Virus specific CTL activity after interferon monotherapy to chronic hepatitis C

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AASLD ABSTRACTS

HEPATOLOGY October 2001

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VIRUS SPECIFIC CTL A C T M T Y AFTER INTERFERON MONOTHERAPY TO CHRONIC HEPATITIS C. Koju Kobayashi, Tohoku Univ Coil of Medical Sciences, Sendal Japan; Masaaki Shiina, Motoyasu Ishii, Tooru Shimosegawa, Tohokn Univ Sch of Medicine, Sendal Japan; Toshio Takizawa, Yoshikazu Kikumoto, Otsuka Tokushima Third Inst, Toknshima Japan; Yoshiynki Ueno, Tohoku Univ Sch of Medicine, Sendal Japan

ARE CYTOKINE RESPONSES PROMISSING SUSTAINED RESPONSE TO INTERFERON-ALPHA ADMINISTERED IN CHRONIC HEPATITIS C PATIENTS ?. Koji Ishii, Naoko Takamura, Mie Shinohara, Hirokazu Shin, Yasuo Takeuchi, Yasunobn Nishio, Takashi Ikehara, Soichiro Hata, Takashi Kawarune, Yasukiyo Sumino, Toho University, School of Medicine, Tokyo Japan; Wataru Yamamuro, Internal Medicine, Saiseikai-kanagawaken Hospital, Yokohama Japan

BACKGROUND: Cytotoxic T lymphocytes (CTLs) against hepatitis C virus (HCV) participate in the eradication of hepatitis C virus. Relationship between CTLs and interferon efficacy needs to be addressed. [AIM] To investigate the role of CTLs in the eradication of HCV, we examined the activity of virusspecific CTLs in response to virus-peptide/HLA complexes. [METHOD] Peripheral blood mononuclear cells were obtained from 10 patients with chronic hepatitis C after interferon monotherapy (6 to 20 months after therapy). Five of these patients were sustained viral responders (SVR), while the remaining 5 patients failed to d e a r the virus (NR). All patients are positive for HLAA'2402. Six kinds of 9-mer virus peptides having HLA-A*2402 binding motif were synthesized. Conformation-stable complexes of peptides (each of 6 different peptides), j82-microglobulin and HLA-A*2402 were made and fixed on 96 well microtiter plate. CD8-positive cells positively selected by anti-CD8 antibody-conjugated magnetic heads from peripheral blood mononuclear cells were cultured on the plate. After incubation for 24 hours, supernatants were harvested, and the concentration of interferon-7 was measured by ELISA. Simultaneously, CD8-positive cells from 5 HLA-A*2402-positive healthy subjects were tested in the same way. RESULTS: In 3 of 5 SVR, their CD8-positive T cells produced interferon-T (73.3 + 26.6 pg/ml; mean ± SD) in response to several peptide/HLA complexes (4 to 6 epitopes per patient). On the other hand, the production of interferon- T was observed in one NR (9.35 pg/ml) in response to single peptide/HLA complex. CD8-positive cells from healthy subjects did not respond to any epitopes. Cytotoxic potential of these CD8-posirive cells after the stimulation with peptide/HLA complexes were further tested in slCr-release assay with HLA-A*2402 positive EBV-transformed B cell line. CD8-positive T cells from SVR that produced interferon- T in response to two peptide/HLA complexes showed cytotoxicity to target cells labeled with the same peptides. Conclusion: HLA-A*2402 is reportedly carried by more than half of Japanese population. CTL epitopes recognized in the context of HLAA ' 2 4 0 2 were defined by this work and it is indicated that peripheral CTLs are present in patients with SVR after interferon monotherapy.

