1. Human immune response to Hepatitis C virus Geert Leroux-Roels Center for Vaccinology Ghent University and Hospital

2. Overview of the presentation The principal actors –Hepatitis C virus or HCV –The human immune system Innate immune system Adaptive immune response Mechanisms of persistence Consequences for vaccine development

3. The HCV genome and expressed polyprotein Lauer et al. NEJM 345:41-52, 2001

4. Hepatitis C virus Envelope proteins E1 (gp31) E2 (gp70) Nucleoprotein - Core (p21) RNA genome

5. B cell Hepatocyte CD8 + CTL CD4+ Th cell Y Y Y Y Y APC NK NKT cells The human immune response

6. Study of the immune response Patient studies Animal models

7. Acute infection Spontaneous clearance Chronic infection 20% 80% Treatment Chronic hepatitis No response Sustained response Patient Studies

8. Patient studies Liver infiltrating lymphocytes PBMC - fresh - cultured Liver infiltrating lymphocytes - fresh - cultured

9. Study of the immune response Patient studies Animal models –chimpanzee (ethics, = human) –mouse models HLA-A2 transgenic mouse HCV transgenic mice huPBL-SCID mouse, Trimera mouse, huHepatocyte-uPA-SCID mouse,..

10. The adaptive immune response to HCV CD4+ Th cell CD8 + CTL TNF-  IFN-  Lysis IFN-  Hepatocyte APC Y Y B cell Y  -E1  -E2  -NS3

11. Kinetics of anti-HCV response in patients with transfusion- associated hepatitis C and resolution of infection Chen et al. Gastroenterology 1999;116:135-143

12. Kinetics of anti-HCV response in patients with transfusion- associated hepatitis C who develop chronic HCV Chen et al. Gastroenterology 1999;116:135-143

13. Target of neutralizing antibodies Envelope proteins E1 and E2 Protective role demonstrated by in vitro neutralization of chimpanzee- infectious HCV with antibody directed against HVR1 and other regions of E2

14. Neutralisation of binding - NOB assay CD81 MOLT 4 CD81 HVR1 E2 E1

15. Are antibodies needed to clear HCV infection ? Human HCV infection can resolve in agammaglobulinemic children –Bjoro et al. NEJM 1194; 331:1607-1611 –Adams et al. Ped Inf Dis J 1997;16:533-534 –Christie et al. Clin Exp Immunol 1997;110:4-8 HCV clearance in chimp occurred in the absence of any antibody response to envelope proteins –Bassett et al. J Virol 1999;73:1118-1126

16. Gerlach et al. Gastroenterology 1999;117:933-941 Proliferative CD4 + T-cell response in the acute phase of disease to recombinant HCV proteins in 38 patients with acute HCV infection

17. Immune response during acute and chronic HCV infections

18. Correlate of protection and disease progression ?

19. B cell CD8 + CTL Hepatocyte CD4 + Th cell Y Y Y Y Y NK NKT cells Immune response to HCV infection : Role largely unknown - early, vigorous, multi- specific response - strong NS3-response in resolving acute HCV - T H 1 in recovery - T H 2 in chronic - vigorous, multi- specific response - CTL exert some control on viral load antibodies to most structural and non-structural viral proteins are made

20. Potential Mechanisms of Viral Persistence Inadequate HCV-specific IR –inadequate innate immune response –insufficient induction of adaptive IR –inability to maintain the adaptive IR Viral evasion mechanisms

21. Potential Mechanisms of Viral Persistence Inadequate HCV-specific IR –inadequate innate immune response NK Cell function Dendritic cell function –insufficient induction of adaptive IR –inability to maintain the adaptive IR

22. Effect of HCV on NK cell function CD81 NK cell (in vitro) HCV Binding of HCV E2 protein to CD81 on NK cell causes inhibition of - cytolytic activity - IFN-  production Crotta et al. JEM 2002;195:35-41 Tseng et al. JEM 2002;195:43-49 E2 E1

23. Effect of HCV on NK cell function Natural cytotoxicity and antibody-dependent cytotoxicity (ADCC) is not impaired in patients suffering from chronic hepatitis C Düesberg U, Schneiders A, Flieger D, Inchauspé G, Sauerbruch T, Spengler U. J Hepatol 2001;35:650-657

24. Potential Mechanisms of Viral Persistence Inadequate HCV-specific IR –insufficient induction of adaptive IR low level of viral antigen expression virus infection of antigen-presenting cells and dendritic cell function inappropriate cytokine profile of T H lack or low frequency of neutralizing antibodies

25. Liver 2x10 11 hepatocytes Kidney Pancreas Spleen & Lymphoid tissue B lymphocyte Monocyte Dendritic cell Liver and extra-hepatic infection sites BDEC ?

