Linda K. Dixon1

1 Institute for Animal Health, Pirbright Laboratory, UK

Current Approaches for African Swine Fever Virus Vaccine Development

New CL4 Laboratory Complex being built at IAH Pirbright

IAH Resources for ASFV Research•High containment (BSL4) laboratory, large animal facilities and insectary•OIE Reference Lab for ASFV•Large collection of ASFV strains and reagents•Interdisciplinary research programmes•3 lines of inbred pigs, colonies of Ornithodoros ticks

African swine fever virus• Large double-stranded DNA virus, genome length 170-190 kbp• Only member of virus family the Asfarviridae• Replicates in the cytoplasm – similar strategy to Poxviruses• Virus particle contains RNA polymerase and other enzymes

needed to start replication cycle – virus DNA is not infectious• Encodes about 151-167 genes including enzymes required for

replication and transcription of the virus genome• Many genes ( ~1/3) are not essential for virus replication in

cells but play an important role in virus survival and transmission

• Replicates mainly in macrophages in vivo• No vaccine

Institute for Animal Health

Nucleo-cytoplasmic large DNA virus superfamily

ASFV structure

•ASFV virions have a complexmultilayer structure•More than 50 proteins are present•Extracellular and intracellularmature virions are both infectious

a) schematic showing layers in extracellular virionsb) extracellular virions buddingc) and d) intracellular virus factories showing immature (IM) and mature (M) virionsc) chemical fixation, d) high pressure freezing

200 nmPippa Hawes IAH

Virus Particle

P72cD2Vp22

p54Proteins on surface of extracellular and intracellular virus particletargets for antibody mediated protection

B438L

Benin 97/1 complete genome MGF360MGF110MGF505/530

MGF100P22evasion

ReplicationStructuralunknown

182 kbp

Virus Genome160-175 genes Many not essential for replication

Genes involved in immune evasion/virulence

Inhibitors of host signalling pathways that block transcription of host immunomodulatory genes

A238L, broad inhibition of host gene transcription.- Inhibitors of IFN -Inhibitor of Toll-like receptors TLR 3 and 4 Adhesion proteins

CD2v, causes binding of infected cells and virus particles to red blood cells, impairs lymphocyte proliferationC-type lectin -resembles NK cell inhibitory

receptors Apoptosis inhibitors – IAP and Bcl2 homologues

Comparison of sequences of non-pathogenic and pathogenic strains

Sharon Brookes, Alex Hyatt

RBC

RBCV

VV

ASFV infected macrophage

Red Blood Cells bound to ASFVinfected macrophages

Extracellular virus particlesbound to Red Blood Cells

“Hides” virus particles and infected cells

Courtesy Sharon Brookes

Pathogenesis• Highly virulent isolates ~100% death of pigs within 5 to 12

days. – High viraemia (> 10 8 ) Apoptosis of lymphocytes Damage to endothelial cells lining blood vesicles, disseminated intravascular coagulation, haemorrhage

• Moderately virulent isolates cause death of 30 to 50 % of pigs. - Disease similar to highly virulent isolates but survivors tend to have lower viraemia (10 4-6). Virus persists in recovered pigs

• Low virulence isolates. Very few deaths. - Occasional low viraemia 10 2-3 and fever. Virus in tissues. Persistent infection in pigs.

• Pigs which recover from infection are protected against challenge with lethal dose of related virulent viruses

• Low virulence isolates provide good model for understanding protection

Left end

Benin 97/1 MGF 360 3HL, IL, LLMGF 360 3CL, DL, EL

MGF 530 3FR, NR

MGF110

MGF110

Non-pathogenic OurT88/3 has deletions and insertions compared to highly pathogenic Benin97/1 isolate

Summary: Genome comparisons Benin 97/1 (highly pathogenic) compared to OUR T88/3 (non-pathogenic)

• Gene deletions at left end of OURT88/3 genome include members of MGF360 (6 copies) and MGF530 (2 copies) implicated in virulence, cell tropism and IFN induction

• CD2v and C-type lectin genes interrupted in OURT88/3. CD2v implicated in impairing lymphocyte activation

• MGF 300 (1 copy) and MGF 110 (2 copies) in Benin not OUR T88/3

• MGF 110 (4 copies) and 4 other ORFs in OUR T88/3 not Benin.• Conserved ORFs encode proteins with 98 to 100% identity.• Two ORFs encode proteins with variable numbers of tandem

repeats.

