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48 Impact of viremic HCV infection on surface expression of NK cell receptors and cytolytic function of NK cells
Parallel Session 5: Hepatitis C - Natural History and Pathophysiology ranks as the leading cause of cirrhosis and hepatocellular carcinoma in the world. However, the efficacy of currently available treatments is limited. We recently reported the effects of combined interferon(IFN)-a and cyclosporin A (CsA) treatment. In the present study, we examined the effects and mechanism of CsA on HCV replication in both clinical and virological settings. Methods: We evaluated the effect of CsA in following clinical settings. We compared reduction rate of serum HCV RNA between combined IFN-C~/CsA group and IFN monotherapy group. We examined the relationship between anti-HCV effect of CsA and its blood levels. We evaluated the effect of CsA on the replication of HCV in vitro using replicon cell system and other cell culture system. We examined the effect of CsA, FK506 and other inhibiturs of the peptidyl-prolyl cis-trans isornerase activity of cyclophilins. The gene expression of three representative cyclophilins (A, B and D) and Pin-1 were knocked down using small interfering RNAs to clarify which cyclophilin(s) is associated with HCV RNA replication. Results: The rate of reduction in serum HCV RNA with combined IFN-c~/CsA treatment was more rapid than that with interferon monotherapy. Keeping high blood level of CsA is essential for anti-HCV effect. CsA and other inhibitors of tile peptidyl-prolyl cis-trans isomerase activity of cyclophilins inhibited HCV RNA replication in HCV replicon cells and in a cell culture system. In contrast, FK506 did not have any inhibitory effect on HCV RNA replication. Conclusions: These findings indicated that cyclophilins are a crucial component ofHCV RNA replication. The results suggested that cyclophilin B, cyclophilin D and other members of the cyclophilins are involved in HCV RNA replication. Cyclophilins are essential for HCV RNA replication, and thus potent inhibitors of the cyclophilin family are promising anti-HCV drugs.
•7] GENETIC
EVOLUTION OF HYPERVARIABLE REGION 1 (HVR1) DURING THE C O U R S E OF ACUTE HEPATITIS C SUGGESTS THAT HVR1 IS NOT THE PRINCIPAL TARGET FOR ANTI-HCV NEUTRALIZING R E S P O N S E S
Y. Morice I , R. Brillet 1, D. Lavillette2, E Penin 3, R Donot2, G. Germanidis4, A. Soulier I , E. Pagkalos 4, G. Sakellariou 4, EL. Cosset2, JM. Pawlotsky I . 1Department of Virology. INSIERM U635, H@ital
Henri Mondog Universitd Paris 12, Crdteil, France," 2Laboratoim de Vectorologie Rdtrooirale et Thdrapie Gdnique, IFR 128 BioSciences Lyon-Gerland, Ecole Normale Suplrieure, Lyon, France; SlBCP, CNRS UMR 5086, Lyon, France," 4Departments of Internal Medicine and Nephrology, Papageorgiou General Hospital, Thessaloniki, Greece Hypervariable region 1 (HVR1) is a 27 amino acid (aa) stretch located at fire N-terminus of the HCV E2 envelope glycoprotein. Although HVR1 is tolerant to amino acid substitutions, the physico-chernical properties of its residues are strongly conserved. HVR1 is thought to be a target of anti-HCV neutralizing responses. Our aim was to study the effect of neutralizing responses on HVR1 genetic evolution at the acute phase o f HCV infection. Using a neutralisation assay based on infectious HCV pseudo-particles, we could classify a cohort of hemoclialysis patients with acute hepatitis C acquired in a dual source HCV nosocomial outbreak into 2 ~oups. (i) Group 1 (n 7) was characterized by a high baseline HCV RNA load evolving toward partial or complete control of viral replication that inversely correlated with the raise o f a strong neutralizing response. HVR1 quasispecies analysis showed that 3 patients experienced a genetic shift of HVR 1 sequences that accompanied the development of the strong nel~tralizing response, concomitantly to HCV RNA load decrease, suggesting selection of escape variants by the neutralizing response. No HVR1 changes were observed in tile remaining 4 patients. (ii) Group 2 was characterized by high, stable HCV RNA levels without any detectable neutralizing response over the course of infection. No patient experienced any change in HVR1 quasispecies, suggesting that viral escape is not necessary to establish chronic infection. (iii) Analysis of patient and prototype HCV strain sequence identities and prediction of antigenic regions
21
suggested that HVR1 is not the principal target for anti-HCV neutralizing responses, whereas other regions of E1E2 envelope glycoproteins appear as more likely candidates. Conclusions: Anti-HCV neutralizing responses drive the genetic evolution of HVR1, but the role of viral escape from neutralizing responses in the establishment of chronic HCV infection is unlikely and HVR1 does not appear to be the principal target for anti-HCV neutralizing responses.
