Hepatitis C Virus

Casey McGrath

BIO 360

• Epidemiology

• Introduction to Hepatitis C Virus

• Immune response

• Novel drug therapies

• Conclusions

Outline

Epidemiology

Hepatitis C Virus (HCV):

• ~170 million people worldwide

• Chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (HCC)

• Transmitted via blood--transfusions, intravenous drug use

Prevalence of HCV by world region

HEPATITIS C ESTIMATED WORLD INFECTION PREVALENCE

(BY WHO REGION)

WHO Region

Total Population (Millions)

Hepatitis C Prevalence

(Rate %)

Infected Population (Millions)

Number of Countries (by WHO Region)

Where Data Unavailable

Africa 602 5.3 31.9 12

Americas 785 1.7 13.1 7

Eastern Mediterranean

466 4.6 21.3 7

Europe 858 1.03 8.9 19

South-East Asia

1500 2.15 32.3 3

Western Pacific

1600 3.9 62.2 11

Total 5811 3.1 169.7 59

Data Source: Weekly Epidemiological Record No. 49 / Dec, 1999 / WHO

Transmission sources

Disease statistics

Infected Individuals

Persistent Infection

Liver Disease

Death

85%

30%

1-5%

Most patients are asymptomatic and unaware they’re infected

HCV research

• Unknowns

• No cell culture system

• No small animal model

• Epidemiology

• Introduction to Hepatitis C Virus

• Immune response

• Novel drug therapies

• Conclusions

Outline

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

Distribution of Hepatitis C genotypes

From Forns and Bukh, 1999.

HCV virion structure

Hypothesized structure:

• Icosahedral lipid membrane with E1/E2 glycoproteins

• Icosahedral nucleocapsid

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

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

Protein F

• Newly discovered protein F

• Produced by ribosomal frameshift mutation around codon 11 of Core protein

• Infected individuals contain antibodies

• Function unknown

Hepatitis C Life Cycle

www.rockefeller.edu/pubinfo/hepc.jpg

CD81?

Outline

• Epidemiology

• Introduction to Hepatitis C Virus

• Immune response

• Novel drug therapies

• Conclusions

Patterns of Viremia

1. Drop after peak successful control

2. Drop followed by rebound chronic infection

3. Consistent HCV chronic infection

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)

PKR PKR

dsRNA

PKR activated

IRFs

phosphorylation of IRFs

IRFs act as transcription factorsto upregulate antiviral gene products

Gene products degrade viral RNAand prohibit protein translation

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)

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

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

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

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 cellhttp://www.wasatchhealth.com/images/NK-Picture.jpg

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

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

Liver Environment

Normal liver:

• “Immuno-silent” state

• CD8+ T cells trapped apoptosis

• Prevents unnecessary immune response to thousands of antigens liver is exposed to

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

Liver Environment

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

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

Outline

• Epidemiology

• Introduction to Hepatitis C Virus

• Immune response

• Novel drug therapies

• Conclusions

Current therapy

• Combination pegylated interferon-α and ribavirin (nucleoside analog)

• Mechanism poorly understood

• Protein synthesis suppression; degradation of plus strand RNA

• 50-80% effective

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

Novel drug therapies

• Non-nucleoside inhibitors (NNIs)

• Protease inhibitor

• TGF-β

• Cyclosporin A

• Arsenic trioxide

• RNA therapieis

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

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

Transforming growth factor-β

• Naturally occurring cytokine induced by core protein

• Direct effect on HCV replication unknown

• Decreased viral load

• Increased fibrosis and cirrhosis

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

Arsenic Trioxide

• Inhibits HCV replication at submicromolar concentrations

• Non-toxic

• Combination with IFN

• Mechanism unknown

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

RNAi

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

RNAi

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

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

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

siRNAs

• siRNAs targeted to cellular cofactors for HCV– La, PTB, hVAP-33

• Blocks HCV replication

• Combats sequence specificity problem

• Adenoviral-mediated expression

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

References• 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.

• 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 Disease 3.• 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.

• Hwang, D. et al (2004). Inhibition of hepatitis C virus replication by arsenic trioxide. Antimicrobial Agents and Chemotherapy 48: 2876-2882.

• Kowdley, K.V. (2005). Hematologic side effects of interferon and ribavirin therapy. Journal of Clinical Gastroenterology 39, 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 Virology 78: 3436-3446.

Slide template picture: http://www.english.bayerconosur.com/noticias/tema008-1.asp

References• Lamarre, D. et al (2003). An NS3 protease inhibitor with antiviral effects

in humans infected with hepatitis C virus. Nature 426: 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 Weekly 131: 231-298.

• Moradpour, D. and Blum, H.E. (2004). A primer on the molecular virology of hepatitis C. Liver International 24: 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. Virology 331: 407-417.

• Nakagawa, M. et al (2004). Specific inhibition of hepatitis C virus replication by cyclosporine A. Biochemical and Biophysical Research Communications 313: 42-47.

• Penin, F., Dubuisson, J., Rey, F.A., Moradpour, D. and Pawlotsky, J. (2004). Structural Biology of Hepatitis C Virus. Hepatology 39: 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. Vaccine 22: 991-1000

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 Microbiology 58: 391-424.

• 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.

• Trujillo-Murillo, et al. (2004). Experimental models for hepatitis C virus (HCV): New opportunities for combating hepatitis C. Annals of Hepatology 3: 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. Virology 320: 135-143.