variation in
inhibition of host cell antiviral response is due to functional variation in
the protease are on going. Results of these studies and analysis of NS3/4A amino
acid sequence evolution over the course of infection will be presented along
with clinical correlation to viral persistence verses clearance following acute
infection.
Abstract 520 – HCV can infect T cells and
cause suppression of IFN-γ signaling
Y. Kondo; K. Machida; M.
Liu; G. Y. Yu; T. B. Macnaughton; M. M. Lai
Background
Several
groups reported that HCV can infect and replicate in primary human T cells and
the established T cell lines. But the influence of possible HCV infection on T
cells is still unclear due to the lack of proper infection and detection
systems. Previously, our group established an SB cell line that produces HCV
particles with preferential lymphotropism.
Aim
To
establish a proper HCV T cell infection system that can be used for studying
the effects of HCV on T cell signaling. [Methods]
(Cell culture) SB culture supernatant was used for infection of Raji (B cell), Molt-4 (T), Jurkat
(T), and primary human peripheral blood cells. We analyzed infected cells
sequentially every 3 days. (Intracellular HCV RNA) Positive and negative strand
HCV RNA was semi-quantified by using rTth RT-PCR and
nested PCR. (HCV RNA in the culture supernatant) HCV RNA in the culture
supernatant was quantified by real-time PCR. (NS5A Detection) NS5A protein was
detected by confocal laser microscopy. (Human primary
T cell) Several kinds of lymphocytes were isolated by FACS sorting and
cultivated under appropriate cytokine conditions.
(IFN-γ
signaling) STAT1α and P-STAT1 proteins were
analyzed by western blotting. Real-time PCR was carried out with established
primers and probed for STAT1 and T-bet mRNAs at several time points after the
IFN-γ stimulation.
Results
We
detected both positive- and negative-strand HCV RNA in T cell lines transiently
after in vitro infection. The titre of HCV RNA in Molt4 culture was similar to
that in Raji culture (B cells) up to day14.
Approximately 80% of Raji, 56% of Molt4 cells were
infected after 14 days in culture. Moreover, infected Molt4 could produce
infectious HCV particles. We thus established that HCV produced from SB cells
could infect and replicate in Molt4 cells. Furthermore, human primary T
lymphocytes, particularly, naïve (CD45RA+CD45RO-) CD4+cells, could also be
infected with HCV. The amount of STAT-1 and phosphorylated
-STAT1 in the HCV-infected Molt4 was much lower than those in the controls,
while the amount of its mRNA remained nearly the same. Moreover T-bet and STAT1
mRNA expression levels after IFN-γ stimulation in HCVinfected
Molt4 were lower than those in the controls, indicating a suppressed response
to IFN-γ.
Conclusion
We
established that T cells could be infected with HCV. HCV replication suppressed
the IFN-γ/STAT1/T-bet signaling due to the
decrease of STAT1 protein. Replication of HCV in T cells might contribute to
disturbance of Th1 commitment or Th1 hyporesponsiveness.
S. Smyk-Pearson;
D. Lezotte; H. Rosen
Background:
In some exposed individuals, the adaptive immune
response can spontaneously eradicate HCV infection. Development of vaccine
candidates to prevent spread of this disease remains a top priority.
Methods:
We synthesized 750 genotype 1a peptides spanning the
entire HCV genome (15 mer-peptides, overlapping by 11
amino acids, aa), and these were pooled into 33
distinct subgenomic pools. Using a highly sensitive
IFN-gamma ELISPOT assay, we characterized total and sub-genomic HCV-specific
CD4+ and CD8+ T cell responses in a cohort of HLA diverse subjects with chronic
(n = 25) and spontaneously resolved (n = 25) infection.
Results:
Receiver operating characteristic (ROC) analyses were
used to display the results of sensitivity and the false positive error rate
(1- specificity) of CD4+ and CD8+ T cell responses as predictors of spontaneous
recovery. Total HCV-specific CD4+ T cell IFN-producing responses were highly
predictive (area under ROC curve = .912, p < .0001) of recovery as were
responses to individual gene products, in particular nonstructural (NS) 3 and
NS5. The cut-off value for total HCV-specific CD4+ T cells where the false
positive error rate is minimum and sensitivity is maximum is 752 HCV-specific
CD4+ T cells//106 total CD4+ T cells).Individuals exposed to HCV who exceeded
this threshold are 23-fold more likely to resolve HCV infection spontaneously
as compared to those individuals who did not. Further, we built a mathematical
model that was highly predictive of recovery (p < .0001, AUROC 0.89) using
CD4+ T cell responses to E1A (aa 193-299), NS3 3H (aa 1173-1279), and NS3 5H (aa
1369-1479). As shown in Table, there were 8 probability levels with different
sensitivity/specificity levels based on the combinations of responses to these three
pools.
