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The identification of the hepatitis C virus

Michael Houghton, PhD Epiphany Biosciences Inc. San Francisco. FLAVIVIRUSES. PESTIVIRUSES. HEPACIVIRUSES. The identification of the hepatitis C virus. A bare tool-box in 1982.

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The identification of the hepatitis C virus

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  1. Michael Houghton, PhD Epiphany Biosciences Inc. San Francisco FLAVIVIRUSES PESTIVIRUSES HEPACIVIRUSES The identification of the hepatitis C virus

  2. A bare tool-box in 1982 • Infected human & chimpanzee materials available but NANBH titers were generally much lower than for the other hepatitis viruses • PCR not yet discovered • No reliable NANBH antigen or antibody defined • No cell culture system • No high-throughput sequencing • Several months to sequence 1kbps and several weeks to synthesise a single 20 oligonucleotide RT primer • No molecular handle available • Since the existence of NANH was first demonstrated in 1974 ( Feinstone et al ), techniques used to identify HAV & HBV had been unsuccessful at identifying NANBH agent(s)

  3. FLAVIVIRUSES PESTIVIRUSES HEPACIVIRUSES But, many talented colleagues & collaborators…..

  4. Colleagues • My lab in the early 1980’s at Chiron Corporation : • Qui-Lim Choo, • Amy Weiner • Kangsheng Wang • Maureen Powers • Josy Yu • Lacy Overby ( consultant & mentor ) • George Kuo’s lab ( adjacent lab. at Chiron working on HBV vaccination ) • Dan Bradley (CDC) collaboration • Chimpanzee materials

  5. Subtractive-hybridisation methods Trying to find a “needle in a hazardous haystack”

  6. Improved sensitivity of ~0.01% was inadequate to detect NANBH • cDNA libraries prepared from NANBH-infected human & chimpanzee livers • Probed in duplicate with highly-radioactive cDNA probes prepared infected (+) or uninfected (-) tissue • Succeeded in identifying NANBH-specific clones present at ~ 0.01% but none deemed to be derived from a putative NANBH genome • Frozen liver pieces needed to be powdered prior to RNA extraction to maintain mRNA integrity • Aerosol hazard • Specially-designed bagged liver crusher • Plague BL3 lab • Millions of clones from different human & chimpanzee livers screened using a total of ~ 500mCi P32 • Concerns from OCEA • Ultra-centrifuges used for infectious NANBH plasma equipped with special hepaire filters

  7. Developed sensitive silver staining methods to identify a high-M.Wt. NANBH genome • 300ml of infectious chimpanzee plasma pelleted , treated with nucleases and then extracted, purified and run through a single gel slot followed by silver staining • No discrete high M.Wt. RNA or DNA band observed

  8. Using known viral genomes as hybridisation probes • HBV • HAV • YFV • BVDV • Oligonucleotides to highly conserved regions of the above • All unsuccessful at identifying a specific nucleic acid in NANBH samples

  9. Is the NANBH agent (s) a relative of the hepatitis delta agent ? • Membranous cytoplasmic tubules observed in NANBH-infected livers are similar to those observed in delta-infected livers • R.Purcell et al 1983 • Mario Rizzetto discovered the delta hepatitis virus as an antigen within the nucleii of HBV-infected livers • M.Rizzetto et al 1977 • RNA molecule associated with delta hepatitis samples • M.Rizzetto, J. Gerin et al 1980 • John Gerin’s Lab cloned & sequenced a small piece of this RNA but found most of the RNA unusually refractory to cloning • K.Denniston et al 1986

  10. Electron micrographs of HDV RNA Source: Wang et al, Nature 1986,102:18544-18549

  11. Self-complementarity of HDV RNA Computer line matrix Covalently-closed, double stranded HDV RNA structure 1003 1170 1337 1504 1671 1 168 335 602 669 836 1 168 335 602 669 836 1003 1170 1337 1504 1671 Source: Wang et al, Nature 1986,102:18544-18549

  12. Nucleotide sequence of HDV RNA encoding delta antigen Source: Wang et al, Nature 1986,102:18544-18549

  13. Autoradiogram contains control HDV RNA and total nucleic acids extracted from high titer NANB plasma hybridized to 32P-labelled HDV cDNA insert DNA under moderate and low stringency conditions Failure to observe specific hybridisation of HDV RNA to NANBH a b 1 2 3 4 1 2 3 4 a a b b c c d d Source: Weiner et al, J Med Virol 1987, Mar 21(3):239-247

