HCV Co-infection

of HBV DNA in patients with lamivn:tdine resistant HBV. In a minority of patients vieemia remained detectable at levels of 10/'\5-10/X6 copies/ml despite > 12 months of treatment

insight into the pathogenesis of liver damage. We tested the hypothesis that vigorous HCVspecific Thl-fike immune responses are correlated with protection from liver damage in persons with H1V/HCV cmnfectton. Methods: Baseline data ti'om a representative subset of subjects with HW and HCV coinfection who were enrolled in ACTG 5071 (n = 102/134 enrolled) were studied. Subjects had liver biopsies and PBMCs collected prior to entering aetwe treatment with interferon/ribavirin based therapy ELISPOT assays were performed on PBMC tbr IENy and IL-10 using HCV antigens (Ag) Corn, NS3 and NS5, recall Ag Candida (Can), and PHA. Data were analyzed using nonparametric methods including Spearman rank correlation and recursive partitioning (CART). Results: There were significant negative correlations between the inflammatory score (IS) and IFN',/production in response to the HCV proteins core (p = 0.02) and NS5 (p = 0.002). Fibrosis scores (FS) were negatively correlated with 1FN',/production m response to NS5 (p = 0.03). There were also strong negative correlations between iFNy production in response to Can and the IS (p = 0001) and PS (p < 0.001). There was no correlation tbr IL-lO production in response to Ag or G'm with either FS or IS. Cut-off values for IFN-core, IFN-Can, IL10-Can and IL10-NS3 were identified by CART that, in combination, correctly identified 62% of those with low 1S (0~4 versus > =5) and 83% of those with high IS (p=0.02, McNemar test). Similarly, cut-off values of lFN-core, IFN-Can, lLl0-Can and 1LI0-NS3 were identified by" CART that correctly identified 52% of those with low FS (0-2 versus 3~6) and 83% of those with high FS (p = 0.002) There. was no association of HOe'-specific response and CD4 count in this cohort (median CD4 count 486, range 177 - 1,001) Conclusions: In this large cohort of patients co-infected with HIV and HCV, HCV-specific Thl responses are correlated with milder inflammation and fibrosis, but joint associations between type 1 and type 2 responses are useful for predicting liver histology.

342

Tenofovir and HBV Mutants After Liver Transplantation Jose Nery, Daryl Lau, Cain Nery, Kamran Safdar, Maria Torres, Guy W. Neff, Mama Montalbano, Doug Meyer, Debbie Weppler, Sylon Britto, Arie Regev, Seigo Nishida, David Levi, Juan Madariaga, Tomaki Kato, Eugene R, Schiff, Andreas Tzakis Aim:Resistant HBV strains develops in approximately 30% of post liver transplant (LTx) recipients treated with lamivudine(LAM) within 2 years trom nine of transplantatinn. Adetbvir(ADV) has recently" been reported to be ettective against mutants, however its use may be limited by nephrotoxicity. We report our experience with tenotbvir(TNV), another nudeotide analogue reverse transcriptase inhibitor, in LTx recipients developing I.AM resistanceMethods:8 pts developed resistance to LAM 10 to 85 mos (median: 26) post-kTx. TNV (300 mg/ day, P.O.) was added 1 to 66 mos after breakthrough (BT), and continued for 2 to 12 mos (mefflan: 4). Prior to receiving TNV, these paUents had been excluded from recewing ADV due to: age> 80 yo. (5), HIV co-infection (1), enhstment in another drug study (1) and potential non-compliance (1). Criteria for BT included elevation of liver chemistries along with reappearance of HBs~g, HBeAg and/or HBVDNA. HBV genotype and "~NIDD variants were identified through DNA sequence analysis prior to and after TNV; sequential HBVDNA levels were measured by hybridization (pts 2,4,6,7&8) or PCR (pts 1,3&5).Results:No adverse reactinn was associated with TNV, The tables below summarize the data pertaining the study pts.Condusion:This preliminary experience indicates that TNV markedly decreases rephcation of L4M-resiatant HBV variants post-kTx These results demonstrate anottaer potential option tbr tfie treatment of HBV L~.lvl resistance

345 Persistence of Hepatitis C After Homologous Monoclonal Re-Challenge Associated with the Emergence of New Virus Variants Jens Bukh, Robert Thimme, William C. Satterfield, Jean-Christophe Meunier, Xavier Fores, Masayuki Yanagi, Snzanne U. Emerson, Hans Christian Spangenberg, Kyong Mi C3"lang,Francis V. Chisari, Robert H. Purcell

TNV Treatment Summarization P~t 1 2 3 4 5 6 7 8

Median Creatinine(rng/dl) Pre-TNV PosbTNVp 131.3 n.s, 1.81.7 n.s 1,41,5 n.s, 1,41.5 n,s, 1,01,1 n,s, 1.01.1 n.s, 1,31.1 n,s 0,9 0.9 n,s,

Median ALT (IU/I) TNV PosI-TNVp 17 65 n,s 71 175 n,s 103 68 n,s 46 38 n.s 113 39 n.s, 122 104 n,s, 60 36 n.s, 95 30 0.001

Lo9 HBV DNA Pre.TNVPost-TNVp 6.98 3.55 0.008 2,21 <-0,3 0.016 4.04 2.570,001 4,51 <~0,3<0,001 >8,707,11 <0,001 >3.78 -0.16 <0,001 3.53 062 0,004 2.92 <-0,3 0.026

