HLA and response to IFN therapy in chronic HCV infection

430A

AASLD ABSTRACTS

HEPATOLOGYO c t o b e r 2 0 0 1

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LONG-TERM USE OF RECOMBINANT INTERLEUKIN 10 (IL-10) IN PATIENTS WITH ADVANCED CHRONIC HEPATITIS C: INSIGHT INTO IMMUNOPATHOGENESIS. David Nelson, Consuelo Soldevila-Pico, Manal Abdelmalek, Yiling Xn, Debbie Sampson, K J Kao, Gary Davis, Univ of Florida, Gainesville, FL

QUANTITATIVE AND QUALITATIVE CHANGES IN HCV-SPECIFIC LIVER INFILTRATING T-CELLS PARALLEL THE PROGRESSION OF LIVER DAMAGE IN CHRONIC HEPATITIS C. Ivana Mullerova, Eirini Rigopoulou, Philip Haigh, Helen Cooksley, Institute of Hepatology, UCL, London United Kingdom; Michaela Lucas, Paul Klenerman, Dept Medicine, J o h n Radcliffe Hospital, Oxford United Kingdom; Debbie Mesogiti, Julian Jenkins, G1axoSmithKline, Stevenage United Kingdom; Roger Williams, Nikolai V Naoumov, Institute of Hepatology, UCL, London United Kingdom Chronic HCV infection is characterized by a lack of spontaneous resolution of persistent viremia and a wide range of liver damage, varying from mild hepatitis to cirrhosis. The immune mechanisms underlying HCV persistence and progression of liver disease remaiu unknown. The aim of the present study was to determine directly (without in vitro expansion) the frequency and functional characteristics of HCV-specificCD4+ and CD8+ T cells in the liver and circulation at different stages of chronic hepatitis C. Methods: Peripheral b|ood mononuclear ceils (P13MC),liver infihrating lymphocytes (LIL) and hepatocytes were isolated from 35 patients with histologically proven chronic hepatitis C (all HCV RNA positive). According to the grade (G) and stage (S) of liver histology, the patients were divided into three groups: mild (G~--3,S 0-1, n= 10); moderate (G~:4, S 2-4, n=t0) and severe (G--~6, S 5-6, n=15) hepatitis. The frequency of HCV-specific CD4+ T cells (producing 1FN-%IL-5 or IL-10in response to HCV antigens - Core, NS3 andNS4) was enumerated using Elispot assays with the corresponding antibodies. In HLA-A2positive patients (n=20), the frequency of HCV-speeific CD8+ T cells was enumerated with peptides, corresponding to HLA-A2-restrietedCore and NS3 epitopes, using an Elispot assay, as well as by staining with an HLA-A2/NS3peptide tetramer and flow eytometric analysis.The CD4/CD8T ceU subsets in both compartments were determined by flow cytometry. The distribution of CD8+ T cells in the liver was visi~afizedby immunohistochemistry. The number of HCV RNA copies per hepatocyte and viremia levels (copies/ml) were determined in paired samples by real time TaqMan® PCR. Results: There was an inverse relationship between the frequency of HCV-specific [FN-',/producing CD4+ and CD8+ T cells in both compartments with the severity of chronic hepatitis C. The highest frequencies of these cells were found in patients with mild hepatitis: the averageproportion of CD4+ T cells specificfor Core, NS3 and NS4 was 0.03% of total CD4+ cells in PBMCand 0.18%of CD4+ cells in L1L;the averageproportion of CD8+ T cells specificfor Core and NS3 was 0.04% of total CD8+ cells in PBMC and 0.08% of CD8+ cells in LIE The low frequency of virus-specificCD8+ T cellswas confirmed by NS3 tetramer staining for both LIL and PBMC. With the progression of liver disease, virus-specific CD4+ and CD8+ T cells, which produce [FN-%decreased significantlyin both compartments (p= 0.01). Imporrant/y, the number of virus-specific T cells producing type 2 eytokines - CD4+ (IL-5, lL-10) and CD8+ (IL-10), showed the opposite changes with 7 to 10 fold greater frequency of IL-10 producing cells in cirrhotic patients than in mild hepatitis C. There was no difference in T cell responses to recall antigens at different stages of hepatitis C. HCV-specificT cells represented a minority of liver infiltrating lymphocytes,par ticularly at the stage of moderate and severe hepatitis. The CD4/CD8 ratio in LIL was reversed from 0.5-+0.1 in mild disease to 1.2-+0.1 in cirrhosis, indicating an increasing predominance of CD4+ T lymphocytes. The number of HCV RNA copies/hepatocyte (mean-+SEM)did not differ between patients with mild (6.0-+2.1), moderate (6.5+-2.1) or severe (5.6-+1.8) hepatitis and there was no difference in viremia levels (P> 0.1). Conclusion: These results provide direct evidence for the low frequency of HCV-specificCD4+ and CD8+ T cells, both in the liver and in circulation, which are unable to control HCV replication at any stage of chronic HCV infection. Furthermore, with the progression of liver damage there is an increasing imbalance of type 1/type 2 T cell reactivity to HCV, which permits chronic viral persistence and may explain the poor response to antiviral treatment in patients with more advanced stage of chronic hepatitis C.