  14. Attempts to culture the NANBH agent(s) • Numerous cell-lines incubated with numerous NANBH chimpanzee & human sera/plasma/liver samples • CPE & EM as read-outs • Adenoviruses & foamyviruses cultured but not specific to NANBH

  15. Is the NANBH agent(s) a retrovirus ? • Various reports of RT activity in pelleted NANBH sera samples • Retroviral foamy-like viruses cultured from NANBH samples • No reproducible RT activity observed and foamy viruses cultured in vitro were not specific to NANBH samples

  16. Ongoing debate ( 1982-1985 ) :Should we screen NANBH cDNA expression libraries using sera from clinically-diagnosed NANBH samples ? • Con : • Too difficult & risky • M.Ptashne; H.Varmus; Others • Personal experience of difficulty with this approach even when using well-characterised and specific Abs • let alone using uncharacterised human & chimp sera in which the existence & titer of NANBH-specific Abs were unknown • Concerns that because of it’s high chronicity rates, NANBH may not elicit robust immune response • Pro : • Highly recommended by George Kuo ( 1985 ) • His quantitative assessment of Ag/Ab binding sensitivities in the context of known NANBH liver titers ( determined by Dan Bradley ) provided an explanation for the failure by the field to have identified specific NANBH antibodies • Also suggested by Dan Bradley • Then working in parallel on this approach with Genelabs

  17. 1985-1987 : Expression screening • Many liver & plasma cDNA libraries screened using numerous different chimpanzee & human NANBH sera as a presumptive source of specific Abs • Using chimpanzee liver & plasma samples from Dan Bradley of relatively high infectivity titer for NANBH • Obtained as a result of a collaborative screening initiative to identify NANBH samples of known,high infectivity titers in chimpanzees • Used many different convalescent & chronic NANBH sera as presumptive source of specific NANBH Abs • Result : Succeeded in cloning MS2 bacteriophage RNA & plenty of host genes but not NANBH

  18. 13-14 years since the demonstration of the existence of NANBH 1987/1988 : Success at last, using serum from a patient with chronic NANH (associated with unusually high ALT levels) as the presumptive source of NANBH Ab

  19. Ultracentrifuge NANBH-Infectious Chimpanzee Plasma NANBH Patients Serum Antibodies Pellet Extract total RNA+DNA False positives Clone 5-1-1 Incubate Bacterial cDNA libraries in "expression" vector gt11 DETERMINE PROPERTIES OF CLONE 5-1-1 • Extra-chromosomal • Derived from RNA (~9600 nt) found only in NANBH samples • Encodes protein that binds antibodies found only in NANBH infections IDENTIFICATION OF HEPATITIS C VIRUS (HCV)

  20. Proof that clone 5-1-1 cDNA really was derived from an etiological agent of NANBH

  21. Hybridization analysis of clone 81 cDNA with host DNA a b 1 2 3 4 5 6 M 10 7 8 9 1 2 M Source: Qui-Lim Choo et al, Science 1989, 244(4902):359-362

  22. Hybridization of clone 81 cDNA to RNA a c d 1 2 3 1 2 1 2 3 a a b b c b 1 2 3 Source: Qui-Lim Choo et al, Science 1989, 244(4902):359-362

  23. Immunoblot assay for PS5 antibodies a 2 1 • b NANBH HBV HAV ALT 9 71 19 17 11 54 9 10 18 106 10 22 C DAY 0 76 118 154 0 42 169 223 0 15 41 129 Source: Choo et al, Science 1989, 244(4902):359-362

  24. Incidence of PS5 antibodies in experimentally infected chimpanzees Chimp Agent Sampling times ALT Counts per minute 1 2 3 4 5 6 7 8 9 10 11 NANBH NANBH NANBH NANBH HBV HBV HBV HAV HAV HAV HAV 0, 76, 118, 154 0, 21, 73, 138 0, 43, 53, 159 0, 55, 83, 140 0, 359, 450 0, 115, 205, 240 0, 42, 169, 223 0, 15, 41, 129 0, 22, 115, 139 0, 26, 74, 205 0, 25, 40, 268 9, 71, 19, 17 5, 52, 13, 13 8, 205, 14, 6 11, 132, 7, 7 12, nd, 6 9, 126, 9, 13 11, 54, 9, 10 18, 106,10, 22 7, 83, 5, 10 15, 130, 8, 5 4, 147, 18, 5 250, 306, 5664, 8301 294, 398, 2133, 8632 152, 349, 392, 3738 349, 267, 392, 2397 804, 660, 656 618, 606, 514, 790 454, 221, 272, 198 256, 597, 266, 295 218, 176, 214, 341 162, 219, 554, 284 333, 453, 419, 358 Source: Choo et al, Science 1989, 244(4902):359-362