To study immunity to HCV, we re-challenged two chimpanzees following resolved monoclonal genotype la infection. After homologous challenge of one ammal HCV persisted despite vigorous anamnestic intrahepatic T cell responses. The entire polyprotein sequence of viruses recovered at weeks 3, 10, 35, 47, 61 and 82 post-challenge was compared with the sequence of the virus recovered during the first inti:ction. Only" a single mutation was identified at week 3, whereas multiple mutations were idemified at each of the subsequent weeks, suggesnng that HCV persisted through an immune escape mechanism. Thus, resolved prinlary infection does not prevent persistence even after homologous challenge. Re-challenge of the second chimpanzee resulted in a different outcome This animal had only" 2 weeks of viremia after the first re
343 An lmmunomodulatory Role for C D 4 + C D 2 5 + T Lymphocytes in Hepatitis C Infection Rnniel Cabrera, Zhengkun Tu, Roberto J. Firpi, Yiling Xu, Hugo Rosen, David R. Nelson Background: Host-varus interactiot~s play a pivotal role in perpetuation or clear,ace of HCV inkctinn. A regulatory" CD4 T cell has been described with the phenotype CD4 + CD25 + that suppre~ T call activation and proinflammatory cytokine production. Aim: To characterize the t;requency, phenotype, and function of CD4 CD25 T cells m HCV. Methods: Mononuclear cells fi'om liver parenchy~la, peri-bepatic lymph nodes and peripheral blood (PBMC) of chronic HCV patients were analyzed tbr CD4 CD25 frequency and phenotype. Peripheral CD4 CD25 T cells kom spontaneously cleared (n = 10), chronic infected (n = 20) and postliver transplant (LT) patients (n = 10) were analyzed for %CD4 cells and eftect on HeYspecific T cell responses. PBMC were depleted or supplemented with CD4 CD25 T cells and tunctionally characterized using IFN-F ELISPOT, CFSE pmliferatinn, and cytokme assay in response to HCV (core, NS3, NS4, NS4/5, NSS) and control antigens (Tr, Pkta.). Viral, biochemical, anti histologm data were collected for correlation analysis. Results: Frequency/ Phenotype: CD4+ CD25+ T cells were higher in the periphery (mean 6.9% lyinphocytes) followed by" peri-bepatic lymph nodes (2.3%) and least abundant in the hver parenchy~la (1%). A higher proportion of CD4 CD25 T ceils were found in chronic infi:ction (mean 2.8%) and early post-LT infection ( 4 1 % ) when compared to self-limited nitection (1 7%). Phenotypm characterization reveals markers of early, differentiated, antigen experienced, and late activated cell population (86% CD45RO, 88% CD27 +, 94% CD28+, 82% CD95 + and 82% CD62L + ). Function: HCV-specifie IFN-F activity was enhanced in PBMC depleted of CD4 CD25 T cells vs baseline [cfirnnic: mean 43.6 vs 8.2, p = 0.03; spontaneous cleared: 328.2 vs 112.8,p = 0.05; and post-LT: 3 2 7 vs 2.4, p =0.03, spotsg2 x I05 PBMCJ Addition of CD4 CD25 cells reversed this ett~:ct. CFSE analysis revealed that removal of CD4 CD25 cells enhanced HCV-specific CD4 proliferation 16 fold Cytokine analysis suggested an inhibitory, role for TGF-Bh Correlation: No correlation was noted with serum AH', HCV RNA, and disease seventy. Conclusion: CD4 + CD25 + T lymphocytes are (1) increased in chronically infected hosts (2) compartmentalized in the peripbery (3) inhibit the 1FN-F viral specific T cell response and (4) mediate immunomodulatory function via cytokine and cellcell mediated pathwa~zs. Implication: CD4 CD25 regulator5, cells appear to play- a role in viral persistence and may be amenable to therapeutic intervention.

346

Signaling for Apoptosis and Repair in Hepatitis C RNA Replicon Model Manuela G. Neuman, Michael J Gale We aimed to characterize m vitro EtOH-signaling and the apoptotic response in Huh7 human bepatoma cells and Huh7 cells harboring an HCV snbgenomm RNA replicon. Experiments were conducted in Huh7-cells harbounng an HCV subgenomic repficon, and m parental Huh7 cells that laek the HCV repficon. ELISA and TUNEL assays assessed apoptosis in cells that were exposed to 1-3 consecutive treatments of 20 - 120 mM EtOH tbr 24 - 72 hours. Additional experiments assessed apoptosis in cells pre-treated with S-AdenosylMethinnine (SAMe) or caspases 3 or 9 inhibitors and than treated with consecutive doses of EtOH. HCV RNA replication was associated with a dose and tinle-dependent increase m apoptosis. Beginning with exposure to 60 mM ETOH, the amount of apoptosis observed m the rephcon cells was at 2-4 times higher for the same treatment in the parental Huh7 cells lacking the HCV replicon. Mitochondrial injury and a reductimx in mitochondrial suceinate dehydrogenase activity were associated with EtOH-induced apoptosis over increasing doses of EtOH exposure. Cells pretreated with SAMe have a lower degree of apoptosis than controls. Caspases 9 and 3 both reduced apoptosis, but the mechanism of action differ m one caspase versus the other and from SAMe. HCV RNA replication significantlyexacerbates the apoptotic effects of EtOH exposure, and this is linked to perturbation of mitochondrial enzyme functions Modulation of mitochondnal damage may have implications HCV pathogenesis and the development of new therapeutic strategies in HCV-infected patients.

344

The Association of Hepatitis C Virus (HCV)-Specific Immune Responses with Liver Histology in Subjects with HIV/HCV Co-Infection Camilla S. Graham, Annalee Wells, Liu Tun, Kem~eth E Sherman, Marion Peters, Raymond Chang, Janet Andersen, Margaret J. Koziel Background: Persons conitected with HIe and HC~/are at increased risk ['or progression to cirrhosis compared with those with HCV monoinfection, but reasons tor this are unclear. The relationship of HCV-specific immune responses to hver injury' in HIV may provide

AASLD Abstracts

A-710