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CD8/CTLs Predict Response to IFN/RBV Therapy
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"Intrahepatic and peripheral blood virus-specific cytotoxic T lymphocyte activity is associated with a response to combination IFN-alpha and ribavirin treatment among patients with chronic hepatitis C virus infection"
Journal of Viral Hepatitis- March 2005
A. J. Freeman 1 , G. Marinos 2 , R. A. Ffrench 3 and A. R. Lloyd 11Department of Pathology, School of Medical Sciences, The University of New South Wales; 2Viral Hepatitis Research, Gastrointestinal and Liver Unit, The Prince of Wales Hospital; and 3Department of Immunology and Infectious Diseases, School of Women's and Children's Health, The University of New South Wales, Sydney, New South Wales, Australia
Summary-
This report describes an association between intrahepatic and peripheral blood cytotoxic T lymphocytes (CTL) activity present prior to receiving treatment, and a response to combination interferon- alpha (IFN- alpha ) and ribavirin therapy for chronic hepatitis C virus (HCV) infection. Recombinant vaccinia virus constructs were used to expand and detect cytotoxic effectors against the entire genotype 1a HCV polyprotein. Six patients with a sustained response to therapy were significantly more likely to display intrahepatic and peripheral blood HCV-specific CTL activity than patients who relapsed or had no treatment response. Limited longitudinal data suggested that rather than combination therapy acting to enhance the CTL response to achieve viral clearance, detectable CTL prior to treatment increases the likehood of the host responding to the direct antiviral activity of IFN- alpha and ribavirin.
INTRODUCTION
Hepatitis C virus (HCV) typically causes persistent infection and patients may ultimately manifest cirrhosis-related morbidity and mortality [1]. Virus-specific CD8+ cytotoxic T lymphocytes (CTL) localize to the liver of patients with chronic HCV infection and may pay a role in limiting viral replication [2-4]. In addition, it has been suggested that the baseline host CTL response may be important in determining the outcome of interferon-alpha (IFN- alpha ) monotherapy [5,6]. However, it has also been demonstrated that the antiviral effect of IFN- alpha is independent of CTL activity [2,7]. Combination treatment with IFN- alpha and ribavirin achieves viral clearance in a greater proportion of patients than with IFN- alpha alone [8]. The aim of this study was to examine subjects with chronic HCV infection who received combination therapy and to explore the relationship between CTL activity and response to treatment.
AUTHOR DISCUSSION
Similar to the results presented here, others have suggested that HCV-specific CTL may play a role in determining the outcome of therapy in patients with chronic HCV infection. In one study, seven of nine patients with detectable bulk intrahepatic CTL activity, as assessed prior to a course of IFN- alpha monotherapy, normalized serum alanine aminotransferase (ALT) levels during therapy, while only one of 10 with no CTL response had a treatment response [5]. In another study, responders demonstrated significantly higher HCV-specific CTL precursor frequencies in peripheral blood by ELISpot assay for TNF- alpha than nonresponders [6].
It has been postulated that treatment may promote viral clearance by enhancing the host CTL response [5,6]. However, against IFN- alpha playing a predominantly immunomodulatory role are the results from one study where CTL precursor frequencies against a range of HCV epitopes did not change during or after a course of IFN- alpha monotherapy [7]. In a related study, the overall CTL activity in 10 subjects decreased after initiation of treatment [2]. The longitudinal data from three treatment responders described here, and a cross-sectional study of HCV-specific CTL responses in patients after completion of a course of treatment (unpublished data), also suggest that CTL activity drops after removal of the antigenic stimulus in subjects that clear HCV viraemia. In addition, because HCV-specific CTL is detectable in the majority of patients with chronic HCV-infection, and spontaneous clearance of HCV is very rare, it is unlikely that the anti-viral activity of CTL alone, even if enhanced by IFN- alpha , is responsible for viral clearance in treatment responders.
Therefore, rather than acting to enhance CTL activity, detectable CTL prior to treatment may control viral replication and thereby make the host more susceptible to the direct antiviral activity of IFN- alpha and ribavirin. Consistent with this notion, HCV-specific CTL activity has been associated with low serum HCV-RNA levels [2-4], and low pretreatment viral load has been shown to predict a response to IFN- alpha monotherapy [10]. However, the small number of subjects investigated, and ethical considerations that prevented liver biopsies being performed during treatment, meant that enhancement of HCV-specific CTL in treatment responders during therapy could not be ruled out. A longitudinal assessment of CTL activity before, during and after combination therapy, incorporating a greater number of subjects, may help elucidate whether IFN- alpha and ribavirin act synergistically with a vigorous CTL response present at the initiation of treatment, or whether treatment is responsible for the induction of such a response. Considering that the association between CTL activity and response to treatment was observed using both intrahepatic and peripheral blood effector cells, conclusions may be able to be made in larger studies examining only CTL activity in blood at multiple time points during treatment. This would also facilitate using alternative assay systems, for example ELISpot assessment of ex vivo cytokine production, in studies of anti-HCV cellular immunity in relation to treatment.