Background: An imbalance in T h l and Th2 cytokine production is implicated in disease progression of chronic HCV. IL-10 is a Th2 cytokine that downregulates the proinflammatory response and may have antifibrotic activity. Aim: To determine the immunomodulating properties of IL-10 in patients with progressive liver disease. Methods: Thirty patients with extensive fibrosis or cirrhosis from HCV who had failed interferon-based therapy were enrolled in a 12-month treatment regimen with SQ 1L-10 given with daily or tiw dosing. Liver biopsies were performed before and at the end of therapy. Serum and PBMC were collected bi-monthly for HCV RNA, ALT, and functional T cell analysis. PBMC were evaluated for their specific response to HCV antigens (core, NS3, NS4, NS5) and HCV peptides (panel of 17 defined A2 and B7 epitopes) by IFN-gamma ELISPOT and 3H proliferation assays. All patients were followed for four months post-therapy. Results: Clinical: Twenty-eight patients completed at least 12 months of IL-10 therapy. IL-10 was well tolerated and led to significant improvement in serum ALT (mean ALT: day 0 = 142 + 17, month 6 = 6 2 + 6 , month 12 = 7 5 +- 10, and end off/u = 110-+16;p<0.05). Hepatic inflammation decreased by at least 2 in 13/28 patients (mean decrease for all paired samples from 4.6+0.3 to 3.7+0.3, p<0.05) and 11/28 showed a reduction in fibrosis (mean change from 5.0-+0.2 to 4.5+0.3, p<0.05). Viral: Mean serum HCV RNA levels increased by 0.5 log during the 12 months of therapy (mean HCV RNA levels: day 0: 12.3 +- 3.0 Meq/ml; 6 months: 25.6 + 6.6 Meq/ml; 12 months: 38 Meq/ml; p<0.05) and returned to baseline at the end of foUow-up (11.0-+2.4 Meq/ml). 5 patients developed viral loads of > 120 Meq/ml and two developed an acute flare in serum ALT (10 x baseline) that was associated with marked reduction in viral load ( > 2 log drop). Immunologic: IL-10 caused a marked decrease (2-4 fold) in the number of HCV-specific C D 4 + and CD8+IFN-gamma secreting T cells and a phenotypic change in PBMC cytokine production upon antigen stimulation from Th0/Thl to a Th2 dominant profile. These changes parallel the improvement in ALT and rise in HCV RNA. This effect was reversible with either discontinuation of the drug, or as in 2 of the 5 patients with viral load > 100 Meq, it occurred spontaneously with an ALT flare. Importantly, this acute ALT flare Corresponded with a 2 log decrease in HCV RNA and a dramatic 10-100 fold rise in HCV-specific CD4/CD8 IFN-gamma activity that was targeted toward NS3 and NS4, similar to that seen in spontaneously resolving acute hepatitis C. Conclusions: (1) Long-term rIL-10 therapy appears to decrease disease activity (improved inflammatory activity within the liver and normalization of ALT), but also leads to increased HCV viral burden via alterations in immunologic viral surveinance. (2) During longitudinal analysis, ALT and virallevel closely reflect the relative activity of the IFN-gamma HCV-specific T-cell response to nonstructural proteins. Implication: This data shows the important role of Thl/Th2 cytokines and the interplay with the HCV-specific T cell response in both viral control and disease progression.

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DIFFERENTIAL INTERFERENCE W I T H ANTIGEN-SPECIFIC T CELL RECOGNITION BY HCV-DERIVED PROTEINS. Anne Wertheimer, David M Lewinsohn, Portland VAMC, Portland, OR; H u g o R Rosen, Portland VAMC/ Oregon Health Sciences University, Portland, OR

HLA AND RESPONSE TO IFN THERAPY IN CHRONIC HCV INFECTION. Marion G Peters, University of California at San Francisco, San Francisco, CA; R a y m o n d Apple, Roche Molecular Systems, Alameda, CA; Heide A Sfirnadel, J o n a t h a n G Sheldon, Roche Discovery Welwyn, W E l w y n Uk; Palavi Patel, W a s h i n g t o n University, St Louis, MO; Jean Charles Ryff, J e a n Depamphifis, Roche Clinical Research a n d The Hepatitis Study Group, Garden City, NJ