[ EXPRESSION ] I OFMVIREMIC P AHCV CINFECTION T ON SURFACE OF NK CELL RECEPTORS AND CYTOLYTIC FUNCTION OF NK CELLS
J. Nattetmann, G. Feldmann, H.D. Nischalke, T. Sauerbruch, U. Spengler.
Department of Internal Medicine L University of Bonn, Bonn, Germany Introduction: Impaired activity of natural killer cells has been proposed
as a mechanism contributing to viral persistence in hepatitis C virus infection. Function of NK cells is regulated by NK cell receptors (NKR). Among these are natural cytotoxicity receptors (NCR; e.g. Nkp30, Nkp 44, Nkp46). They are the primary mediators of activating signals in response to binding to as-yet-undefined cell surface ligand(s). Another important group of NKR are file C-type lectins comprising activating NKG2C/NKG2D receptors as well as the inhibitory NKG2A receptor, which specifically interacts with HLA-E. Here, we analyzed whether the decreased NK cell cytolytic function in chronic hepatitis C corresponds to a dysregulated expression of NKR. Material and Methods: Expression of the NK cell receptors was flowcytumetrically analyzed on lymphocytes from HCV RNA-positive subjects (n 30), patients who became HCV RNA-negative under antiviral therapy (n= 10), and 10 healthy individuals. As a Nrther control we analyzed NK cells from 3 HBV-infected patients. Cytolytic fimction of NK cells was studied in a re-directed lysis assay and in a standard 51chromium release cytotoxicity assay, respectively. Results: In patients with chronic hepatitis C, freshly drawn NK cells expressed significantly decreased surface densities of NKp46 and NKp30 NCR as compared to healthy HCV RNA@) subjects (n 10). The low surface density of NCR as compared to uninfected donors was also confirmed in in-vitro-activated NK cell populations derived from HCV patients. In contrast, patients who had cleared HCV RNA( ) under antiviral combination therapy (n= 10) showed a normal expression of Nkp44, Nkp30, and Nkp46. Functional tests confirmed that reduced NCR expression in chronic hepatitis C occurred in parallel with a decrease of NCRmediated killing of target cells. Furthermore, the proportion of NKG2Aexpressing NK cell was significantly increased in HCV RNA-positive patients (52.3%±5.2%) in comparison to healthy controls (16.7%±3%; p < 0.001). More importantly, NK cells from patients with persistent HCV infection showed markedly reduced cytolytic activity against cells pulsed with peptide HCV core aa35-44, which is known to stabilize HLA-E expression. Conclusion: The present study indicates that defective expression of NKR represents a novel mechanism contributing to impaired NK cell function in chronic hepatitis C.
