Hepatitis C Virus

1. Hepatitis C Virus Casey McGrath BIO 360

2. Outline • Epidemiology • Introduction to Hepatitis C Virus • Immune response • Novel drug therapies • Conclusions

3. Epidemiology Hepatitis C Virus (HCV): • ~170 million people worldwide • Chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (HCC) • Transmitted via blood--transfusions, intravenous drug use

4. Prevalence of HCV by world region

5. Transmission sources

6. Disease statistics Infected Individuals 85% Persistent Infection Most patients are asymptomatic and unaware they’re infected 30% Liver Disease 1-5% Death

7. HCV research • Unknowns • No cell culture system • No small animal model

8. Outline • Epidemiology • Introduction to Hepatitis C Virus • Immune response • Novel drug therapies • Conclusions

9. HCV • Genus Hepacivirus • Family Flaviviridae, with classical flaviviruses and animal pestiviruses • 6 genotypes worldwide, many subtypes and isolates based on nucleotide diversity • Quasispecies within individual

10. Distribution of Hepatitis C genotypes From Forns and Bukh, 1999.

11. HCV virion structure Hypothesized structure: • Icosahedral lipid membrane with E1/E2 glycoproteins • Icosahedral nucleocapsid

13. HCV Genome • 9.6 kb positive strand RNA genome • 5’ (with IRES) and 3’ noncoding regions • Open reading frame encoding polyprotein of ~3000 amino acids

14. C Core protein (nucleocapsid) 5’ UTR E1 Envelope glycoprotein-1 HVR-1 E2 Envelope glycoprotein-2 HVR-2 Viroporin ? p7 NS2 Zn-dependent proteinase ORF NS3 Zn-dependent proteinase, serine protease, helicase NS4A NS3 cofactor ER-derived membranous web formation NS4B NS5A Unknown function; component of replicase? 3’ UTR NS5B RNA dependent RNA polymerase

15. Protein F • Newly discovered protein F • Produced by ribosomal frameshift mutation around codon 11 of Core protein • Infected individuals contain antibodies • Function unknown

16. Hepatitis C Life Cycle CD81? www.rockefeller.edu/pubinfo/hepc.jpg

17. Epidemiology Introduction to Hepatitis C Virus Immune response Novel drug therapies Conclusions Outline

18. Patterns of Viremia • Drop after peak  successful control • Drop followed by rebound  chronic infection • Consistent HCV  chronic infection

19. Innate Immune Response 2 days after infection: • Protein kinase R (PKR) • Interferon regulatory factors (IRFs) • Antiviral gene products (type I IFN-inducible genes and immune TFs)

20. IRFs dsRNA PKR PKR PKR activated phosphorylation of IRFs IRFs act as transcription factors to upregulate antiviral gene products Gene products degrade viral RNA and prohibit protein translation

21. Innate Immune Response • Regardless of infection outcome • Viral resistance • Targeting by HCV proteins? • NS5A and E2 (PKR) • Core (JAK-STAT pathway) • NS3/4A (phosphorylated IRF-3)

22. Adaptive Immune Response Individuals who control virus: • IFN-γ preferentially expressed in liver • Induces expression of • genes encoding chemokines that attract T cells into inflamed tissues • proteins associated with antigen processing and presentation

23. CD8+ and CD4+ T cells • More vigorous CD8+ and CD4+ T cell responses in all individuals that controlled infection • Chronic infections occur when • unable to mount HCV-specific T cell responses • strong response that results in viral RNA clearance, followed by contraction in CD8+/CD4+ and rebound in viremia

24. Chronic HCV infection • Low frequencies and reduced capacity of HCV-specific CD8+ cells • Dendritic cells do not mature normally and have impaired stimulatory activity • CD4+ cells have reduced IL-2 production and proliferation http://www.lbl.gov/Publications/Currents/Archive/Oct-03-2003.html

25. Chronic HCV Infection • Impairment of Natural Killer (NK) cell cytotoxic activity • Reversible in patients responsive to IFN-α drug therapy • Frequency of NKT cells decreased NKT cells (orange) attacking an infected cell (pink) http://www.spectroscopynow.com/ftp_images/killertcells.jpg Natural Killer cell http://www.wasatchhealth.com/images/NK-Picture.jpg

