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

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Presentation on theme: "The identification of the hepatitis C virus Michael Houghton, PhD Epiphany Biosciences Inc. San Francisco PESTIVIRUSES FLAVIVIRUSES HEPACIVIRUSES."— Presentation transcript:

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

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

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

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:

11 Self-complementarity of HDV RNA Source: Wang et al, Nature 1986,102: Computer line matrixCovalently-closed, double stranded HDV RNA structure

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

13 Source: Weiner et al, J Med Virol 1987, Mar 21(3): 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 32 P-labelled HDV cDNA insert DNA under moderate and low stringency conditions 123 a a b d 4 c 123 b a b d 4 c

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 ( ) :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 : 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 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 years since the demonstration of the existence of NANBH

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

20 Proof that clone cDNA really was derived from an etiological agent of NANBH

21 Hybridization analysis of clone 81 cDNA with host DNA Source: Qui-Lim Choo et al, Science 1989, 244(4902): M10 ab 78912M

22 Hybridization of clone 81 cDNA to RNA Source: Qui-Lim Choo et al, Science 1989, 244(4902): a a b c 12 c a b 123 b 13 d 2

23 Immunoblot assay for PS5 antibodies Source: Choo et al, Science 1989, 244(4902): a ALT DAY NANBH HBV HAV C b

24 Incidence of PS5 antibodies in experimentally infected chimpanzees Source: Choo et al, Science 1989, 244(4902): Chimp Agent NANBH HBV HAV Sampling times 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 ALT 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 Counts per minute 250, 306, 5664, , 398, 2133, , 349, 392, , 267, 392, , 660, , 606, 514, , 221, 272, , 597, 266, , 176, 214, , 219, 554, , 453, 419, 358

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 exhibited distant sequence identity with Dengue virus

26 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

27 Detection of HCV antibodies in proven infectious blood samples - the Harvey Alter panel ( 1st generation assay ) Source: George Kuo et al, Science 1989, 244: (PT-NANBH) 2 (PT-NANBH) 3 (PT-NANBH) Acute NANBH patients Proven infectious in chimp 31,962 22,871 25, ,883 25,812 31,495 32,107 17,483 20, ,521 23,512 30,907 32,121 21,623 21, ,870 26,476 33,723 28,584 19,863 20, ,526 23,723 33,043 Acute PT-NANBH patient Implicated blood donor Unproven infectivity in chimp 1, , , Pedigreed normal controls Alcoholic hepatitis Primary biliary cirrhosis Disease controls , SerumCounts per minute Implicated blood donors Blood donors Chronic NANBH patients

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

29 RNA-binding nucleocapsid O pen R eading F rame (~9050nt) (Py) n 5' 3' CgpE1gpE2p7NS2NS3NS4aNS4bNS5a HCV Zn-dep. proteinase IRES (341 nt)UTR (~200 nt) Proteolysis: Host signalase Functions: Envelope glycoproteins Ion channel & Virion secretion Zn-dep. proteinase /Ser protease/ helicase Ser protease co-factor Membranous web Virion secretion RNA-dep. replicaseZn-dep. proteinase HCV NS3/NS4a Ser protease NS5b C F Frame shift Organization of the HCV genome

30 Number Acute infectionChronic infection 3126 (84%)5 (16%) 2424 (100%)15 (62%)Controls Number that developed: Vaccinees P = < Combined results from homologous HCV-1 and heterologous HCV-H challenges ( 1a viruses that predominate in USA ) Recombinant gpE1/gpE2 vaccine protects chimpanzees against challenge with homologous and heterologous HCV 1a viruses (Houghton & Abrignani (2005) Nature ) Note: Controls data pooled from Chiron + NIH

31 (J.Bukh et al; NIH)

32

33 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 GroupNo. of Carriers Controls8/14 (57%) Vaccine3/19 (16%) P=0.02

34 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 ;

35 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

36 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

37 Specific binding of Shimizu antibody to NANBH-infected hepatocytes Source: Shimizu et al, PNAS USA 1985, 82: Chimpanzee Chimpanzee 38 Time after inoculation (weeks) Alanine aminotransferase activity (Karmen units) Time after inoculation (weeks) Cytoplasmic fluorescence present Cytoplasmic fluorescence absent Normal activity = 30 Karmen units

38 Ultrastructural localization of the Shimizu antigen Source: Shimizu et al, PNAS USA 1985, 82: Immunoperoxidase EM Bar = 500 nm

39 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

40 Detection of HCV antibody in NANBH patients from the United States ( 1st generation assay ) Source: G.Kuo et al, Science 1989, 244(4902): Transmission Blood transfusion No identifiable source (community acquired) Total patients Percent positive 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.

41 Detection of HCV antibody in NANBH cases from Italy and Japan ( 1st generation assay ) Source: Kuo et al, Science 1989, 244(4902): Country Italy (F.Bonino) Japan(T.Miyamua) Japan(T.Miyamura) Number of patients Disease Chronic Acute, resolving Percent positive 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.

42 1st International Meeting on HCV & Related Viruses F.Bonino Venice, Italy 1992

43 Colleagues Lacy Overby, Amy Weiner – Chiron Corporation Jang Han – Chiron Corporation Karen McCaustland – CDC


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