446 Contribution of auto-immune reactions involving cytochrome P4502E1 to hepatocyte injury during chronic hepatitis C

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and remodelling. Ang-2 expression contributes to tumoral formation and progression (Tanaka, JCI 1999) and it is also related to inflammatory processes (Kim et al., JCB 2000). Aim: Thus, we analyzed the expression of Ang-2 in chronic hepatitis C (CHC) liver samples to better understand the pathophysiologic role of the neoangiogenic process in this progressive liver disease. Methods: Liver biopsies from patients with CHC (n 10) and controls, non-autoimmune cholestasis (n 3) and healthy individuals (n 2), were OCT included and frozen stored. Ang-2 expression was assessed either by RIPA extraction and Western Blotting or by immunohistochemistry. Ang-2 mRNA expression was also evaluated in controls, cholestatic and CHC patients biopsies by RT-PCR. To accurate equal gel loading, tubulin (Western Blot) and beta-Actin (RT-PCR) expression assays were also performed and densitometrically analyzed. Results: Immunohistochemical assays revealed weak or absent Ang-2 expression in control and cholestatic liver samples opposed to that observed in CHC, where Ang-2 staining was clearly evident in vascular structures and sinusoidal spaces of inflamed portal and periportal areas. These results were confirmed by densitometric analysis of Western Blot assays images, where we could appreciate a marked induction of Ang-2 expression in CHC samples (25-fold induction). RT-PCR experiments showed specific Aug-2 mRNA expression in most CHC samples but not in the cholestatic or control ones. Conclusions: Expression of Ang-2 in liver samples from CHC patients, in addition to other proangiogenic factors, presents stronger evidence about the existence of vascular remodelling associated to this chronic liver disease. The Ang-2 expression could represent an important factor in CHC disease progression and could facilitate the hepatocarcinoma development.

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Q U A S I S P E C I E S EVOLUTION IN C H R O N I C HEPATITIS C PATIENTS A F T E R IMMUNIZATION WITH THE T H E R A P E U T I C VACCINE IC41

C. Sarrazin 1, A. Wohnsland 1, H. Wedemeyel 2, V. Bfirger 3, W. Zauner 3, E. Tauber3, C.S. Klade 3. lInnere Medizin II, Saarland University

Hospital, Homburg, Germany," 2Abteilung Gastroenterologie, Medizinisehe Hoehsehule Hannover, Germany," 3Intercell, Wien, Austria Introduction: IC41 is a therapeutic peptide vaccine, which can induce HCV specific IFN-?-secreting CD4+ helper- and CD8+ cytotoxic T-cells (CTL) in patients with chronic HCV infection and failure to previous standard therapy. In individual patients a significant decline of HCV RNA concentration and subsequent rebound was observed concomitant with T cell responses. Here we investigate the emergence of mutations within the HCV non-structural (NS)3 protein corresponding to an immunodominant CTL epitope of IC41. Methods: Sequencing and cloning for analyses of HCV quasispecies within the NS3 gene was performed in 5 patients at 4 time points before, during and after treatment with IC41. Results: Overall a low quasispecies diversity and complexity within a mean number of 17 isolates of the NS3 gene per time point and patient was observed. In the 5 patients a mean decline of 0.75 log HCV RNA IU/mL from baseline to week 16 of therapy was observed. In only one of the 5 patients a significant change of a single amino acid within the NS3 epitope possibly affecting binding was observed. The functional consequences of this exchange are currently under investigation. In the remaining 4 patients no significant amino acid exchanges or emergence of a significant number of new isolates within NS3 quasispecies was detected. Conclusion: Although epitope escape mutations within dominant HCV T cell epitopes may contribute to viral rebound in rare cases, most patients with declining RNA during therapeutic vaccination showed stable sequences and no shift in quasispecies. Further, sequencing studies addressing the different HCV peptide binding regions are needed.