Background: Interferon (1FN) therapy is only approved therapy to eliminate hepatitis C virus (HCV). IFN-alphahas direct anti-viral effects and immunomodulatoryeffects. We investigated whether immune responses to lFN-alpha administration in patients with chronic hepatitis C (CHC) related with successfuleliminationof HCV from body. PATIENTSAND METHODS:The present study included 31 CHC patients.SerumHCV typeswere dividedinto: type 1 (genotypela, lb), type 2 (genotype2a, 2b), and unclassifiedtypes.Theywere detectedin 12, 17, and 2 patients, respectively.In addition, they were negativefor serologicalmarkers of hepatitis Bvirus infection (absenceof ribs antigenand anti-HBcantibody)and pathologicallydiagnosed.Accordingto serum HCV-RNAquantifiedusing AmpficorPCR (Version1), CHC patients with equal or less than 100 k copies/mLwere 14, and those with more than 100 k copies/mLin serum HCV-RNAwere 17. Twenty-ninepatients receivednatural lFN-alphaor recombinantalpha-2b, 5-10 MU everyday for 2 weeksand 3 times a week for 22 weeks,and were followedup after treatment.IFN therapieswere completedin 24 of 29 patients and they were followedup 24 weeks after cessationof IFN. They were divided into 2 groups, according to the response to the IFN. Responder (12 patients) was defined as undetectableserum HCV-RNAat the end of follow-up(week 48). Non-responder (12 patients) was defined as detectablein serum HCV-RNAuntil week 48. Beforeand 2 weeks after IPN-alpha administration, serum interleukin (IL)-I beta, IL-6, lL-10, IL-18, TNF-alpha, and intracellular cytokine in CD4 positive T cells as described by Jung et al. (J Immunol Method 159:t97-207,1993) were measured.Flow cytometricdeterminationof IFN gammaand IL-4in the cytoplasm of peripheral CD4 positive T cells were performed, and relative prevalence of IFN gamma+ and IL-4- (Thl), IFN gamma-and IL-4+ (Th2), IFN gamma+ and IL-4+ (Th0), and 1FNgamma-and IL-4- (mature) cellswas countedby FACS.RESULTS:Serumlevelsof IL-18were significantly(p < 0.05, by Wilcoxon'ssign rank tesOelevatedto 113 +/- 79 (mean +/- SD) pg/mL 2 weeks after IFN-alphaadministrationfrom 47 +/- 46 pg/mLbefore treatmentin the responders, but not significantlychanged (from 55 +/- 64 pg/mL to 55 +/- 69 pg/mLin the non-responders. Serum levelsof IL-1beta were insignificantlychangedbefore (0.65 +/- 0.72 pg/mL)and 2 weeks after IFN-alphaadministration(0.35 +/- 0.20 pg/mL)in the responders, and the non-responders (1.17 +/- 2.37, 0.50 +/- 0.64 pg/mL, respectively).Serum levels of IL-6 were insignificantly changedbefore (1.33 +/- 1.61 pg/mL)and 2 weeksafter lFN-alphaadministration(1.49 +/- 1.51 pg/mL) in the responders, and the non-responders(1.31 +/- 1.50, 1.54 +/- 1.53 pg/mL,respectively). Serum levels of IL-10 were insignificantlychanged before (3.59 +/- 3.30 pg/mL)and 2 weeks after IFN-alpha administration (2.48 +/- 1.91 pg/mL) in the responders, and the nonresponders (2.06 +/- 1.90, 1.74 +/- 1.08 pg/mL, respectively).Serum levels of TNF-alpha were insignificantlychangedbefore (5.41 +/- 4.92 pg/mL)and2 weeks after IFN-alphaadministration (4.52 +/- 3.26 pg/mL) in the responders, and the non-responders (10.47 +/- 11.43, 10.50 +/10.29 pg/mL, respectively).The relative prevalence of Th0, Thl, or Th2 ceils 2 weeks after IFN-alpha administrationwas significantly(p<0.05, Wfleoxon'ssigh rank test) more increased than that before IFN-alphain the responders, but not in the non-responders. The relativeprevalence of CD4 positive T cell subsets in peripheral blood was not correlated with pathological gradingor staging.CONCLUSIONS:Sustainedresponse to IFN-alphatherapy maybe predictiveif the serum IL-18in patients with CHC is induced 2 weeks after 1FN-alphaadministrationas a host immuneresponse, therebyinterferongammain the CD4positiveT (Th0 and Thl) cellsis induced, and Th2 cells are also induced by cross-regulationin peripheralblood.

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INTERFERON PHARMACOKINETICS AND HOST FACTORS: EFFECTS ON HEPATITIS C GENOTYPE 1 VIRAL KINETICS DURING TREATMENT W I T H CONSENSUS INTERFERON. Avidan U Neumann, Bar-flan Univ, Ramat-Gan Israel; K. Rajender Reddy, University of Miami, Miami, FL; Rachel S Levy-Drummer, Harel Dahari, Bar-Ilan Univ, Ramat-Gan Israel; Shekman Wong, Amgen, Inc, Thousand Oaks, CA; F. Blaine Hollinger, Baylor Coll of Medicine, Houston, TX; Jennifer Poulakos, Amgen, Inc, Thousand Oaks, CA; Thomas J Layden, Univ of Illinois, Chicago, IL

EVALUATION OF RIBAVIRIN DOSAGE IN COMBINATION W I T H PEGINTRON IN THE TREATMENT OF PATIENTS W I T H CHRONIC HEPATITIS C. Juif F Jen, Mark Laughlin, Carol Chung, Samuel Heft, Melton B Affrime, Gerald Hajian, Schering-Plough Research Institute, Kenilworth, NJ