In summary, among patients with chronic HCV infection, the presence of HCV-specific CTL prior to treatment is associated with viral clearance after a course of IFN- alpha and ribavirin. Sustained responders previously infected with HCV genotype 3 demonstrated significant CTL activity against genotype 1 proteins. This finding may have important implications for therapeutic vaccine development. If CTL responses to regions conserved across HCV genotypes can be demonstrated to be important in facilitating viral clearance, strategies to enhance activity against such epitopes prior to initiating antiviral treatment may improve outcomes for patients with chronic HCV infection.
RESULTS
Eight men and four women with chronic HCV infection were treated for 48 weeks with combination IFN- alpha and ribavirin therapy (Table 1). Eleven patients became serum HCV-RNA negative during treatment. Of these, six achieved a sustained response (SR) with absence of viraemia 6 months after cessation of therapy and five relapsed (RR). One patient showed no response (NR) with persistently detectable serum HCV-RNA.
By comparison with RR/NR patients, there was a nonsignificant trend for SR patients to be younger (mean age 39 vs 47 years, P = 0.06), have a lower viral load (median 5.7 vs 6.1 log10 copies/mL serum, P = 0.25; 4.6 vs 5.5 log10 copies/mg liver, P = 0.19) and display a greater number of lobular CD8+ T lymphocytes (mean 10.2 vs 3.3 lobular CD3+ cells/0.1 mm2, P = 0.06; mean 7.7 vs 4.4 lobular CD8+ cells/0.1 mm2, P = 0.31). There were no significant differences between the two groups with regard to sex, source and duration of HCV infection, alcohol consumption, anti-HCV antibody enzyme-linked immunosorbent assay (ELISA) sample/cutoff ratio, HCV genotype, serum transaminase levels and hepatic histology. Prior to treatment, patients who gained a SR were more likely to display intrahepatic (12/12 vs 6/12 assays positive, P = 0.01; 6/6 vs 2/6 patients with two positive assays, P = 0.06) and peripheral blood (10/12 vs 1/12 assays positive, P < 0.001; 5/6 vs 0/6 patients with two positive assays, P = 0.02) CTL responses.
Liver tissue was available to assess HCV-specific CTL activity in five patients 6 months after completing treatment (Table 2). Among three SR patients, there was a nonsignificant trend towards fewer positive assays and lower specific lysis (Fig. 1). In two RR patients detection of CTL activity was unchanged. In a larger cross-sectional study of CTL responses after treatment, there were no significant differences in terms of number of positive assays and specific lysis between SR, RR and NR patients (unpublished data).
METHODS
Patient population
Treatment naïve subjects with chronic HCV infection were recruited and investigated by immunohistochemistry and for HCV-specific CTL responses in liver and peripheral blood as reported previously [4]. If indicated, therapy with combination IFN- alpha -2a and ribavirin (Roche, Basel, Switzerland) was prescribed for 48 weeks. Response to treatment was based on absence of serum HCV-RNA as detected by RT-PCR (Amplicor; Roche Diagnostic Systems, Branchburg, NJ, USA). Return of viraemia within 6 months after cessation of therapy indicated relapse. In some patients, liver biopsies were performed 6 months after completing treatment. The local institutional ethics committee approved this study. All patients gave written, informed consent.
Assay for HCV-specific CTL activity
As described [4], intrahepatic and peripheral blood mononuclear cells (PBMC) were isolated and re-stimulated in bulk culture using recombinant HCV-vaccinia virus (vv) constructs encoding the entire genotype 1a HCV polyprotein (core-NS2 and NS2-NS5, gift from C.E. Rice) [9]. CTL activity was assessed on day six by chromium-release assay using autologous Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line targets infected with the same HCV-vv constructs and 'cold' target inhibition of EBV and vv reactivity. As described [4], intrahepatic CTL assays were considered positive if lysis was greater than 36% [effector:target cell ratio (E:T 50:1)] and peripheral blood assays positive if lysis was greater than 25% (E:T 100:1). Different cutoff per cent specific lysis for a positive assay and E:T ratios between liver and blood related to lower HCV CTL precursor frequencies in blood and the limited number of mononuclear cells available from liver biopsy specimens.
Statistics
Statistical analyses were performed using Mann-Whitney's nonparametric rank sum test for continuous variables, and Fisher's exact test for dichotomous variables. Data was analysed using Stata 7.0 (Stata Corporation, College Station, TX, USA). A P-value < 0.05 was considered significant.
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