Background: Hepatitis C virus (HCV) infection becomes chronic in the majority of cases, a n d the m e c h a n i s m s u n d e r l y i n g the evasion of the i m m u n e response a n d viral persistence remain poorly understood. Because we have observed diminished HCV-specific CD4 + T cell responses in subjects chronically infected with HCV, we hypothesized that HCV-derived proteins m a y interfere with M H C class II d e p e n d e n t antigen presentation. W e have developed a highly reproducible model system to ask functionally if the expression of the various HCV proteins results in an altered ability of antigen-specific T cells to recognize antigen. Methods: Recombinant vaccinia viruses expressing various HCV antigens (core, NS3, NS4, NS5a, NS5b) a n d m o n o c l o n a l antibodies to these antigens were obtained from C h i r o n (core, NS3, NS4, NS5b) or Virostat (NS5a). Cell line LCL d160, derived from B cells, was used as antigen presenting ceils. These cells were infected with wild type or r e c o m b i n a n t vaccinia viruses, pulsed with specific antigen (mycobaeterial derived MTb39), then cultured with autologous C D 4 + T cell clones reactive with MTb39. IFNg a m m a p r o d u c t i o n was measured b y ELISPOT. Western blots confirmed that infection of LCL cells with vaccinia containing the r e c o m b i n a n t HCV constructs resulted in the expression of the intended HCV proteins. Results: ELISPOT assays revealed that interferon g a m m a p r o d u c t i o n in response to specific antigen was consistently reduced b y at least 20% in the presence of vaccinia constructs expressing HCV NS5a a n d NSSb proteins, b u t not core, NS3 or NS4 proteins. Further experiments revealed the effects of the antigen dose a n d the multiplicity of infection (MOI) of the vaccinia constructs. The reduction in antigen specific recognition was detected at various antigen concentrations including 5ug, 2.5ng, 1.25ug, a n d 0.65ug; MOI levels r a n g i n g from 5 to 1 h a d a similar effect. Using FACS, we f o u n d that the expression of M H C Class I, Class II, CD 40, CD20 a n d CD19 was not altered a m o n g the various LCLs studied (uninfected, infected with wild type vaccinia a n d infected with vaccinia constructs expressing HCV NS5a a n d NS5b proteins). In addition, viability, as measured b y PI staining, was comparable. Conclusions: O a r data suggest that NS5a a n d NS5b are potent inbibitors of class II restricted antigen presentation. W o r k is ongoing to define potential mechanisms. Supported in part b y VA R.E.A.P. P r o g r a m Project

BACKGROUND: Chronic hepatitis C is the major cause of chronic hepatitis in the United States and an important cause of end-stage liver disease and hepatocellular carcinoma. The response of patients with chronic Hepatitis C infection to therapy depends upon both host and viral factors. Host factors that play a role include young age, female gender and absence of cirrhosis. Clearance of virus depends upon the rapid and appropriate recognition of viral antigens in conjunction with human histocompatability antigens (HLA) on the surface of antigen presenting cells or hepatocytes. AIM: We investigated the association between HLA and response to therapy with interferon. PATIENTS AND METHODS: 162 patients with chronic hepatitis C were studied. They were receiving interferon ALFA-2a (Hoffmann-La Roche Ltd, Basel, Switzerland)6MU for 3 mos, then 3 MU tiw for 3 or 9 mos. DNA was isolated from peripheral blood mononuclear ceils. HLA gentoyping for Class II DRB1, DQAI, DQB1 and DPB1 and Class I HLA-A locus was performed using sequence specific oligonucleotide hypbridizafion (PCR-SSO). Allele frequencies in the study population were recorded. Differences in allele and haplotype frequencies between sustained virologic responders (SR) and non-responders (NR) at end of treatment (ETR) and 6 months after cessation of therapy (SVR) were assessed using logistic regression modeling. Alleles with a frequency of greater than 5 were included the analysis. Viral genotype was included in the model as a covariate. RESULTS: There was no statistical difference in age, weight, ALT at entry between sustained virologic responders and nonresponders. Responders tended to be younger, thinner with higher pre-therapy ALT. Overall, HLA allele frequencies varied between SR and NR. Prevalence of haplotype DRBI*1301-DQAI*0103DQB1 *0603 was significantlyassociated with ETR and SVR as was DPBl*0401, both adjusted for viral genotype. The DPBI*0401 allele is uncommon in black subjects and remained strongly associated with SVR when adjusted for race. CONCLUSION: This study clearly demonstrated the importance of human genetic factors in treatment response. Beside HCV genotype, the most important indicators of SVR was the presence of DPBl*401 allele and the extended haplotype. This highlights the importance of host factors, including HLA genotype in determining the response to therapy.

.parameter *Haplotype DPBI*0461 Viral genoiype

Response,,,,, OR ETR 4.847 SVR 4.604 ETR 4,734 SVR 7.193 ETR 3.051 SVR 2,583

95% CI _ Ch!-square 1,406 16.713 6.2474 1.339 15.827 5,8732 1,890 tl.861 11,0108 2.247 23.021 11,0508 1.804 5.t60 17.3091 1.469 4,400 10,9768

p-value 0.0124 0,0154 0,0009 0,0009 0.0001 0,0009