  25. Additional Proof • PS5 antibodies observed in the majority of clinically-diagnosed NANBH cases and not in controls • Small cohorts obtained from Gary Gitnick ( UCLA ) et al • PS5 antibodies induced following post-transfusion NANBH infection • Samples from Gary Tegtmeier ( Kansas City Blood Bank ) • Overlapping clones of clones 5-1-1 exhibited distant sequence identity with Dengue virus

  26. Qui-Lim Choo, George Kuo, Amy Weiner, Lacy Overby, Daniel Bradley & Michael Houghton 1st public disclosure at UCSF in early 1988 Etiological role in NANBH now proven - Agent now termed the hepatitis C virus (HCV)

  27. Detection of HCV antibodies in proven infectious blood samples - the Harvey Alter panel ( 1st generation assay ) Serum Counts per minute Proven infectious in chimp 1 (PT-NANBH) 2 (PT-NANBH) 3 (PT-NANBH) Acute NANBH patients 1 2 3 31,962 22,871 25,381 909 40,883 25,812 31,495 32,107 17,483 20,983 726 33,521 23,512 30,907 32,121 21,623 21,039 767 35,870 26,476 33,723 28,584 19,863 20,047 580 34,526 23,723 33,043 Chronic NANBH patients Implicated blood donors Unproven infectivity in chimp Acute PT-NANBH patient Implicated blood donor 1,207 590 740 469 1,786 477 1,489 461 Pedigreed normal controls 1 2 3 4 5 998 887 591 634 584 775 632 446 533 531 647 561 459 758 553 584 469 327 649 429 Blood donors Disease controls Alcoholic hepatitis Primary biliary cirrhosis 842 915 571 1,118 586 741 566 750 Source: George Kuo et al, Science 1989, 244:362-364

  28. The HCV Discovery Team ( Nature Medicine 2000 ) : George Kuo, Qui-Lim Choo, Daniel Bradley, Michael Houghton

  29. Organization of the HCV genome IRES (341 nt) UTR (~200 nt) 5' Open Reading Frame (~9050nt) 3' (Py)n Host signalase HCV Zn-dep. proteinase HCV NS3/NS4a Ser protease Proteolysis: C gpE1 gpE2 p7 NS2 NS3 NS4a NS4b NS5a NS5b Envelope glycoproteins Ion channel & Virion secretion Zn-dep. proteinase /Ser protease/ helicase Ser protease co-factor Virion secretion Functions: RNA-binding nucleocapsid C Zn-dep. proteinase Membranous web RNA-dep. replicase F Frame shift

  30. Recombinant gpE1/gpE2 vaccine protects chimpanzees against challenge with homologous and heterologous HCV 1a viruses(Houghton & Abrignani (2005) Nature ) Combined results from homologous HCV-1 and heterologous HCV-H challenges ( 1a viruses that predominate in USA ) Number that developed: Number Acuteinfection Chronic infection Vaccinees 31 26(84%) 5(16%) P = <0.001 Controls 24 24(100%) 15(62%) Note: Controls data pooled from Chiron + NIH

  31. (J.Bukh et al; NIH)

  32. Summary of chimpanzee prophylactic data using heterologous HCV-H challenges • Naïve chimpanzees immunised with rec.HCV-1 gpE1/gpE2 & challenged 2-4 weeks later with heterologous HCV-H (both 1a genotypes) • No sterilising immunity achieved Group No. of Carriers Controls 8/14 (57%) Vaccine 3/19 (16%) P=0.02

