Lucilla Steinaa, Principal Scientist, ILRI
International Symposium on Animal Functional Genomics & Health
19 Nov 2020
Towards a vaccine for African swine fever

• Caused by a large double stranded DNA
virus.
• Between 150-167 genes, 22 genotypes
based on the p72 sequence.
• Belongs to the family Asfaviridae, nearest
relatives are the poxviruses.
• There is a wildlife reservoir : warthogs,
bush pigs. Soft ticks are involved in
transmission.
African swine fever

• ASF is now a huge problem in Eastern
Europe, Russia, China, Asia, and Africa.
• ASF is present in about 26 countries in
Africa. Multiple genotypes are present.
• ASF is a major problem for potential pig
farmers, who are discouraged from
entering the pig business due to ASF.
African swine fever

The architecture of the virus
Wang et al. 2019

Neighbor-Joining tree depicting the p72 gene relationships
and geographical distribution of the major ASFV genotypes
CAM/1/02
CAM/4/85
GHA/1/02
LIS/60
NIG/1/01
ANG/70
IC/2/96
BEN/1/97
SPEC/205
SPEC/209
Mkuzi/79
I
MOZ/1/02
MOZ/1/05
LUS1/93
MOZ/2/02
MOZ/1/03
II
RSA/5/95
SPEC/257
RSA/3/03
RSA/Warmbaths
III
MOZ/1979
MAL/1/02
MOZ/1960
Tengani/62
V
MOZ/8/94
SPEC265
MOZ/1/94
VI
RSA/3/04/
RSA/Warthog
RSA/1/99/W
IV
RSA/2/96
RSA/2/03
RSA/3/96
RSA/1/04
XIX
Lillie
RSA/1/95
XX
XIIIRSA/1/03
RSA/4/95
RSA/1/98
SPEC/154
VII
XIVNYA/12
TAN/2/03
TAN/1/03
XVI
XVTAN/1/01
XIIISUM/14/11
XIIMZI/1/92
XIKAB/6/2
MCH/1/89
MCH/3/89
BAN/1/91
DED/1/89
LIL/20/2
DOWA
CHJ/1/89
KLI/2/88
PHW/1/88
TMB/1/89
VIII
UGA/1/95
KEN/5/01 IX
BUR/1/84
BUR/1/90
Kenya/50
X
100
79
74
57
91
0.005
p72 gene
Contribution: Livio Heath (ARC-OVI)
Distribution of genotypes in Africa
Genotype I
Genotype II,III, IV, V,
VI, VII, XIX, XX, XIII
Genotype VIII, XI, XII, XIII,
XV, XVI, XIV
Genotype IX, X

Engineering of African Swine Fever Virus
for Production of Live Attenuated Vaccine
Candidates

Engineering of African Swine Fever Virus for
Production of Live Attenuated Vaccine Candidates
• Establishment of vaccine platforms for making attenuated
African swine fever vaccine candidates
• Gene modification by CRISPR-Cas.
• Gene modification by using a synthetic approach – reverse genetics system
• International Livestock Research Institute, J. Craig Venter Institute and
Friedrich Loeffler Institute.

• The virus
• Genotype IX isolated from an outbreak area
between Uganda and Kenya. Genomic
sequence available.
• Viruses from current outbreak areas in the
region (Kenya, Uganda, (Tanzania)) have been
sequenced at ILRI. All are genotype IX and X,
and genomes are almost identical to the one
we have. Genotype IX and X have been
detected in outbreaks in Congo.
Virus backbone for modification of ASFV
Gallardo C et al. A.J. Biotech 2011
Onzere C. et al. Virus Genes 2018

CRISPR approaches for gene modification

CRISPR-modification pathway (NHEJ)
Stable CAS
transfected cell line
(WSL)
Transfection with
guide RNA
Infect with
GFPvirus
Harvestsup./cells
with virus
10-1
10-2 10-3
10-4
10-5 10-6
Checkfor genome
modifications
Dilutions
Virusclones
Genome cleavage
detection kit
OR
DNA Sequencing of
modified region

Genome cleavage detection
uncleaved
cleaved
Mismatched
heterogenous
duplex
Denature &
Reanneal
GeneArt™GenomicCleavageDetection Kit
Digestw/ detection
enzyme
Cleaved
heteroduplex
Electrophoresis
T C
T-Treated
C- Control
PCRamplify
targeted locus
Banddensitometryfor
cleavageefficiency
G1 G2 C
1 2 3
500
PCRamplify
targetedlocus
1 2 3
Denature& Reanneal
Digest w/ detectionenzyme
Electrophoresis
CRISP-modification of two genes (SingleguideRNA)
500
300
200
1 2 3 1 2 3
negative enzyme
control

Clones tested with genome cleavage kit
PCR amplify targeted locus (500bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 W 15 16 17 18 19 20 21 22 23 24 25 26 28 29 W27
Spike with wildtype DNA amplicon:
Denature & Reanneal and Digest w/ detection enzyme
1 2 3 4 5 6 7 8 9 10 11 12 13 14 W15 16 17 18 19 20 21 22 23 24 25 26 28 29 W27
Assorted clones: Denature & Reanneal and Digest w/ detection enzyme
2 3 9 10 11 12 16 19 21 25 29 W23
Sequence clones 2, 3, 9, 11, 12, 16, 19, 29… 25 and wt to confirm editing

