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Casey McGrath- "Hepatitis C Virus


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Casey McGrath- "Hepatitis C Virus

  1. 1. Hepatitis C Virus Casey McGrath BIO 360
  2. 2. <ul><li>Epidemiology </li></ul><ul><li>Introduction to Hepatitis C Virus </li></ul><ul><li>Immune response </li></ul><ul><li>Novel drug therapies </li></ul><ul><li>Conclusions </li></ul>Outline
  3. 3. Epidemiology <ul><li>Hepatitis C Virus (HCV): </li></ul><ul><li>~170 million people worldwide </li></ul><ul><li>Chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (HCC) </li></ul><ul><li>Transmitted via blood--transfusions, intravenous drug use </li></ul>
  4. 4. Prevalence of HCV by world region Data Source: Weekly Epidemiological Record No. 49 / Dec, 1999 / WHO 59 169.7 3.1 5811 Total 11 62.2 3.9 1600 Western Pacific 3 32.3 2.15 1500 South-East Asia 19 8.9 1.03 858 Europe 7 21.3 4.6 466 Eastern Mediterranean 7 13.1 1.7 785 Americas 12 31.9 5.3 602 Africa Number of Countries (by WHO Region) Where Data Unavailable Infected Population (Millions) Hepatitis C Prevalence (Rate %) Total Population (Millions) WHO Region HEPATITIS C ESTIMATED WORLD INFECTION PREVALENCE (BY WHO REGION)
  5. 5. Transmission sources
  6. 6. Disease statistics Infected Individuals Persistent Infection Liver Disease Death 85% 30% 1-5% Most patients are asymptomatic and unaware they’re infected
  7. 7. HCV research <ul><li>Unknowns </li></ul><ul><li>No cell culture system </li></ul><ul><li>No small animal model </li></ul>
  8. 8. <ul><li>Epidemiology </li></ul><ul><li>Introduction to Hepatitis C Virus </li></ul><ul><li>Immune response </li></ul><ul><li>Novel drug therapies </li></ul><ul><li>Conclusions </li></ul>Outline
  9. 9. HCV <ul><li>Genus Hepacivirus </li></ul><ul><li>Family Flaviviridae, with classical flaviviruses and animal pestiviruses </li></ul><ul><li>6 genotypes worldwide, many subtypes and isolates based on nucleotide diversity </li></ul><ul><li>Quasispecies within individual </li></ul>
  10. 10. Distribution of Hepatitis C genotypes From Forns and Bukh, 1999.
  11. 11. HCV virion structure <ul><li>Hypothesized structure: </li></ul><ul><li>Icosahedral lipid membrane with E1/E2 glycoproteins </li></ul><ul><li>Icosahedral nucleocapsid </li></ul>
  12. 13. HCV Genome <ul><li>9.6 kb positive strand RNA genome </li></ul><ul><li>5’ (with IRES) and 3’ noncoding regions </li></ul><ul><li>Open reading frame encoding polyprotein of ~3000 amino acids </li></ul>
  13. 14. C E1 E2 p7 NS2 NS3 NS4A NS4B NS5A NS5B Core protein (nucleocapsid) Envelope glycoprotein-1 Envelope glycoprotein-2 Viroporin ? Zn-dependent proteinase Zn-dependent proteinase, serine protease, helicase NS3 cofactor ER-derived membranous web formation Unknown function; component of replicase? RNA dependent RNA polymerase HVR-1 HVR-2 5’ UTR 3’ UTR ORF
  14. 15. Protein F <ul><li>Newly discovered protein F </li></ul><ul><li>Produced by ribosomal frameshift mutation around codon 11 of Core protein </li></ul><ul><li>Infected individuals contain antibodies </li></ul><ul><li>Function unknown </li></ul>
  15. 16. Hepatitis C Life Cycle CD81?