Conclusions:
By performing whole HCV genome mapping, this study
provides the first clear evidence that a quantitative T cell threshold exists
above which spontaneous recovery occurs following HCV infection. Regions
identified as highly immunogenic might represent indispensable components of a
prophylactic vaccine.
|
Probability
level |
E1 A
response |
NS3 3H
response |
NS3 5H
response |
Positive
Predict. value |
|
.998 |
- |
+ |
+ |
100% |
|
.96 |
+ |
+ |
+ |
100% |
|
.931 |
- |
+ |
- |
100% |
|
.903 |
- |
- |
+ |
95% |
|
.390 |
+ |
+ |
- |
90% |
|
.306 |
+ |
- |
+ |
77% |
|
.17 |
- |
- |
- |
52% |
|
.01 |
+ |
- |
- |
50% |
Abstract 522 – Binding of the
HCV E2 protein to CD81 impairs migration of dendritic cells towards the lymphotropic chemokine CCL21
J. Nattermann;
H. Zimmermann; M. von Lilienfeld-Toal; L. Leifeld; T. Sauerbruch; U. Spengler
Background:
Dendritic
cells (DC) are crucially involved in the initiation of immune responses.
Reports on DC dysfunctions in chronic hepatitis C are controversial but may be
the result of interaction of HCV E2 with the tetraspanin
CD81. Recruitment of mature DC to lymphatic tissues, which requires
interactions between CCR7 and the lymphoid chemokine
CCL21, is a crucial step for presentation of antigens and priming of T-cells.
However, little is known about the effects of HCV infection on the migratory
capacity of DC.
Methods:
Monocyte-derived
dendritic cells (MODC) were generated following
standard protocols and stimulated with anti-CD81 or HCV E2 protein. DC
migration towards CCL21 was analyzed using a nitrocellulose filter micro
chamber system. Markers of DC maturation and differentiation as well as surface
expression of integrins were analyzed by flowcytometry.
Results:
We
found that cross-linking of CD81 by HCV E2 dramatically impaired migration of DC
towards CCL21, a chemokine importantly involved in DC
trafficking to lymph nodes. This effect could be blocked by pre-incubation with
anti-CD81 mAb. HCV E2-induced impairment of DC
migration was neither due to altered maturation of dendritic
cells nor due to down-modulation of CCR7, the recepetor
for CCL21. Furthemore, surface expression of the integrins CD29, CD18 and CD11a,which are critically
involved in DC migration, where also not affected by CD81 binding of HCV E2.
Conclusion:
We
found that cross-linking of CD81 by HCV E2 dramatically reduces the migratory
responses of dendritic cells to CCL21 by a hitherto
unidentified mechanism. This effect reduces recruitment of DC to lymphoid
tissues in hepatitis C possibly contributing to impaired priming of T cells.
R. S. Brown; I. M. Jacobson;
N. Afdhal; B. Freilich; F.
Regenstein; S. Flamm; P. Kwo;
M. Pauly; L. H. Griffel; C.
A. Brass
Background:
Genotype 2 (G2) and 3 (G3) chronic
hepatitis C virus (HCV) have high sustained virologic response (SVR) rates to peginterferon (PEG-IFN) alfa and ribavirin and require
shorter duration of treatment. The optimal dosing of ribavirin and differences
between SVR in G2 and G3 is undefined.
Objective:
To determine the predictors of
response to PEG-IFN alfa-2b plus ribavirin in patients with HCV G2 and G3.
Methods:
WIN-R, a multicenter,
randomized, open-label, investigator-initiated trial in 225 US sites between
12/00 and 6/05 randomized treatment-naïve adults HCV patients with compensated
liver disease to receive PEG-IFN alfa-2b 1.5 μg/kg/week
plus FD ribavirin (800 mg/day) or WBD ribavirin (800 mg/day for weight <65
kg, 1,000 mg/day for 65–85 kg, 1,200 mg/day for >85–105 kg, 1,400 mg/day for
>105–125 kg). G2 and G3 patients were also randomized to receive 24 or 48
weeks of therapy. HCV RNA was assessed at weeks 0, 24, 48 and 72. The primary
endpoint was SVR (absence of detectable HCV RNA at week 72) in patients
≥65 kg.
