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PESTIVIRUSES The identification of the hepatitis C virus Michael Houghton, PhD Epiphany Biosciences Inc. San Francisco 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) PESTIVIRUSES But, many talented colleagues & collaborators….. 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 Trying to find a “needle in a hazardous haystack” Subtractive-hybridisation methods 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 Developed sensitive silver staining methods to identify a highM.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 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 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 Electron micrographs of HDV RNA Source: Wang et al, Nature 1986,102:18544-18549 Self-complementarity of HDV RNA 1671 1504 1337 1170 1003 836 669 602 335 168 1 Computer line matrix Covalently-closed, double stranded HDV RNA structure 1 168 335 602 669 836 1003 1170 1337 1504 1671 Source: Wang et al, Nature 1986,102:18544-18549 Nucleotide sequence of HDV RNA encoding delta antigen Source: Wang et al, Nature 1986,102:18544-18549 Failure to observe specific hybridisation of HDV RNA to NANBH 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 a 1 2 3 4 b 1 a a b b c c d d 2 3 4 Source: Weiner et al, J Med Virol 1987, Mar 21(3):239-247 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 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 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 wellcharacterised 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 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 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 13-14 years since the demonstration of the existence of NANBH Ultracentrifuge NANBH-Infectious Chimpanzee Plasma NANBH Patients Serum Antibodies Pellet Extract total RNA+DNA False positives Clone 5-1-1 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) Incubate Proof that clone 5-1-1 cDNA really was derived from an etiological agent of NANBH 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 Hybridization of clone 81 cDNA to RNA a 1 2 c 3 1 2 d 1 2 3 a a b b c b 1 2 3 Source: Qui-Lim Choo et al, Science 1989, 244(4902):359-362 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 DAY 0 76 118 154 0 42 169 223 0 15 41 129 C Source: Choo et al, Science 1989, 244(4902):359-362 Incidence of PS5 antibodies in experimentally infected chimpanzees Chimp Agent Sampling times 1 NANBH 0, 76, 118, 154 9, 71, 19, 17 250, 306, 5664, 8301 2 NANBH 0, 21, 73, 138 5, 52, 13, 13 294, 398, 2133, 8632 3 NANBH 0, 43, 53, 159 8, 205, 14, 6 152, 349, 392, 3738 4 NANBH 0, 55, 83, 140 11, 132, 7, 7 349, 267, 392, 2397 5 HBV 0, 359, 450 12, nd, 6 804, 660, 656 6 HBV 0, 115, 205, 240 9, 126, 9, 13 618, 606, 514, 790 7 HBV 0, 42, 169, 223 11, 54, 9, 10 454, 221, 272, 198 8 HAV 0, 15, 41, 129 18, 106,10, 22 256, 597, 266, 295 9 HAV 0, 22, 115, 139 7, 83, 5, 10 218, 176, 214, 341 10 HAV 0, 26, 74, 205 15, 130, 8, 5 162, 219, 554, 284 11 HAV 0, 25, 40, 268 4, 147, 18, 5 333, 453, 419, 358 ALT Counts per minute Source: Choo et al, Science 1989, 244(4902):359-362 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 Etiological role in NANBH now proven Agent now termed the hepatitis C virus (HCV) Qui-Lim Choo, George Kuo, Amy Weiner, Lacy Overby, Daniel Bradley & Michael Houghton 1st public disclosure at UCSF in early 1988 Detection of HCV antibodies in proven infectious blood samples the Harvey Alter panel ( 1st generation assay ) Serum Proven infectious in chimp 1 (PT-NANBH) Chronic NANBH patients 2 (PT-NANBH) 3 (PT-NANBH) Acute NANBH patients 1 Implicated blood donors 2 3 Unproven infectivity in chimp Acute PT-NANBH patient Implicated blood donor Pedigreed normal controls 1 2 Blood donors 3 4 5 Disease controls Alcoholic hepatitis Primary biliary cirrhosis Counts per minute 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 1,207 590 740 469 1,786 477 1,489 461 998 887 591 634 584 775 632 446 533 531 647 561 459 758 553 584 469 327 649 429 842 915 571 1,118 586 741 566 750 Source: George Kuo et al, Science 1989, 244:362-364 The HCV Discovery Team ( Nature Medicine 2000 ) : George Kuo, Qui-Lim Choo, Daniel Bradley, Michael Houghton Organization of the HCV genome IRES (341 nt) UTR (~200 nt) 5' Open Reading Frame (~9050nt) Proteolysis: C HCV Zn-dep. proteinase Host signalase gpE1 gpE2 Envelope glycoproteins RNA-binding nucleocapsid Functions: C p7 NS2 Ion channel & Virion secretion NS3 F Frame shift HCV NS3/NS4a Ser protease NS4a NS4b Zn-dep. Ser proteinase protease /Ser co-factor protease/ helicase Zn-dep. proteinase (Py)n NS5a NS5b Virion secretion Membranous web RNA-dep. replicase 3' 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: Vaccinees Number Acute infection Chronic infection 31 26 (84%) 5 (16%) P = < 0.001 Controls 24 Note: Controls data pooled from Chiron + NIH 24 (100%) 15 (62%) (J.Bukh et al; NIH) 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 Controls Vaccine No. of Carriers 8/14 (57%) 3/19 (16%) P=0.02 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 ; Evidence for multiple, blood-transmissible NANB hepatitis agents • • • Different incubation times in humans & infected chimpanzees – Silent or occult HBV infections - was NANB really an altered form of HBV – Dan Bradley et al, CDC ; Bob Purcell et al, NIH Filtration studies indicated that the enveloped tfa was <80nM and the chloroform-resistant , nonenveloped agent was ~ 30nM – • Yohko Shimizu et al , Japan Solvent-sensitive ( ie, the presumed enveloped tfa ) and solvent-resistant ( non-enveloped ) agents could be transmitted to chimpanzees – • 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)” – • Bob Purcell et al, NIH ; Blaine Hollinger et al, Houston 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 deltalike hepatitis agent or a very novel type of virus – Dan Bradley et al, CDC ; Bob Purcell et al, NIH 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 Specific binding of Shimizu antibody to NANBH-infected hepatocytes Alanine aminotransferase activity (Karmen units) Chimpanzee 61 Chimpanzee 38 300 150 200 100 100 50 0 5 10 15 20 25 30 35 50 155 0 Time after inoculation (weeks) 5 10 15 20 25 30 35 55 60 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 Ultrastructural localization of the Shimizu antigen Immunoperoxidase EM Bar = 500 nm Source: Shimizu et al, PNAS USA 1985, 82:2138-2142 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 Detection of HCV antibody in NANBH patients from the United States ( 1st generation assay ) Total patients Percent positive Blood transfusion 24 71* No identifiable source (community acquired) 59 58† Transmission • • * 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 Detection of HCV antibody in NANBH cases from Italy and Japan ( 1st generation assay ) Number of patients Disease Percent positive Italy (F.Bonino) 32 Chronic 84* Japan(T.Miyamua) 23 Chronic 78† Japan(T.Miyamura) 13 Acute, resolving 15† Country • * 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 1st International Meeting on HCV & Related Viruses F.Bonino Venice, Italy 1992 Colleagues • Lacy Overby, Amy Weiner – Chiron Corporation • Jang Han – Chiron Corporation • Karen McCaustland – CDC