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The identification of the hepatitis C virus
Michael Houghton, PhD
Epiphany Biosciences Inc.
San Francisco
PE
ST
IVIR
US
ES
FLAVIVIRUSES
HEPACIVIRUSES
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)
But, many talented colleagues &
collaborators…..
PE
ST
IVIR
US
ES
FLAVIVIRUSES
HEPACIVIRUSES
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 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
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
Source: Wang et al, Nature 1986,102:18544-18549
1 16
8
33
5
60
2
66
9
83
6
10
03
11
70
13
37
15
04
16
71
1
168
335
602
669
836
1003
1170
1337
1504
1671
Computer line matrix Covalently-closed, double stranded HDV RNA structure
Nucleotide sequence of HDV RNA encoding delta antigen
Source: Wang et al, Nature 1986,102:18544-18549
Source: Weiner et al, J Med Virol 1987, Mar 21(3):239-247
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
1 2 3a
a
b
d
4
c
1 2 3b
a
b
d
4
c
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 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
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
Pellet
NANBHPatients
Serum Antibodies
Bacterial cDNA libraries in "expression" vector gt11
False positives
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
Incubate
Extract total RNA+DNA
Clone5-1-1
IDENTIFICATION OF HEPATITIS C VIRUS (HCV)
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
Source: Qui-Lim Choo et al, Science 1989, 244(4902):359-362
1 2 3 4 5 6 M 10
a b7 8 9 1 2 M
Hybridization of clone 81 cDNA to RNA
Source: Qui-Lim Choo et al, Science 1989, 244(4902):359-362
1 2 3a
a
b
c
1 2c
a
b
1 2 3b
1 3d 2
Immunoblot assay for PS5 antibodies
Source: Choo et al, Science 1989, 244(4902):359-362
2a
1
9 71 19 17
0 76 118 154
ALT
DAY
NANBH
11 54 9 10
0 42 169 223
HBV
18 106 10 22
0 15 41 129
HAV
C
b
Incidence of PS5 antibodies in experimentally infected chimpanzees
Source: Choo et al, Science 1989, 244(4902):359-362
Chimp
1
2
3
4
5
6
7
8
9
10
11
Agent
NANBH
NANBH
NANBH
NANBH
HBV
HBV
HBV
HAV
HAV
HAV
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, 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
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 )
Source: George Kuo et al, Science 1989, 244:362-364
1 (PT-NANBH)2 (PT-NANBH)3 (PT-NANBH)
Acute NANBH patients123
Proven infectious in chimp31,96222,87125,381
90940,88325,81231,495
32,10717,48320,983
72633,52123,51230,907
32,12121,62321,039
76735,87026,47633,723
28,58419,86320,047
58034,52623,72333,043
Acute PT-NANBH patientImplicated blood donor
Unproven infectivity in chimp1,207
590740469
1,786477
1,489461
12345
Pedigreed normal controls998887591634584
775632446533531
647561459758553
584469327649429
Alcoholic hepatitisPrimary biliary cirrhosis
Disease controls842915
5711,118
586741
566750
Serum Counts per minute
Implicated blood donors
Blood donors
Chronic NANBH patients
The HCV Discovery Team ( Nature Medicine 2000 ) : George Kuo, Qui-Lim Choo, Daniel Bradley, Michael Houghton
RNA-binding nucleocapsid
Open Reading Frame (~9050nt) (Py)n5' 3'
C gpE1 gpE2 p7 NS2 NS3 NS4a NS4b NS5a
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
NumberAcute
infection Chronic infection
31 26 (84%) 5 (16%)
24 24 (100%) 15 (62%)Controls
Number that developed:
Vaccinees
P = < 0.001
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
(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 No. of Carriers
Controls 8/14 (57%)
Vaccine 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– 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
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
Source: Shimizu et al, PNAS USA 1985, 82:2138-2142
Chimpanzee 61
300
200
100
0 5 10 15 20 25 30 35 50 155
150
100
50
Chimpanzee 38
Time after inoculation (weeks)
0 5 10 15 20 25 30 35 55 60Ala
nin
e a
min
otr
an
sfe
rase
act
ivity
(K
arm
en
un
its)
Time after inoculation (weeks)
Cytoplasmic fluorescence present
Cytoplasmic fluorescence absent
Normal activity = 30 Karmen units
Ultrastructural localization of the Shimizu antigen
Source: Shimizu et al, PNAS USA 1985, 82:2138-2142
Immunoperoxidase EM
Bar = 500 nm
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 )
Source: G.Kuo et al, Science 1989, 244(4902):362-364
Transmission
Blood transfusion
No identifiable source (community acquired)
Total patients
24
59
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.
Detection of HCV antibody in NANBH cases from Italy and Japan ( 1st generation assay )
Source: Kuo et al, Science 1989, 244(4902):362-364
Country
Italy (F.Bonino)
Japan(T.Miyamua)
Japan(T.Miyamura)
Number of patients
32
23
13
Disease
Chronic
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.
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