26. Janeway-Immunobiology Dendritic cell precursor - Monocyte IL-4 + GM-CSF LPS/TNF  Dendritic cell maturation

27. Reduced capacity of mature DC from HCV patients to induce allogeneic T cell proliferation. Bain et al. Gastroenterology 120:51-524, 2001

28. IL-2 production and percentages of CD4+/CD25+ cells in response to HCV core or TT antigens in HCV patients Sarobe et al. J.Virol 76:5062-5070, 2002

29. Potential Mechanisms of Viral Persistence Inadequate HCV-specific IR –inadequate innate immune response –insufficient induction of adaptive IR –inability to maintain the adaptive IR Viral evasion mechanisms

30. Potential Mechanisms of Viral Persistence Viral evasion mechanisms –replication in immune privileged sites –viral interference with antigen processing –viral suppression of host immune response –viral sequence variation –viral insusceptibility to cytokine mediated inhibition of replication and gene expression

31. Potential Mechanisms of Viral Persistence Viral evasion mechanisms –replication in immune privileged sites –viral interference with antigen processing –viral suppression of host immune response –viral sequence variation –viral insusceptibility to cytokine mediated inhibition of replication and gene expression

32. HCV core controversy The Journal of Immunology, 2001, 167: 5264-5272. Hepatitis C Virus Core Protein Inhibits Human T Lymphocyte Responses by a Complement-Dependent Regulatory Pathway 1,2 12 Zhi Qiang Yao, Duong Tony Nguyen, Apostolos I. Hiotellis and Young S. Hahn 3 3 Journal of Virology, February 2002, p. 990-997, Vol. 76, No. 3 Hepatitis C Virus Genotype 1b Core Protein Does Not Exert Immunomodulatory Effects on Virus-Induced Cellular Immunity Zhang-Xu Liu, 1 Hiroshi Nishida, 1 Jian-Wen He, 1,2 Michael M. C. Lai, 1,2 Ni Feng, 1 and Gunther Dennert 1**

33. Potential Mechanisms of Viral Persistence Viral evasion mechanisms –replication in immune privileged sites –viral interference with antigen processing –viral suppression of host immune response –viral sequence variation escape from humoral immune response escape from cellular immune response –viral insusceptibility to cytokine...

34. Variability of HCV 6 major genotypes more than 50 subtypes Quasispecies 5’UT CE1E2 p7 NS2 NS4B NS3 NS5A NS5B 3’ Hypervariable region - HVR1 384 410 cross-reactivity (%) R9QTTVVGGSQSHTVRGLTSLFSPGASQN60 F78QTHTTGGGAGHQAHSLTGLFSPGAKQN70 M122QTTTTGGSAHAVSSLTGLFSPGSKQN44 G31TTHTVGGSVARQVHSLTGLFSPGPQQK77 H1QTHTTGGVVGHATSGLTSLFSPGPSQK42 D6QTTTTGGQVSHATHGLTGLFSLGPQQK66

35. Potential Mechanisms of Viral Persistence Viral evasion mechanisms –replication in immune privileged sites –viral interference with antigen processing –viral suppression of host immune response –viral sequence variation –viral insusceptibility to cytokine mediated inhibition of replication and gene expression

36. Antagonism of IFN by HCV proteins IFN Protein Kinase PKR inactive Initiation Factor eIF-2  Phosphorylated Initiation Factor eIF-2  P Phosphatase (soluble) Pi mRNA translation inhibition Protein Kinase PKR active dsRNA ssRNA HCV E1 HCV NS5A

37. Development of HCV vaccines badly needs Better understanding of mechanisms of immune protection and clearance Development of tissue culture system and small animal model of HCV infection

39. Dendritic cell maturation Jacques Banchereau et al. Immunobiology of Dendritic Cells Annu. Rev. Immunol. 2000. 18:767-811.