A clone virus with desired mutation
1 2 3 1 2 3
gRNA target
Stop Codon

17th Oct 2019
Reflections on the NHEJ pathway
• Bias towards in-frame indels using NHEJ, one guide RNA approach.
• Difficulties in cloning due to lack of marker for modification.
Mitigation:
• Use HDR approach
NHEJ pathway

Homology directed repair pathway
ASFV gene
RHALHA
LHA RHA
Plasmid
GFP in ASF
genome
ASF
genome

GFP plasmid pGFPΔA238L for integration of GFP
in the ASF genome
17th Oct 2019
pGFPΔA238L, synthesised by GenScript
circular
linear
Infection /transfection experiments in WSL– Cas cell line
Kenya 1033 (WT) + plasmid
Kenya 1033 (WT) + amplicon
Kenya 1033 (∆CD2v) + plasmid
Kenya 1033 (∆CD2v) + amplicon

Microscopy after transfection of pGFPΔA238L
in Kenya1033-ΔCD2v-DsRed
Super-imposed images
DsRed eGFP
Two

PCR confirmation of GFP genome integration
17th Oct 2019
Amplicon sizes:
F1R1 (5′) = 1488 bp
F1R2 = 3740 bp (KO),
F1R2 = 3337 bp (wt),
F2R2 (3′) = 1681 bp
Expected band sizes for both amplicon and plasmid
transfections in Kenya1033 & Kenya1033-ΔCD2v
Kenya1033
amplicon
Kenya1033
plasmid
1033-ΔCD2v
plasmid
1033-ΔCD2v
amplicon
F1R1
F2R2
F1R2
F1R1
F2R2
F1R2
F1R1
F2R2
F1R2
F1R1
F2R2
F1R2
5Kb
3Kb
1.5Kb
PCR to check integration of the plasmid/amplicon into the virus genome
F1 R1 F2 R2

Viruses produced by homology directed
repair (CRISPR)
Backbones
WT-ASF1033 ASF1033 ΔCD2v
1 gene deletion 1 gene deletion
3 gene deletion 3 gene deletion
6 gene deletion 6 gene deletion
Ready for animal experiments

Towards a subunit vaccine for African
swine fever

Toward a subunit vaccine
• Identification of antigens for use in a subunit vaccine
• Screening of peptide library in ELISpot
• Production of recombinant viral vectors
• Adenovirus/MVA prime-boost
• Testing of pools of viral vectors in pigs for protection
Adenovirus Vaccinia

Immunisation using attenuated ASF virus
ASF1033 ΔCD2v
• 10 European breed animals
• 10 local breed animals

Clinical scoring/temperature after immunization
using attenuated ASF virus ASF1033ΔCD2v

CD2v knockout
• The virus is attenuated.
• 80-90% of pigs survives 103 HAD.
• Clearly, another mutation is needed.
• We are soon testing if pigs immunized
with ASF1033ΔCD2v are protected
against wild-type.

Cellular immune responses after immunization
using attenuated ASFΔCD2v
0
200
400
600
800
1000
1200
1400
PB188 PB189 PB190 PB191 PB192 PB195 PB196 PB197
Media ASFV-1033 P72V CD2v Adeno
SFU/106PBMC
IFN-gamma ELIspot

Antibody responses in pigs after
immunization with attenuated ASFΔCD2v
0
20
40
60
80
100
120
0 5 10 15 20 25 30
PB188 PB190 PB191 PB192 PB195 PB196 PB197
Days post infection
%inhibition
Cut off

Antibody responses in pigs after
immunization with attenuated ASFΔCD2v
D0D28 D0D28 D0D28
0
50
100
%Blocking
Local breed
European breed II
European breed I

Recognition by PBMC of overlapping peptides
spanning the entire proteome
immunization with attenuated ASFΔCD2v
• 217 gene pools in all
• 42 pools recognized in all 3
screenings.
• 62 pools not recognized
Genepool

Peptide pools recognized in ELISpot using full
proteome library
immunization with attenuated ASFΔCD2v
CD8 cells
PBMC

Single antigen recognition by number of animals
CD8 cells
Best recognition
of single Ag:
6/22 animals
PBMC
Best recognition
of single Ag:
17/22 animals

Production of viral vectors with ASF genes
• Ten genes from the peptide
screen were selected
• Each of the 10 genes
produced in both
adenovirus (HuAd5) and
Modified Vaccinia virus
Ankara (MVA).
• Next, to test pools in the
animal model

Virus batch for challenge experiments
0 2 4 6 8 10
0
50
100
Survival - Groups
Day after challenge
Percentsurvival
102
103
104
105
0 2 4 6 8
38
40
42
Body temperature - Groups
Day after challenge
Bodytemperature
102
103
104
105
0 2 4 6 8 10
0
5
10
15
Clinical score - Groups
Day after challenge
Clinicalscore
102
103
104
105
Establishment of minimal lethal challenging dose
HAD-titer: 102, 103, 104, 105
5 animals per group
Scoring system: King et al.

33
ILRI
Hussein Abkallo
Hanneke Hemmink
Nicholas Svitek
Jeremiah Khayumba
Anna Lacasta
Elias Awino
Rosemary Saya
Bernard Odour
Emanuel Khazalwa
Lucilla Steinaa
Collaborators
Sanjay Vashee, Assoc prof.
J.Craig Venter Instisitute
Walter Fuchs, Group leader
Friedrich Loeffler Institute
Acknowledgements

This presentation is licensed for use under the Creative Commons Attribution 4.0 International Licence.
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