  33. Non-A, Non-B Hepatitis (NANBH) in the early 1980’s • Post-transfusion NANB hepatitis occurred in up to 10% transfusions • Harvey Alter et al, NIH ; Jim Mosley et al, Transfusion-Transmitted Virus Study Group • Also occurred frequently as sporadic, non-transfusion-associated NANB hepatitis • Miriam Alter et al, CDC • Often resulted in persistent hepatitis and could develop into liver cirrhosis • Harvey Alter et al, NIH : Leonard Seeff et al VA • NANB hepatitis could be transmitted to chimpanzees following experimental i/v challenge using human sera or blood products • Daniel Bradley et al, CDC; Bob Purcell et al , NIH ;

  34. Evidence for multiple, blood-transmissible NANB hepatitis agents • Different incubation times in humans & infected chimpanzees • Bob Purcell et al, NIH ; Blaine Hollinger et al, Houston • Silent or occult HBV infections - was NANB really an altered form of HBV • Christian Trepos et al, Lyon ; Christian Brechot et al, Paris • One NANBH agent caused characteristic ~200nM membranous tubules in infected chimpanzee livers - the “tubule-forming agent (tfa)” • Yohko Shimizu et al , Japan • Solvent-sensitive ( ie, the presumed enveloped tfa ) and solvent-resistant ( non-enveloped ) agents could be transmitted to chimpanzees • Dan Bradley et al, CDC ; Bob Purcell et al, NIH • Filtration studies indicated that the enveloped tfa was <80nM and the chloroform-resistant , non-enveloped agent was ~ 30nM • Dan Bradley et al, CDC; Bob Purcell , Steve Feinstone et al, NIH • Physico-chemical characteristics suggested that the tfa might be a togavirus or flavivirus or a delta-like hepatitis agent or a very novel type of virus • Dan Bradley et al, CDC ; Bob Purcell et al, NIH

  35. Enter the “Shimizu antibody” • B cells from NANBH patients were immortalised and then screened for specificity of binding to NANBH liver sections • NANBH-specific antibodies identified • Y. Shimizu et al 1985

  36. Specific binding of Shimizu antibody to NANBH-infected hepatocytes Chimpanzee 61 Chimpanzee 38 300 150 200 Alanine aminotransferase activity (Karmen units) 100 100 50 0 5 10 15 20 25 30 35 55 60 0 5 10 15 20 25 30 35 50 155 Time after inoculation (weeks) Time after inoculation (weeks) Normal activity = 30 Karmen units Cytoplasmic fluorescence present Cytoplasmic fluorescence absent Source: Shimizu et al, PNAS USA 1985, 82:2138-2142

  37. Ultrastructural localization of the Shimizu antigen Immunoperoxidase EM Bar = 500 nm Source: Shimizu et al, PNAS USA 1985, 82:2138-2142

  38. Shimizu antibodies • Many isolated in-house at Chiron • Later found to be binding to host antigenic sequences and not binding to NANBH-specific sequences • Yohko Shimizu et al • Subsequently, unable to identify the Shimizu cDNA by expression screening

  39. Detection of HCV antibody in NANBH patients from the United States ( 1st generation assay ) Transmission Total patients Percent positive Blood transfusion No identifiable source (community acquired) 24 59 71* 58† • * Between one and three serum samples assayed from patients who had received transfusions and • who were diagnosed with chronic NANBH on the basis of clinical symptoms, elevations of serum ALT for >6 months, serologic exclusion of infection with other agents and the exclusion of other apparent causes of liver injury. • † Sequential serum samples obtained prospectively up to 3 years after the onset of clinical hepatitis associated with elevated serum ALT in the absence of serologic markers for other agents and other identifiable causes of liver injury. Source: G.Kuo et al, Science 1989, 244(4902):362-364

  40. Detection of HCV antibody in NANBH cases from Italy and Japan ( 1st generation assay ) Country Number of patients Disease Percent positive Italy (F.Bonino) Japan(T.Miyamua) Japan(T.Miyamura) 32 23 13 Chronic Chronic Acute, resolving 84* 78† 15† • * Serum samples assayed in triplicate from each patient with transfusion-related chronic NANBH • † A prospective study in which sequential serum samples were assayed for at least 6 months after the onset of acute NANBH. The serum ALT of acute, resolving patients returned to normal and stable levels, whereas chronic patients displayed abnormal levels for at least 6 months. Source: Kuo et al, Science 1989, 244(4902):362-364

  41. F.Bonino Venice, Italy 1992 1st International Meeting on HCV & Related Viruses

  42. Colleagues • Lacy Overby, Amy Weiner • Chiron Corporation • Jang Han • Chiron Corporation • Karen McCaustland • CDC

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