ASFV Multigene families

• 5 Multigene Families (MGFs)– A set of genes derived by duplication of an ancestral gene

followed by independent mutational events resulting in a series of independent genes

• Constitute ~17% - 25% of the coding capacity

• Lack similarity to other known genes, functions unknown

• Vary in gene number between ASFV isolates:– MGF 100: 2-3 genes per genome– MGF 110: 5-13 genes per genome– MGF 300: 3-4 genes per genome– MGF 360: 11-19 genes per genome– MGF 530: 8-10 genes per genome

Deletion of MGF360 and MGF530 reduces virus growth in macrophages and virulence in pigs

macrophagereplication

virulence in pigs

IFNinduction

tickreplication

+

+++

+102-103

+ +

+

+

+

102-103

102-103

NT

NT

NT

NT

NT

NT

NT

-

Zsak et al., 2001, Neilan et al., 2002, Afonso et al, Burrage et al.,2004

Note these MGF 360 and 530 genesare also deleted from non-pathogenic isolate (Chapman et al., 2008)

Prospects for vaccine development

• Survivors of ASF can resist challenge by related virulent viruses (eg De Tray 1957, Malmquist 1963, Handy and Dardiri 1983) - therefore prospects for ASFV vaccine development are good

Obstacles to ASFV vaccine development• Inactivated ASF virions do not induce protection• Serially passaged ASFV vaccine strain used in Portugal

and Spain in 1960s caused post-vaccination reactions in 128,684 of 550,000 vaccinated -Loss in confidence and need for extensive tests of vaccine emphasised

• Complexity of virus (~160-175 genes encoded. Virus particles contain > 50 proteins in several concentric layers)

• Neutralising antibodies are not effective• Genetic complexity. Many virus genotypes (22) have

been defined by sequence of the gene encoding the major capsid protein.

However -• Highest ASFV diversity is in natural hosts (warthogs

and O. moubata ticks) in E and S Africa. Spread of genotypes to domestic pigs is limited and in some endemic areas a single genotype is circulating

• In addition cross-protection can be induced between genotypes (King et al., 2011)

• ASFV is a large DNA virus with more accurate replication than RNA viruses. This results in a relatively stable genome.

Pigs can be protected:

• Survivors of ASF can resist challenge by related virulent viruses (eg De Tray 1957, Malmquist 1963, handy and Dardiri 1983)

Pigs are protected when:• Inoculated with viruses attenuated by passage in tissue

culture, eg E75CV (Ruiz Gonzalvo et al., 1986, Gomez-Puertas et al., 1998)

• Inoculated with natural low virulence isolates, eg NHP68, OurT88/3 ( Leitao et al., 2001, Boinas et al., 2004, Denyer et al., 2006)

- Low sporadic or no viraemia detected, protection close to 100%.

• Inoculated with recombinant virus with single genes deleted (Lewis et al., 2000, Neilan et al., 2004).

- Viraemia 10 3-6 over ~20 days. High percentage protection

.

Understanding mechanisms of protection

• Identification of correlates of protection for vaccine development

• Identification of protective immune mechanisms directs strategies for vaccine development

Mechanisms of protection induced by attenuated viruses: A role for CD8+ T cells• CD8+ T cells are necessary. Depletion of CD8+ T cells

abrogates protection induced by OURT88/3 (Oura et al., 2004)

• Protection correlates with frequency of ASF specific IFN-gamma producing memory T cells

• Ability of different virus isolates to stimulate lymphocytes from OURT88/3 immune pigs correlates with cross-protection (King, et al., Vaccine 2011)

• Key virus antigens involved in inducing immunity mediated by T cells not defined.

