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