•7] GENETIC
EVOLUTION OF HYPERVARIABLE REGION 1 (HVR1) DURING THE C O U R S E OF ACUTE HEPATITIS C SUGGESTS THAT HVR1 IS NOT THE PRINCIPAL TARGET FOR ANTI-HCV NEUTRALIZING R E S P O N S E S
Y. Morice I , R. Brillet 1, D. Lavillette2, E Penin 3, R Donot2, G. Germanidis4, A. Soulier I , E. Pagkalos 4, G. Sakellariou 4, EL. Cosset2, JM. Pawlotsky I . 1Department of Virology. INSIERM U635, H@ital
Henri Mondog Universitd Paris 12, Crdteil, France," 2Laboratoim de Vectorologie Rdtrooirale et Thdrapie Gdnique, IFR 128 BioSciences Lyon-Gerland, Ecole Normale Suplrieure, Lyon, France; SlBCP, CNRS UMR 5086, Lyon, France," 4Departments of Internal Medicine and Nephrology, Papageorgiou General Hospital, Thessaloniki, Greece Hypervariable region 1 (HVR1) is a 27 amino acid (aa) stretch located at fire N-terminus of the HCV E2 envelope glycoprotein. Although HVR1 is tolerant to amino acid substitutions, the physico-chernical properties of its residues are strongly conserved. HVR1 is thought to be a target of anti-HCV neutralizing responses. Our aim was to study the effect of neutralizing responses on HVR1 genetic evolution at the acute phase o f HCV infection. Using a neutralisation assay based on infectious HCV pseudo-particles, we could classify a cohort of hemoclialysis patients with acute hepatitis C acquired in a dual source HCV nosocomial outbreak into 2 ~oups. (i) Group 1 (n 7) was characterized by a high baseline HCV RNA load evolving toward partial or complete control of viral replication that inversely correlated with the raise o f a strong neutralizing response. HVR1 quasispecies analysis showed that 3 patients experienced a genetic shift of HVR 1 sequences that accompanied the development of the strong nel~tralizing response, concomitantly to HCV RNA load decrease, suggesting selection of escape variants by the neutralizing response. No HVR1 changes were observed in tile remaining 4 patients. (ii) Group 2 was characterized by high, stable HCV RNA levels without any detectable neutralizing response over the course of infection. No patient experienced any change in HVR1 quasispecies, suggesting that viral escape is not necessary to establish chronic infection. (iii) Analysis of patient and prototype HCV strain sequence identities and prediction of antigenic regions
21
suggested that HVR1 is not the principal target for anti-HCV neutralizing responses, whereas other regions of E1E2 envelope glycoproteins appear as more likely candidates. Conclusions: Anti-HCV neutralizing responses drive the genetic evolution of HVR1, but the role of viral escape from neutralizing responses in the establishment of chronic HCV infection is unlikely and HVR1 does not appear to be the principal target for anti-HCV neutralizing responses.
[ EXPRESSION ] I OFMVIREMIC P AHCV CINFECTION T ON SURFACE OF NK CELL RECEPTORS AND CYTOLYTIC FUNCTION OF NK CELLS
J. Nattetmann, G. Feldmann, H.D. Nischalke, T. Sauerbruch, U. Spengler.
Department of Internal Medicine L University of Bonn, Bonn, Germany Introduction: Impaired activity of natural killer cells has been proposed
as a mechanism contributing to viral persistence in hepatitis C virus infection. Function of NK cells is regulated by NK cell receptors (NKR). Among these are natural cytotoxicity receptors (NCR; e.g. Nkp30, Nkp 44, Nkp46). They are the primary mediators of activating signals in response to binding to as-yet-undefined cell surface ligand(s). Another important group of NKR are file C-type lectins comprising activating NKG2C/NKG2D receptors as well as the inhibitory NKG2A receptor, which specifically interacts with HLA-E. Here, we analyzed whether the decreased NK cell cytolytic function in chronic hepatitis C corresponds to a dysregulated expression of NKR. Material and Methods: Expression of the NK cell receptors was flowcytumetrically analyzed on lymphocytes from HCV RNA-positive subjects (n 30), patients who became HCV RNA-negative under antiviral therapy (n= 10), and 10 healthy individuals. As a Nrther control we analyzed NK cells from 3 HBV-infected patients. Cytolytic fimction of NK cells was studied in a re-directed lysis assay and in a standard 51chromium release cytotoxicity assay, respectively. Results: In patients with chronic hepatitis C, freshly drawn NK cells expressed significantly decreased surface densities of NKp46 and NKp30 NCR as compared to healthy HCV RNA@) subjects (n 10). The low surface density of NCR as compared to uninfected donors was also confirmed in in-vitro-activated NK cell populations derived from HCV patients. In contrast, patients who had cleared HCV RNA( ) under antiviral combination therapy (n= 10) showed a normal expression of Nkp44, Nkp30, and Nkp46. Functional tests confirmed that reduced NCR expression in chronic hepatitis C occurred in parallel with a decrease of NCRmediated killing of target cells. Furthermore, the proportion of NKG2Aexpressing NK cell was significantly increased in HCV RNA-positive patients (52.3%±5.2%) in comparison to healthy controls (16.7%±3%; p < 0.001). More importantly, NK cells from patients with persistent HCV infection showed markedly reduced cytolytic activity against cells pulsed with peptide HCV core aa35-44, which is known to stabilize HLA-E expression. Conclusion: The present study indicates that defective expression of NKR represents a novel mechanism contributing to impaired NK cell function in chronic hepatitis C.