The identification of the hepatitis C virus

1
The identification of the hepatitis C virus

• Michael Houghton, PhD
• Epiphany Biosciences Inc.
• San Francisco

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..

4
Colleagues

• My lab in the early 1980s at Chiron Corporation
  
• Qui-Lim Choo,
• Amy Weiner
• Kangsheng Wang
• Maureen Powers
• Josy Yu
• Lacy Overby ( consultant mentor )
• George Kuos 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 Gerins 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 1986,10218544-18549
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 1986,10218544-18549
12
Nucleotide sequence of HDV RNA encoding delta
antigen
Source Wang et al, Nature 1986,10218544-18549
13
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
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 its 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
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

19
Ultracentrifuge NANBH-Infectious Chimpanzee
Plasma
NANBH Patients
Serum Antibodies
Pellet
Extract total RNADNA
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
3
2
a
a
b
b
c
1
2
3
b
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
9
71
19
17
ALT
11
54
9
10
18
106
10
22
C
0
76
118
154
DAY
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 HA
V
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
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 )
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,
244362-364
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
HCV Zn-dep. proteinase
Host signalase
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
Membranous web
RNA-dep. replicase
Zn-dep. proteinase
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
31
26 (84)
5 (16)
Vaccinees
P lt 0.001
24
24 (100)
15 (62)
Controls
Note Controls data pooled from Chiron NIH
31
(J.Bukh et al NIH)
32
(No Transcript)
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

Group
No. of Carriers
Controls
8/14 (57)
Vaccine
3/19 (16)
P0.02
34
Non-A, Non-B Hepatitis (NANBH) in the early 1980s

• 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 lt80nM 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
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, 822138-2142
38
Ultrastructural localization of the Shimizu
antigen
Immunoperoxidase EM Bar 500 nm
Source Shimizu et al, PNAS USA 1985, 822138-2142
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 )
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 gt6 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
41
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
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