26. Antibodies • Role of antibodies unclear and poorly studied • Virus can be cleared in absence of detectable antibody responses • Neutralizing antibodies target E2, which is highly variable and able to evade

27. Immune-mediated liver injury • Mechanisms responsible for liver injury poorly understood • Host immune response and not viral replication • High CD8+ in liver  immunopathogenesis and liver injury

28. Liver Environment Normal liver: • “Immuno-silent” state • CD8+ T cells trapped  apoptosis • Prevents unnecessary immune response to thousands of antigens liver is exposed to

29. Liver Environment HCV-infected liver: • Type I IFN production • Release of chemokines that promote infiltration of NK cells • Induced IFN-γ production in NK cells • Expression of chemokines that recruit activated T cells to liver

30. Liver Environment Depletion of NK cells before hepatotropic viral infection leads to inhibition of virus-specific T cell response and liver injury

31. Immune-mediated liver injury • HCV infects only 1-10% of hepatocytes • IFN-γ and TNF-α from CD8+ destroy nearby non-infected hepatocytes (“bystander killing”) • HCC occurs mainly due to high turnover rate in hepatocytes http://medicalimages.allrefer.com/large/hepatocellular-cancer-ct-scan.jpg

32. Epidemiology Introduction to Hepatitis C Virus Immune response Novel drug therapies Conclusions Outline

33. Current therapy • Combination pegylated interferon-α and ribavirin (nucleoside analog) • Mechanism poorly understood • Protein synthesis suppression; degradation of plus strand RNA • 50-80% effective

34. Current therapy Side effects: • Flu-like symptoms, tiredness, hair loss, trouble with thinking, moodiness, and depression • Hematologic • Anemia • bone marrow suppression by IFN  neutropenia, thrombocytopenia • ribavirin directly toxic to red blood cells  hemolysis • Worsening of liver disease

35. Novel drug therapies • Non-nucleoside inhibitors (NNIs) • Protease inhibitor • TGF-β • Cyclosporin A • Arsenic trioxide • RNA therapieis

36. Non-nucleoside Inhibitors • Target RdRp • Discovery method • Structurally distinct: • Benzothiadiazine • Disubstituted phenylalanine • 2 benzimidazole derivatives • Allosteric inhibition • Distinct binding sites http://www.replizyme.com/images/rev_rna_hep_c.gif

37. Protease Inhibitor • BILN 2061—NS3 protease inhibitor • Peptidomimetic • Oral ingestion • Clinical trial: • Rapid decline in viral load • Rebound 4-11 days after treatment http://web.chemistry.gatech.edu/~williams/bCourse_Information/6521/protein/images/hcvmac1.gif

38. Transforming growth factor-β • Naturally occurring cytokine induced by core protein • Direct effect on HCV replication unknown • Decreased viral load • Increased fibrosis and cirrhosis

39. Cyclosporin A • Immunosuppressive drug • Mechanism unknown • FK506 does not suppress HCV replication • CsA binds to cyclophilins and blocks calcineurin  inhibits stimulation of genes essential for T cell activation • Combination with IFN http://www.alexis-corp.com/files/formula/lkt-c9611.gif

40. Arsenic Trioxide • Inhibits HCV replication at submicromolar concentrations • Non-toxic • Combination with IFN • Mechanism unknown

41. RNA treatments • Treatments that use RNA to halt viral replication • Three treatments in development: • RNA interference (RNAi) to degrade viral RNA • Small RNAs to bind to viral proteins • RNAs to outcompete viral proteins for binding to cellular proteins

42. RNAi http://www.life.uiuc.edu/shapiro/RNAipathway.jpg

43. RNAi http://www.life.uiuc.edu/shapiro/RNAipathway.jpg

44. RNAi • Inhibits HCV replication • Highly sequence specific (to 1 nt) • Multiple siRNAs to target different sites of viral genome • Short hairpin RNAs targeting conserved motifs encoded by retroviruses

45. Small RNAs • Overexpression of viral RNA elements • Bind to viral regulatory proteins and prevent binding of viral RNA  inhibits gene expression • RNAs analogous to 5’ UTR inhibited IRES-mediated translation • Combats sequence specificity problem

46. siRNAs • siRNAs targeted to cellular cofactors for HCV • La, PTB, hVAP-33 • Blocks HCV replication • Combats sequence specificity problem • Adenoviral-mediated expression