  16. 17. Outline <ul><li>Epidemiology </li></ul><ul><li>Introduction to Hepatitis C Virus </li></ul><ul><li>Immune response </li></ul><ul><li>Novel drug therapies </li></ul><ul><li>Conclusions </li></ul>
  17. 18. Patterns of Viremia <ul><li>Drop after peak  successful control </li></ul><ul><li>Drop followed by rebound  chronic infection </li></ul><ul><li>Consistent HCV  chronic infection </li></ul>
  18. 19. Innate Immune Response <ul><li>2 days after infection: </li></ul><ul><li>Protein kinase R (PKR) </li></ul><ul><li>Interferon regulatory factors (IRFs) </li></ul><ul><li>Antiviral gene products (type I IFN-inducible genes and immune TFs) </li></ul>
  19. 20. PKR PKR dsRNA PKR activated IRFs phosphorylation of IRFs IRFs act as transcription factors to upregulate antiviral gene products Gene products degrade viral RNA and prohibit protein translation
  20. 21. Innate Immune Response <ul><li>Regardless of infection outcome </li></ul><ul><li>Viral resistance </li></ul><ul><li>Targeting by HCV proteins? </li></ul><ul><ul><li>NS5A and E2 (PKR) </li></ul></ul><ul><ul><li>Core (JAK-STAT pathway) </li></ul></ul><ul><ul><li>NS3/4A (phosphorylated IRF-3) </li></ul></ul>
  21. 22. Adaptive Immune Response <ul><li>Individuals who control virus: </li></ul><ul><li>IFN- γ preferentially expressed in liver </li></ul><ul><li>Induces expression of </li></ul><ul><ul><li>genes encoding chemokines that attract T cells into inflamed tissues </li></ul></ul><ul><ul><li>proteins associated with antigen processing and presentation </li></ul></ul>
  22. 23. CD8+ and CD4+ T cells <ul><li>More vigorous CD8+ and CD4+ T cell responses in all individuals that controlled infection </li></ul><ul><li>Chronic infections occur when </li></ul><ul><ul><li>unable to mount HCV-specific T cell responses </li></ul></ul><ul><ul><li>strong response that results in viral RNA clearance, followed by contraction in CD8+/CD4+ and rebound in viremia </li></ul></ul>
  23. 24. Chronic HCV infection <ul><li>Low frequencies and reduced capacity of HCV-specific CD8+ cells </li></ul><ul><li>Dendritic cells do not mature normally and have impaired stimulatory activity </li></ul><ul><li>CD4+ cells have </li></ul><ul><li>reduced IL-2 </li></ul><ul><li>production and </li></ul><ul><li>proliferation </li></ul>
  24. 25. Chronic HCV Infection <ul><li>Impairment of Natural Killer (NK) cell cytotoxic activity </li></ul><ul><ul><li>Reversible in patients responsive to IFN-α drug therapy </li></ul></ul><ul><li>Frequency of NKT cells decreased </li></ul>NKT cells (orange) attacking an infected cell (pink) Natural Killer cell
  25. 26. Antibodies <ul><li>Role of antibodies unclear and poorly studied </li></ul><ul><li>Virus can be cleared in absence of detectable antibody responses </li></ul><ul><li>Neutralizing antibodies target E2, which is highly variable and able to evade </li></ul>
  26. 27. Immune-mediated liver injury <ul><li>Mechanisms responsible for liver injury poorly understood </li></ul><ul><li>Host immune response and not viral replication </li></ul><ul><li>High CD8+ in liver  immunopathogenesis and liver injury </li></ul>
  27. 28. Liver Environment <ul><li>Normal liver: </li></ul><ul><li>“ Immuno-silent” state </li></ul><ul><li>CD8+ T cells trapped  apoptosis </li></ul><ul><li>Prevents unnecessary immune response to thousands of antigens liver is exposed to </li></ul>
  28. 29. Liver Environment <ul><li>HCV-infected liver: </li></ul><ul><li>Type I IFN production </li></ul><ul><li>Release of chemokines that promote infiltration of NK cells </li></ul><ul><li>Induced IFN-γ production in NK cells </li></ul><ul><li>Expression of chemokines that recruit activated T cells to liver </li></ul>
  29. 30. Liver Environment <ul><li>Depletion of NK cells before hepatotropic viral infection leads to inhibition of virus-specific T cell response and liver injury </li></ul>