Results:
1829 G2/3 patients were enrolled and
randomized to WBD (24 wks n=317, 48 wks n=602) or FD (24 wks n=322, 48 weeks
n=588). SVR rates were similar in the WBD and FD groups (62 vs
60%, respectively) and were higher in the 24-week group (68 vs. 65%) than the
48 weeks (60 vs. 58%, respectively) due to a higher dropout rate after 24 weeks
of therapy with missing data in that group (SVR not known, treated as NR). G2
had a higher SVR and lower relapse rate than G3 (72 vs. 63% with 24 weeks of
WBD therapy and 5 vs. 10%, respectively). G3 patients had higher SVR with WBD
(57 vs. 52% in 24 week group) but this difference was not statistically
significantly. Relapse rates were highest in G3 high viral load patients
treated for 24 weeks (16%). Multivariate analyses revealed G2, less advanced
fibrosis, and 24 weeks of therapy as significant predictors of SVR. Safety and
rates of drug discontinuation were similar between the groups.
Conclusions:
Compared to Genotype 1 patients, WBD
ribavirin and 48 weeks of therapy offers less advantage to FD in combination
with PEG-IFN alfa-2b, in patients with HCV genotype 2 and 3. Compared to G2
patients, SVR rates are lower and relapse rates are higher for G3 patients. G3
patients may benefit from higher ribavirin dosing.
Abstract 524 – In vivo-IFNγ−mediated sensitization prevents TLR4
tolerance in monocytes of patients with chronic HCV
infection.
A. Dolganiuc;
G. Szabo
We reported that monocytes
from patients with chronic HCV infection produce elevated levels of
pro-inflammatory cytokines upon stimulation with lipopolysaccharide
(LPS), a Toll-like receptor (TLR) 4 ligand. Here we
hypothesized that TLR-mediated tolerance, defined as a decline in inflammatory
cytokine production after repeated challenges with a TLR ligand,
is lost in monocytes of HCV infected patients.
TLR-mediated tolerance normally prevents persistent inflammation.
We recruited 18 treatment-naïve
patients with chronic HCV infection and 24 controls. Serum endotoxin
was determined using a Limulus Assay. Monocytes were
pre-treated with LPS and re-challenged with LPS 24 hrs later. TNF production
was analyzed in ELISA, cellular proteins by western blot and RNA levels by
real-time PCR.
Unlike normal monocytes
(MO), Mo from HCV-infected patients failed to decrease LPS-induced TNFα production upon re-challenge with LPS suggesting
loss of TLR4 tolerance. This was not due changes in expression of the TLR4
receptor complex. Messenger RNA but not protein levels of TLR4, its adaptor
MyD88, and its co-receptors CD14 and MD-2 were elevated in patients’ MO
compared to controls. The negative regulators of TLR4 signaling,
including ST2, SIGIRR, PI3, SHIP, TWIST and TTP were comparable in MO of HCV
patients and controls suggesting that neither TLR4 receptor complex expression
nor these negative regulators of TLR contribute to increased cytokine
production in HCV MO. However, the level of IRAK-M, a monocyte-specific
negative regulator of TLR4, was significantly lower upon LPS stimulation in HCV
patients’ MO compared to controls. Furthermore, we found increased expression
of the IFNγ-regulated chemokine
genes, CXCL9, 10 and 11, in HCV patients’ MO. More important, IFNγ levels were increased in the sera of HCV patients
compared to controls. Sensitization of normal monocytes
with IFNγ resulted in elevated LPS-induced TNFα production, increased CXCL9, 10 and 11 mRNA
expression and loss of tolerance to LPS which was similar to responses observed
in HCV patients’ MO. Finally serum endotoxin levels
were also increased in HCV patients.
These data suggest that TLR4 signaling is affected at different levels in patients with
chronic HCV infection. Elevated serum IFNγ may
sensitize monocytes to elevated serum LPS, which
together with reduced induction of the negative regulator, IRAK-M, likely
contribute to loss of TLR4 tolerance and increased pro-inflammatory cytokine
production by monocytes of HCV infected patients.
Our results add valuable insights to
the origin of chronic, non-specific liver inflammation seen in patients with
chronic HCV infection.