47. Conclusions • HCV is a major worldwide health concern • Much remains unknown about HCV • Current drug therapy is inadequate and insufficient • Novel therapies offer IFN-resistant patients and those with serious side effects hope of elimination of hepatitis C infection http://www.english.bayerconosur.com/noticias/tema008-1.asp

48. References • Ahmad, A. and Alvarez, F. (2004). Role of NK and NKT cells in the immunopathogenesis of HCV-induced hepatitis. Journal of Leukocyte Biology76: 743-759. • CDC FAQ: http://www.cdc.gov/ncidod/diseases/hepatitis/c/faq.htm • Forns, X. and Bukh, J. (1999). The Molecular Biology of Hepatitis C Virus: Genotypes and Quasispecies. Clinics in Liver Disease3. • Guo, J., Sohn, A., Zhu, Q. and Seeger, C. (2004). Mechanism of the interferon alpha response against hepatitis C virus replicons. Virology325: 71-81. • Hwang, D. et al (2004). Inhibition of hepatitis C virus replication by arsenic trioxide. Antimicrobial Agents and Chemotherapy48: 2876-2882. • Kowdley, K.V. (2005). Hematologic side effects of interferon and ribavirin therapy. Journal of Clinical Gastroenterology39, Suppl 1: S3-S8. • Kronke, J., Kittler, R., Buchholz, F., Windisch, M.P., Pietschmann, T., Bartenschlager, R. and Fresei, M. (2004). Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs. Journal of Virology78: 3436-3446. Slide template picture: http://www.english.bayerconosur.com/noticias/tema008-1.asp

49. References • Lamarre, D. et al (2003). An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature426: 186-189. • Liver Foundation: http://www.liverfoundation.org/db/articles/1028 • Mercer D, Schiller D, Elliot J, Douglas DN, Hao C, Rinfret A, Addison WR. (2001) Hepatitis C virus replication in mice with chimeric human livers. Nat Med 7: 927-933. • Moradpour, D., Cerny, A., Heim, M.H. and Blum, H.E. (2001). Hepatitis C: an update. Swiss Medical Weekly131: 231-298. • Moradpour, D. and Blum, H.E. (2004). A primer on the molecular virology of hepatitis C. Liver International24: 519-525. • Murata, T., Ohshima, T., Yamaji, M., Hosaka, M., Miyanari, Y., Hijikata, M. and Shimotohno, K. (2005). Suppression of hepatitis C virus replicon by TGF-β. Virology331: 407-417. • Nakagawa, M. et al (2004). Specific inhibition of hepatitis C virus replication by cyclosporine A. Biochemical and Biophysical Research Communications313: 42-47. • Penin, F., Dubuisson, J., Rey, F.A., Moradpour, D. and Pawlotsky, J. (2004). Structural Biology of Hepatitis C Virus. Hepatology39: 5-19. • Puig, M., Major, M.E., Mihallik, K. and Feinstone, S.M. (2004). Immunization of chimpanzees with an envelope protein-based vaccine enhances specific humoral and cellular immune responses that delay hepatitis C virus infection. Vaccine22: 991-1000

50. References • Ray, P.S. and Das, S. (2004). Inhibition of hepatitis C virus IRES-mediated translation by small RNAs analogous to stem-loop structures of the 5’-untranslated region. Nucleic Acids Research 32: 1678-1687. • Sarisky, R.T. (2004). Non-nucleoside inhibitors of the HCV polymerase. Journal of Antimicrobial Chemotherapy 54: 14-16. • Shoukry, N.H., Cawthon, A.G. and Walker, C.M. (2004). Cell-mediated immunity and the outcome of hepatitis C virus infection. Annual Reviews in Microbiology58: 391-424. • Sun, J., Li, K., Shata, M.T. and Chan, T. (2004). The immunologic basis for hepatitis C infection. Current Opinions in Gastroenterology20: 598-602. • Trujillo-Murillo, et al. (2004). Experimental models for hepatitis C virus (HCV): New opportunities for combating hepatitis C. Annals of Hepatology3: 54-62. • World Health Organization (WHO) (1999). Weekly Epidemiological Record No. 49, December. • Zhang, J., Yamada, O., Sakamoto, T., Yoshida, H., Iwai, T., Matsushita, Y., Shimamura, H., Araki, H. and Shimotohno, K. (2004). Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus. Virology320: 135-143.