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FUNCTION A N D P H E N O T Y P E OF PEPTIDE-VACCINE I N D U C E D HCV-SPECIFIC CD8+ T CELLS VS. ACUTE A N D C H R O N I C HEPATITIS C

V. Schlaphoff1, S.B. Jelovcan 2, J. Wiegand 1, M. Cornberg 1, B. Jilma 3, M.R Manns 1, H. Wedemeyer 1, C.S. Klade 2. gDept. Gastroenterology,

Hepatology, Endocrinology, Hannover Medical School, Hannover, Germany," 2Intercell AG, Vienna, Austria," 3Dept. Clinical Pharmacology, Medical University Vienna, Vienna, Austria Different subsets of CD8+ T cells have been determined based on their expression of CCR7 and CD45: naive (CCR7+/CD45RA+), central-memory (Tcm; CCR7+/CD45RA-), effector-memory (Tem; CCR7-/CD45RA-) and CD45RA+ effector-memory (Temra; CCR7-/CD45RA+) cells. We investigated HCV- and CMV-specific CD8+ T cells in HLA-A2-positive patients with acute hepatitis C (n 8), chronic hepatitis C (n 60) and healthy individuals (n 128). The latter two groups were vaccinated with IC41, a vaccine consisting of five synthetic peptides harboring relevant HCV T cell epitopes and the synthetic Thl/Tcl adjuvant poly-L-Arginine (Manns et al., AASLD 2004; Jilma et al. AASLD 2004). Almost all HCV-specific T cells in acute hepatitis C displayed the Tem or Temra phenotype while the mean percentages of Tcm, Tem and Temra in chronic hepatitis C were 31% (range 12 69%), 41% (8 83%) and 14% (0 42%), respectively. In contrast, 80 90% of CMV-specific T cells were CCR7-negative in patients with chronic hepatitis C with approximately 60% of those being Temras. Importantly, we did not find significant differences for CMV-specific CD8+ T cells between healthy controls and chronic HCV patients (>80% CCR7-negative) which contradicts in part to a recent report from the UK. In healthy controls, IC41 peptide vaccination induced mainly CCR7+ HCV-specific T cells (57%, range 30% to 82%), about one third displayed the Tem phenotype while 10% were Temras. HCV-specific tetramer-positive cells induced by vaccination in healthy individuals produced significantly more often interferon gamma (12 73%) than HCV-specific T cells of patients with chronic hepatitis C (0 27%, p<0.001). Longitudinal analysis during IC41 vaccination of patients with chronic hepatitis C showed a transient down-regulation of CCR7 in individual patients accompanied by a slight increase in interferon gamma production per cell. Conclusion: (i) the relative loss of mature (CCR7-) HCV-specific CD8+ T cells in chronic hepatitis C may be less pronounced than previously reported although still significant differences between HCV- and CMVspecific CD8+ T cells are evident and HCV-specific T cells produce less IFN gamma; (ii) peptide-vaccination-induced HCV-specific T cells in healthy individuals are mainly central memory cells; (iii) HCV has only a minor pervasive influence on CMV-specific T cells. Funding: Austrian Fond zur FSrderung der Forschung; German Hep-Net.

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C O N T R I B U T I O N OF A U T O - I M M U N E R E A C T I O N S INVOLVING C Y T O C H R O M E P4502E1 TO HEPATOCYTE I N J U R Y D U R I N G C H R O N I C HEPATITIS C

M. Vidali 1, G. Occhino 1, A. Ivaldi 1, R. Serino 1, S. Moia 1, C. Rigamonti 1, M. Sartori 2, E. Albano 1. 1Dept. of Medical Seiences, University

"A. Avogadro" of East Piedmont, Novara, Italy," 2Gas'troenterology Unit. Ospedale Maggiore e della Car#& Novara, Italy Background and Aims: Chronic hepatitis C (CHC) is often associated with manifestations of auto-immune reactions including the presence of circulating auto-antibodies. Among these auto-antibodies, LKM-1 autoantibodies directed against cytochrome P4502D6 (CYP2D6) are detectable in about 5 10% of CHC patients. In the light of the well documented capacity of alcohol intake to promote the progression of CHC, we have investigated the possible presence of auto-reactivity against ethanolinducible cytochrome P4502E1 (CYP2E1) isoenzyme in patients with HCV infection with and without alcohol consumption.