  30. 31. Immune-mediated liver injury <ul><li>HCV infects only 1-10% of hepatocytes </li></ul><ul><li>IFN-γ and TNF-α from CD8+ destroy nearby non-infected hepatocytes (“bystander killing”) </li></ul><ul><li>HCC occurs mainly </li></ul><ul><li>due to high turnover </li></ul><ul><li>rate in hepatocytes </li></ul>
  31. 32. Outline <ul><li>Epidemiology </li></ul><ul><li>Introduction to Hepatitis C Virus </li></ul><ul><li>Immune response </li></ul><ul><li>Novel drug therapies </li></ul><ul><li>Conclusions </li></ul>
  32. 33. Current therapy <ul><li>Combination pegylated interferon-α and ribavirin (nucleoside analog) </li></ul><ul><li>Mechanism poorly understood </li></ul><ul><li>Protein synthesis suppression; degradation of plus strand RNA </li></ul><ul><li>50-80% effective </li></ul>
  33. 34. Current therapy <ul><li>Side effects: </li></ul><ul><li>Flu-like symptoms, tiredness, hair loss, trouble with thinking, moodiness, and depression </li></ul><ul><li>Hematologic </li></ul><ul><ul><li>Anemia </li></ul></ul><ul><ul><li>bone marrow suppression by IFN  neutropenia, thrombocytopenia </li></ul></ul><ul><ul><li>ribavirin directly toxic to red blood cells  hemolysis </li></ul></ul><ul><li>Worsening of liver disease </li></ul>
  34. 35. Novel drug therapies <ul><li>Non-nucleoside inhibitors (NNIs) </li></ul><ul><li>Protease inhibitor </li></ul><ul><li>TGF-β </li></ul><ul><li>Cyclosporin A </li></ul><ul><li>Arsenic trioxide </li></ul><ul><li>RNA therapieis </li></ul>
  35. 36. Non-nucleoside Inhibitors <ul><li>Target RdRp </li></ul><ul><li>Discovery method </li></ul><ul><li>Structurally distinct: </li></ul><ul><ul><li>Benzothiadiazine </li></ul></ul><ul><ul><li>Disubstituted phenylalanine </li></ul></ul><ul><ul><li>2 benzimidazole derivatives </li></ul></ul><ul><li>Allosteric inhibition </li></ul><ul><li>Distinct binding sites </li></ul>
  36. 37. Protease Inhibitor <ul><li>BILN 2061—NS3 protease inhibitor </li></ul><ul><li>Peptidomimetic </li></ul><ul><li>Oral ingestion </li></ul><ul><li>Clinical trial: </li></ul><ul><ul><li>Rapid decline in viral load </li></ul></ul><ul><ul><li>Rebound </li></ul></ul><ul><ul><li>4-11 days </li></ul></ul><ul><ul><li>after </li></ul></ul><ul><ul><li>treatment </li></ul></ul>
  37. 38. Transforming growth factor-β <ul><li>Naturally occurring cytokine induced by core protein </li></ul><ul><li>Direct effect on HCV replication unknown </li></ul><ul><li>Decreased viral load </li></ul><ul><li>Increased fibrosis and cirrhosis </li></ul>
  38. 39. Cyclosporin A <ul><li>Immunosuppressive drug </li></ul><ul><li>Mechanism unknown </li></ul><ul><li>FK506 does not suppress HCV replication </li></ul><ul><li>CsA binds to cyclophilins and blocks calcineurin  </li></ul><ul><li>inhibits stimulation </li></ul><ul><li>of genes essential </li></ul><ul><li>for T cell activation </li></ul><ul><li>Combination with IFN </li></ul>
  39. 40. Arsenic Trioxide <ul><li>Inhibits HCV replication at submicromolar concentrations </li></ul><ul><li>Non-toxic </li></ul><ul><li>Combination with IFN </li></ul><ul><li>Mechanism unknown </li></ul>
  40. 41. RNA treatments <ul><li>Treatments that use RNA to halt viral replication </li></ul><ul><li>Three treatments in development: </li></ul><ul><ul><li>RNA interference (RNAi) to degrade viral RNA </li></ul></ul><ul><ul><li>Small RNAs to bind to viral proteins </li></ul></ul><ul><ul><li>RNAs to outcompete viral proteins for binding to cellular proteins </li></ul></ul>
  41. 42. RNAi
  42. 43. RNAi
  43. 44. RNAi <ul><li>Inhibits HCV replication </li></ul><ul><li>Highly sequence specific (to 1 nt) </li></ul><ul><li>Multiple siRNAs to target different sites of viral genome </li></ul><ul><li>Short hairpin RNAs targeting conserved motifs encoded by retroviruses </li></ul>