We have recently shown that early hepatitis C viral (HCV) kinetics during interferon alfa-2b (IFN-a) treatment follows a biphasic decline (Neumann et al. Science;1998:282:103-107). However, the drug, viral, and host factors that actually determine these viral dynamics parameters are not yet fully understood. Here, early viral kinetics and pharmacokinetics during consensus interferon (CIFN, Interferon alfacon-1, Infergen ®) treatment were studied in 31 patients infected with HCV genotype 1. The effectiveness of CIFN in blocking viral production in the first 24 hours was significantly (P<0.005) dose dependent, with a mean of 90.8% for 9 mcg of CIFN, 90.4% for 7.5 mcg BID, and 98.8% for 15 mcg of CIFN. A significant correlation (R=0.54, P<0.006) was found between the mean CIFN blood level at 6-12 hours after injection and the drug effectiveness. Also, a significant negative correlation occurred between body weight and both CIFN mean level and effectiveness in blocking virion production. There was no statistically significant effect of age, gender, baseline viral load, or baseline ALT on CIFN effectiveness. The HCV virion clearance (half-life of 2.7 hours) and HCV-infected cell loss rate (half-life of 1-70 days) during CIFN treatment were similar to results published with IFN~a2b. Age, but not gender or weight, impacted both the clearance rate of free virions (R= 0.4, P<0.03) and the loss rate of infected cell (R = 0.6, P<0.002). Neither the CIFN dose nor schedule had any effect on the clearance rate of free virions. However, the CIFN half-life in serum and frequency of injections strongly correlated with the second slope viral decline. These results clearly demonstrate the beneficial effect of higher CIFN levels on its effectiveness in blocking viral production as well as the importance of a stable CIFN level for the 2nd phase slope. Among host factors, weight negatively impacted the drug effectiveness, while older patients had slower virion and cell decay rates, possibly due to a weakening immune response. These results are important for understanding the mechanism behind clinical differences in treatment responses and in designing future treatment strategies.

Background: Sustained virologic response and hematological toxicity showed positive association with ribavirin concentrations in the patients with chronic hepatitis C (CHC) treated with PEG-Intron/Intron A + ribavirin. Pharmacokinetically, body weight was the most influential covariate for the apparent clearance of ribavirin. Some ribavirin dosing strategies should be carefully investigated in order to improve the treatment of patients with CHC. Objectiv_ee:To evaluate the efficacy and safety profiles of several ribavirin dosing strategies including fixed-dose and body-weight-based doses. Method: Simulations on the response and toxicity models were used to assess ribavirin dosing strategies. Four ribavirin dose regimens were considered: ($1) 800 rag/day for every patient; ($2) 1000/1200 mg/day based on WT --<75 / > 7 5 kg; ($3) 13 mg/kg/day; and ($4) 800/1000/1200 rag/day based on WT < 6 5 / 65-85 / > 8 5 kg. The response was the clearance of serum HCV-RNA at or after follow-up week 12 (HCV-RNA/qPCR < 100 copies/mL). A toxicity event was defined as the hemoglobin level at treatment week 4 being below 10.5 g/dE Results: The efficacy and safety profiles of the four ribavirin dosing strategies are shown in the Table. Efficacy and Safety profiles of $1-$4. Simulation results showed that the predicted response rate of $4 increased 6.3 o'~ over that of $1, the fixed 800 mg/day dose. The improved sustained response rate was larger (+7.4%) in the patients with HCV genotype one compared to +3.8% in the patients with HCV genotype non-one. Predicted hematological toxicity showed a mild increase (+2.5%) for $4 over $1. The toxicity event rate was lower than those observed in the treatment arms including ribavirin at 1000/ ]200 nag/day. Conclusion: Based upon the model simulations, ribavirin should be dosed according to body weight. The dosing strategy $4 (800/1000/1200 rag/day based on WT <65/65-85/>85 kg), achieved a moderate improvement in sustained response over $1 and a decrease in hematological toxicity over $2 (the approved regimen in combination with Intron A).

Dose Regimen $1:800 mg/day $2:t000/1200 mg/day $3:t3 rnglkg/day $4:800/1000/1200mgtday

ResponseRate Toxicity Event Rate Genotype Genotype1 Overall Non-1 58.3% 83.8% 46,3% 5,6% 66.8% 88,8% 56,3% 10.3% 65,0% 87.6% 54,3% 7.8% 64,6% 67.6% 53,7% 8.1%

Overall