05C. Viral Hepatitis'

(e) Hepatitis' C

Methods: The presence of auto-antibodies against recombinant human CYP2E1 was investigated by solid phase immunoenzymatic test (ELISA) and western blotting in 47 abstinent and in 62 CHC patients with moderate (~<50g ethanol/day; n 39) and high (>50g ethanol/day; n 23) alcohol consumption. Fifty-nine HCV-free blood donors were used as controls. Results: IgG reactivity against CYP2E1 was significantly (p <0.0001) increased in CHC patients as compared to controls. Titres of these autoantibodies above the 97.5 percentile in the controls were evident in 42 (39%) of HCV infected patients. Among anti-CYP2El-positive patients only 5 (12%) showed reactivity towards CYP2D6 in ELISA. Moreover, competition experiments revealed that CYP2E1 recognition was not due to cross-reactivity with CYP2D6. The presence of anti- CYP2E1 IgG was unrelated to alcohol consumption and no difference in age, disease duration, aminotrasferase levels and virus genotype were evident among the patients with or without anti-CYP2E1 auto-reactivity. The overall scores for steatosis and the histological grading and staging according to Ishak were also comparable in the two groups. However, irrespectively from alcohol consumption, high anti-CYP2E1 titres were significantly (p 0.025) associated with piecemeal necrosis (A scores >1). A marginally significant association (p 0.07) was also evident between anti-CYP2E1 auto-reactivity and focal necrosis and focal inflammation (C score >1). Conclusions: An auto-immune response against CYP2E1 is evident in a significant fraction of patients with HCV infection and because of the plasma membrane localization of CYP2E 1 might contribute to hepatocyte injury during chronic hepatitis C.

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R E I N F E C T I O N WITH THE HEPATITIS C VIRUS: KINETICS OF VIRAL C L E A R A N C E A N D M U L T I - P A R A M E T E R ANALYSIS OF CD4+ T-CELL R E S P O N S E

L. Weseslindtner1, J.H. Aberle 1, C. Gurguta2, P. Steindl-Munda 2, T. Popow-Kraupp1, R Ferenci 2, H. Holzmann 1. 1Clinical Institute of

Virology, Medical University of Vienna, Austria," 2Department of Internal Medicine IV, Gastroenterology and Hepatology, General Hospital Vienna, Medical University of Vienna, Vienna, Austria Background and aim: The CD4+ T-cell response could be identified as decisive factor for the outcome of the acute Hepatitis C Virus (HCV) infection. When chimpanzees which previously cleared the virus are rechallenged with HCV, CD4+ T-cells respond rapidly and intensely in recall to viral antigens. In humans functional capabilities of CD4+ T-cells responding to HCV upon rechallenge have not been characterized because reinfection is rarely observed and can be hardly verified. Our aim was to determine the kinetics of viral clearance in relation to HCV-specific CD4+ T-cell response in patients with HCV reinfection. Methods and patients: This study included patients with clinical evidence of HCV reinfection (previous complete recovery, sustained HCV RNA negativity for a long observation period and acute disease after documented reexposure to HCV) (n 3), patients with acute (n 17) and chronic (n 30) HCV infection. Peripheral CD4+ T-cell responses to HCV core and non-structural proteins (NS3, NS4) quantified by multi-parameter flow cytometry and related serum HCV RNA levels were compared among the patient groups. For functional characterization activation (CD25+), T-helper cells typel (TH1) cytokines (interferon-gamma [IFN-?], tumor necrosis factor-alpha [TNF-alpha) and proliferation (protein Ki-67+) were simultaneously assessed on a single cellular level. Results: In comparison to patients with acute and chronic HCV infection, reinfected patients showed significantly higher frequencies of CD4+ T-cells which upon stimulation with NS3 and NS4 were activated, produced TH1 cytokines (IFN-?, TNF-alpha) and proliferated (p < 0.05). Furthermore, NS3-stimulation induced simultaneous IFN-?-production and proliferation in CD4+ T-cells especially from patients with reinfection. With a maintained strong THl-response against NS3 and NS4 all reinfected patients rapidly cleared (within 28 days) and subsequently showed sustained loss of the virus.