Mechanisms of protection induced by attenuated viruses: The role of antibodies• Pigs can be protected by passive transfer of antibodies from

immune pigs (Onisk et al., 1994). Higher viraemia observed than in pigs protected by attenuated virus

• Mechanism by which antibodies protect:- pre virus entry (neutralisation), targets identified p54 (E183L), p30 (CP204L), p72 (B646L)

- post virus entry (infection inhibition), mechanism and targets not known

• Inhibition of infection in vitro by immune serum correlates with cross-protection observed in vivo against different isolates

OURT88/3 i.m. 10 4

non-virulent genotype I

OURT88/1 i.m. 10 4

virulent genotype I

or OURT88/3Benin 97/1 i.m. 10 4

virulent genotype I

Blood sampling –serum and whole blood

Introduction of non-immune pigs

Termination of experiment

0 d567 14 21 28 36 41 49

Temperature and clinical scores

Experimental vaccination with attenuated ASFV strain OURT88/3

ASFV viraemia and clinical score in vaccinated compared to control pigs

0

5.0 x 106

1.0 x 107

1.5 x 107

2.0 x 107

2.5 x 107

Days post OURT88/3 inoculation

Copy

num

ber p

er m

l

VR89VR90VR92VR97VR98VR99VS00

OURT88/3 OURT88/1 Benin 97/1

ASFV detected only from non-immune pigs

02468

101214161820

1 0 1 2 3 4 5 6 7 8 9 10

Clin

ical

scor

e VR89VR90VR92VR97VR98VR99VS00

Non-immune pigs

Benin 97/1

Days post Benin 97/1challenge

VR89

0

200

400

600

800

1000

0 1 2 3 4 5 6 80

20000

40000

60000

80000

W-0 W-1 W-2 W-3 W-4 W-5 W-6 W-8

0

20000

40000

60000

80000

100000

120000

W-0 W-1 W-2 W-3 W-4 W-5 W-6 W-8

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20000

40000

60000

80000

100000

W-0 W-1 W-2 W-3 W-4 W-5 W-6 W-8

VR90

0

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400

600

800

1000

0 1 2 3 4 5 6 8

VR92

0

200

400

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800

1000

0 1 2 3 4 5 6 8

8

6

4

2

0

3H-TdR

uptake (∆ cpmX

104)

12

10

8

6

4

2

0

10

8

6

4

2

00 1 2 3 4 5 6 8

0 1 2 3 4 5 6 8

0 1 2 3 4 5 6 8

Week post first vaccination

VR89

VR90

VR92

IFN-γ ELISPOT Proliferation assay

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A

B

C

D

E

F

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ASFV

spe

cific

IFN

-γpr

oduc

tion

frequ

ency

per

10

6 PB

MC

Frequency of IFNγ producing cells increases after 1st immunisation and is boosted after 2nd

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

0 10 20 30 40 50 60

1803

1826

1834

1845

0.00

2.00

4.00

6.00

8.00

10.00

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14.00

0 10 20 30 40 50 60

1809

1811

1829

1837

1844

1822

Anti-

ASFV

ant

ibod

y tit

reAn

ti-AS

FV a

ntib

ody

titre

Days post 1st immunisation

A

B

Anti- ASFV p72 antibody responses Exp 2

Anti-p72 response rises to day 20 and is boosted by 2nd immunisation. Infection inhibition assays showed low inhibition of infection in vitro (up to 10 2 )

• OURT88/3, OURT88/1 immune pigs protected against virulent African isolates ASFV Benin 97 and Uganda challenge. No cross-protection to Malawi, only partial protection to Lisbon 57

0

20

40

60

80

100

120

140

VR89 VR90 VR92

% c

ross

-reac

tivity

Cross reactivity to OURT88/3

OURT88/3

Benin -5

Lisbon

malawi

malta

uganda

OURT 1-6

% C

ross

-rea

ctiv

ity

Pig number

Recognition of diverse strains of ASFV by lymphocytes from OURT immune pigs correlates with protection

Good correlation between IFN-γ cross-reactivity and cross-protection

OURT88/3 Type I

Benin 97 Type 1

Lisbon 57 Type 1

Malawi Type VIII

Uganda Type X

Malta 78 Type I

OURT88/1 Type I

Cross-reactivity of OURT88/3 immune pigs PBMC to other ASFV isolates : IFN-g ELISPOT Assay

Comparison of complete genomes of Georgia 2007/1 isolate with other ASFV isolates

