Posters – Tuesday May 23, 2006  8:00AM  Hepatitis C

 

HCV Virology, Pathogenesis and Treatment

 

Abstract 519 – Comparative Studies of HCV NS3/4A Protease Regulation of Host Antiviral Response through Cleavage of IPS-1 in Spontaneous Clearance Verses Progression to Chronic Disease Following Acute Infection.

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.


Abstract 521 – Differential Antigenic Hierarchy and T Cell Threshold Associated With Spontaneous Recovery From HCV Infection: Implications For Vaccine Design

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.


Abstract 523 – Differences In Treatment Outcome To Antiviral Therapy Based On Genotype And Viral Load In Hepatitis C Genotypes 2 And 3 In The WIN-R Trial

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.