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

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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

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