Kenya 69Malawi 88

Georgia 2007/1Mkuzi 79

OURT88/3BA71VBenin 97/1E70

Tengani 62Warmbaths

Pr 96/4 Warthog

W. AfricaEurope

E. and S.Africa

Comparison of the concatenated sequences of 125 conserved genes (~40,000 amino acids)shows the Georgia 2007 isolate is in the same clade as those from Europe and W. Africa

but more distantly related -Chapman et al., Emerging Infectious Diseases 2011

0.004

0 5 100

20

40

60

80

100Immune - Benin

Benin

Days post challenge

Percen

t surviv

al

0 5 100

20

40

60

80

100

Benin

Uganda

Immune - Uganda

Immune - Benin

Days post challenge

Perce

nt su

rvival

0 5 10 15 200

20

40

60

80

100

Benin

OURT88/3 - OURT88/1 - BeninOURT88/3 x 2 - Benin

Days post challenge

Percen

t surviv

al

Exp 1

Exp 2

Exp 3

Challenge of immunepigs with different ASFVisolates: % survivalExp 1 IAH, UK Exp 2 ANSES, France –SPFExp 3 ANSES, FranceExps 1 and 3 100% immunised pigs survived challenge with genotype 1Benin 97/1Exp 2 60%immunised pigs survived challenge with genotype 1Benin 97/1 and 100% genotype X Uganda Some adverse effects of immunisation in experiments in France

Survival of pigs challenged with ASFV isolates form genotype I and X

Challenges for attenuated vaccines

• Safety concerns about release of replicating virus vaccine

• High containment required for production• Optimised cell culture required for growth of vaccine

strain• Current strains may not be sufficiently attenuated• Additional genes involved in virulence deleted from

attenuated strains

Benin 97/1 complete genome MGF360MGF110MGF505/530

MGF100P22evasion

ReplicationStructuralunknown

182 kbp

Virus Genome160-175 genes Many not essential for replication

Effect of ASFV gene deletionsGene Function Effect on

virulenceEffect on replication in culture

Conserved in isolates

dUTPase,ThymidineKinase

Nucleotide metabolism

Reduced Reduced replication in macrophages

Yes

9GL Virionmorphogenesis

Reduced Reduced Yes

MGF 360/530

UnknownIFN induction?

Reduced Reduced No

CD2V Binding red blood cells, lymphocytefunction

Delayeddissemination no reduction in mortality

No effect No

DP71L PP1 regulator Can reduce (short form)

No effect Present as long or short form

Effect of ASFV gene deletions

Gene Function Effect on virulence

Effect on replication

Conserved in isolates

A238L Inhibitor of host transcription

None None Yes

C-Type lectin Inhibition of MHC class I presentation

None None No

IAP Apoptosis inhibition

None None Yes

UK Unknown Reduced None Yes

Subunit vaccines

• Partial protection achieved with recombinant proteins expressed in baculovirus:

- a mixture of proteins p30 and p54 (Gomez-Puertas et al., 1996) – NB Neilan et al., 2004 reported no protection

- CD2-like protein (or haemmaglutinin) (Ruiz-Gonzalvo et al., 1999)

• Delay in onset of disease signs and viraemia, some pigs recover from infection and clear virus

Challenges for subunit vaccines

• Identification of additional protective antigens especially dominant antigens recognised by CD8+T cells

• Identification of vaccine delivery systems for pigs to induce cell-mediated and antibody responses eg host restricted virus vector such as swinepox or avipox

Rapid vaccine development platform• Collaboration Kathy Sykes, Bert Jacobs, Biodesign

Institute, Arizona State University, IAH Pirbright UK• Genome wide screen of ORFs encoded by Georgia

2007 ASFV isolate to rank proteins for induction of cell mediated and antibody responses in pigs

• Genes delivered in pools of 20- 40 to pigs by DNA/prime recombinant vaccinia virus boost

• Antibody and cell mediated immune responses to individual antigens measured using individual in vitro translated proteins

• Test smaller pools “best” antigens for ability to protect pigs against lethal ASFV challenge

Strategy for ASFV Library Construction

Genome wide screen for protective ASFV antigensCollaboration IAH- Biodesign Institute, Arizona State University

Immunize pigs with expression libraries in pools by DNA prime recombinant vacciniavirus boost

Assay sera, PBMC, RNA for immune responses

Sort and Rank all ORFs

Ab Iso-type

Th1 Th2 Cyto-kine

ORF10 ORF5 ORF69 ORF50 ORF100

ORF113 ORF811 ORF98 ORF63 ORF39

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

Test in Pig Challenge-Protection Assays

Select antigens to test

TermORF HisT7 ATGRBS TRX

Magnetic beads for capture and purification proteins

In vitro synthesis of proteins

Proteome-scale protein production and purification

Linear DNAs for in vitro transcription/translation

1. Pool top antigens from each bin and immunize pigs with these pools of antigens by DNA prime and recombinant vaccinia virus boost.