  44. 45. Small RNAs <ul><li>Overexpression of viral RNA elements </li></ul><ul><li>Bind to viral regulatory proteins and prevent binding of viral RNA  inhibits gene expression </li></ul><ul><li>RNAs analogous to 5’ UTR inhibited IRES-mediated translation </li></ul><ul><li>Combats sequence specificity problem </li></ul>
  45. 46. siRNAs <ul><li>siRNAs targeted to cellular cofactors for HCV </li></ul><ul><ul><li>La, PTB, hVAP-33 </li></ul></ul><ul><li>Blocks HCV replication </li></ul><ul><li>Combats sequence specificity problem </li></ul><ul><li>Adenoviral-mediated expression </li></ul>
  46. 47. Conclusions <ul><li>HCV is a major worldwide health concern </li></ul><ul><li>Much remains unknown about HCV </li></ul><ul><li>Current drug therapy is inadequate and insufficient </li></ul><ul><li>Novel therapies offer IFN-resistant patients and those with serious side effects hope of elimination of hepatitis C infection </li></ul>
  47. 48. References <ul><li>Ahmad, A. and Alvarez, F. (2004). Role of NK and NKT cells in the immunopathogenesis of HCV-induced hepatitis. Journal of Leukocyte Biology 76 : 743-759. </li></ul><ul><li>CDC FAQ: http:// </li></ul><ul><li>Forns, X. and Bukh, J. (1999). The Molecular Biology of Hepatitis C Virus: Genotypes and Quasispecies. Clinics in Liver Disease   3 . </li></ul><ul><li>Guo, J., Sohn, A., Zhu, Q. and Seeger, C. (2004). Mechanism of the interferon alpha response against hepatitis C virus replicons. Virology 325 : 71-81. </li></ul><ul><li>Hwang, D. et al (2004). Inhibition of hepatitis C virus replication by arsenic trioxide. Antimicrobial Agents and Chemotherapy 48 : 2876-2882. </li></ul><ul><li>Kowdley, K.V. (2005). Hematologic side effects of interferon and ribavirin therapy. Journal of Clinical Gastroenterology 39 , Suppl 1: S3-S8. </li></ul><ul><li>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 Virology 78 : 3436-3446. </li></ul>Slide template picture:
  48. 49. References <ul><li>Lamarre, D. et al (2003). An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature 426 : 186-189. </li></ul><ul><li>Liver Foundation: </li></ul><ul><li>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. </li></ul><ul><li>Moradpour, D., Cerny, A., Heim, M.H. and Blum, H.E. (2001). Hepatitis C: an update. Swiss Medical Weekly 131 : 231-298. </li></ul><ul><li>Moradpour, D. and Blum, H.E. (2004). A primer on the molecular virology of hepatitis C. Liver International 24 : 519-525. </li></ul><ul><li>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. Virology 331 : 407-417. </li></ul><ul><li>Nakagawa, M. et al (2004). Specific inhibition of hepatitis C virus replication by cyclosporine A. Biochemical and Biophysical Research Communications 313 : 42-47. </li></ul><ul><li>Penin, F., Dubuisson, J., Rey, F.A., Moradpour, D. and Pawlotsky, J. (2004). Structural Biology of Hepatitis C Virus. Hepatology 39 : 5-19. </li></ul><ul><li>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. Vaccine 22 : 991-1000 </li></ul>
  49. 50. References <ul><li>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. </li></ul><ul><li>Sarisky, R.T. (2004). Non-nucleoside inhibitors of the HCV polymerase. Journal of Antimicrobial Chemotherapy 54 : 14-16. </li></ul><ul><li>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 Microbiology 58 : 391-424. </li></ul><ul><li>Sun, J., Li, K., Shata, M.T. and Chan, T. (2004). The immunologic basis for hepatitis C infection. Current Opinions in Gastroenterology 20 : 598-602. </li></ul><ul><li>Trujillo-Murillo, et al. (2004). Experimental models for hepatitis C virus (HCV): New opportunities for combating hepatitis C. Annals of Hepatology 3 : 54-62. </li></ul><ul><li>World Health Organization (WHO) (1999). Weekly Epidemiological Record No. 49, December. </li></ul><ul><li>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. Virology 320 : 135-143. </li></ul>