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Conclusion: Corresponding to the chimpanzee model, CD4+ T-cells respond vigorously when patients who previously cleared the virus get reinfected. Simultaneous IFN-?-production and proliferation of CD4+ T-cells, as well as a rapid loss of HCV can be detected in these patients indicating their TH1 are functionally efficient and capable of eradicating the virus. Insights about the induction of CD4+ T-cell memory upon rechallenge with viral antigens in patients with HCV reinfection could contribute to the development of HCV vaccines. (LW and JHA contributed equally to this work.)

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DECLINE OF HCV-SPECIFIC CD8+ T CELLS D U R I N G I N T E R F E R O N - A L P H A T H E R A P Y OF ACUTE HEPATITIS C (HCV): ROLE OF A P O P T O S I S VS, DIFFERENTIAL REGULATION OF C H E M O K I N E R E C E P T O R S

J. Wiegand 1'3, H. Wedemeyer 1'3, A. Ciner 1, V. Schlaphoff1, P. Buggisch2, E. Jaeckel 1, M.R Manns 1'3, M. Cornberg 1'3. 1Dept. of Gastroenterology,

Hannover Medical School, Germany," 2Universitdtsklinikum Eppendorf Hamburg, Germany," 3Kompetenznetz Hepatitis' (Hep-Net), Germany HCV-specific T cell responses are associated with spontaneous clearance of acute HCV. However, several studies described a decline of HCVspecific CD8+ T cells during antiviral suggesting that the high success of acute HCV therapy with interferon (IFN)-alpha is independent of adaptive immunity. Here we show that different mechanisms may contribute to this decline of virus-specific CD8+ T cells. T cells of patients with a sustained virological response demonstrated an early upregulation of chemokine receptor expressions (CXCR-3) indicating homing to the liver. In contrast, a patient with initial response but virological relapse did not significantly upregulate CXCR-3. HCV-specific CD8+ T cells of the relapse patient displayed a significantly higher staining for Annexin-V as a marker of apoptosis as compared to tetramer-negative CDS+ T cells. Annexin-Vstaining increased even more after the initiation of IFN treatment. This increase of apoptosis was followed by a complete loss of peripheral virus-specific CD8+ T cells by week 12. A different Annexin-V-staining pattern was observed in patients with sustained virological response with lower Annexin-V-stainings at week 0 and only a minor increased staining during IFN-alpha therapy (similar Annexin-V fluorescence intensity between HCV-specific and total CD8+ T cells). Low levels of HCV-specific CD8+ T cells remained detectable throughout treatment and follow-up in sustained responders. Thus, the decline of HCV-specific T cells seen in all patients maybe a consequence of both, apoptosis and homing. However, the balance between cell death vs. regulation of chemokine receptors such as CXCR-3 may have significant impact on the fate of HCV-specific T cells during IFN-alpha therapy potentially leading to different long-term outcomes.

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HEPATITIS C VIRUS S T R U C T U R A L PROTEINS A N D ACTIVATION OF Toil-LIKE R E C E P T O R S 2 A N D 4

M. Hoffmann1, M.B. Zeisel 1, H. Barth 1, B. Gissler1, N. Jilg 1, G. Paranhos-Baccal~ 2, T. Wakita 3, D.T. Golenbock4, H.E. Blum 1, R Henneke 5, T.E Baumert 1. 1Dept. Medicine II, University of Freiburg,

Freiburg, Germany," 2UMR2 714, CNRS-b ioMdrieux-CERVI-IFR128, Lyon, France," 3Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan; 4Division of Infectious Diseases and Immunology, University of Massaehusetts Medical School, Worcester, USA," 5Children ~' Hospital, University of Freiburg, Freiburg, Germany Background and aim: Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Toll-like receptors (TLRs) are pathogen recognition molecules that activate the innate immune system. Recognition of pathogen-associated molecular patterns (PAMPs) by TLRs induces the activation of genes important for an effective host defence. Previous studies have shown that TLRs are involved in the human host defence against