2. Pool top 5-10 antigens from positive bins, and immunize pigs.

3. Re-test and validate vaccine candidates

Immunize

Challenge ?Survival readout

Challenge/protection experiments

Immune responses in pigs immunised with pools of antigens: Antigen pool complexity does not reduce T cell response level

0

100

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300

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600

3H-T

dR u

ptak

e, c

pm

2 antigens

050

100150200250300350400450

ASFV

002

ASFV

004

ASFV

006

ASFV

011

ASFV

012

ASFV

037

ASFV

052

ASFV

053

ASFV

054

ASFV

068

ASFV

070

ASFV

07…

ASFV

07…

ASFV

083t

ASFV

105

ASFV

111

ASFV

122

ASFV

128

ASFV

167

ASFV

179

ASFV

113

ASFV

127

PHA

med

ium3H

-TdR

upt

ake,

cpm

Pool of 22

0

100

200

300

400

500

600

700

ASFV

113

ASFV

163

ASFV

127

ASFV

105

ASFV

145

ASFV

154

ASFV

083

ASFV

006

ASFV

194

ASFV

132

ASFV

205

ASFV

170

PHA

med

ium

3H-T

dR u

ptak

e, c

pm

Pool of 12

Proliferation Assays: Stimulation of lymphocytes from immunised pigs with individual antigens

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

393 396 398 404 405 406

Group 1 (pool of 22) vs. VP30127 pre127 post

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

397 407 409 411 412 424

Group 2 (pool of 22) vs. VP30127 pre127 post

0

0.2

0.4

0.6

0.8

1

1.2

1.4

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2

394 395 410 419 420

Group 3 (pool of 12) vs. VP30127 pre127 post

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

403 417 418 421 423

Group 4 (pool of 2) vs. VP30

127 pre127 post

Antigen pool complexity does not reduceantibody response

Pig #

Pig #

Pig #

Pig #

ELISA assays

Summary of Progress: genome wide antigen screen

• DNA vaccine and protein expression libraries complete

• rVV library 47 complete• Immunome screening in pigs – conditions

optimised and 47 antigens tested by DNA prime rVV boost

• T cell and antibody assays used to rank ORFs for immune responses

• Challenge experiments in progress

Future Priorities Vaccines

• Attenuated vaccines: Rational strategy for attenuation

• Better knowledge of cross-protection between genotypes- antigens involved in cross-potection

• Optimised cell culture• Subunit vaccines: Identification of protective

antigens especially those which induce CD8+ T cell responses

• Incorporation and testing in host-restricted gene delivery systems

Future work vaccines

• Subunit vaccines – complete screen for protective antigens

• Test in pools in challenge experiments• Select best antigens and clone in host-

restricted vaccine delivery vector

AcknowledgementsIAH UK

• Linda Dixon• Dave Chapman• Lynnette Goatley• Fuquan Zhang• Charles Abrams• Emma Fishbourne• Pam Lithgow• Derah Arav

• Geraldine Taylor• Haru Takamatsu• Katherine King• Chris Netherton• Josie Golding• Pippa Hawes

• Don King• Chris Oura• Carrie Batten• Geoff Hutchings

Univ. Victoria, Canada

ANSES Ploufragan, France• Marie-Frederique le Potier• Evelyne Hutot• Roland Carriolet

Biodesign Institute Arizona State University

Center for Infectious Diseases • Bert Jacobs• James Jankovich• Greg GoldenCenter for Innovations in Medicine• Kathy Sykes• Mark Robida

• Chris Upton www.virology.ca

Genotypes of ASFV isolates

Penrith et al., 2007Data from partial sequence of geneencoding p72 capsid protein

Antibody response following DNA prime rVV boost compared to infection

0

0.1

0.2

0.3

0.4

0.5

0.6

cont 01 cont 04 60 76 105 184

Uninfected and ASFV-infected pigs vs. VP72

0

0.5

1

1.5

2

2.5

cont 01 cont 04 60 76 105 184

Uninfected and ASFV-infected pigs vs. VP30

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0.2

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0.8

1

1.2

1:100 1:500 1:2500

Pre/Post Immunization vs. VP72

pre VP72

post VP72

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1

1.2

1:100 1:500 1:2500

Pre/Post Immunization vs. VP30

pre VP30

post VP30