Presentation by Lucilla Steinaa at an International Veterinary Vaccinology Network webinar, 30 November 2021.

1. Better lives through livestock
New developments in vaccines against
African swine fever
Lucilla Steinaa
Principal Scientist
Animal and Human Health Program, ILRI
International Veterinary Vaccinology Network Webinar
30 November 2021

2. 2
Spread of African Swine Fever Virus
1957
1960
2018
2007
2018
2018
Dixon et al., Antiviral Research 2019

3. 3
African Swine Fever Status (2016-2020)

4. 4
African Swine Fever in Africa
• Large DNA virus, Asfaviridae family
• Approximately 160 genes, number depending on
isolate.
• 2 genotypes present in China, one in Europe.
• ASFV present in about 26 African countries.
• All 24 genotypes are present in Africa.
• There is a wildlife reservoir: warthogs and bush pigs.
• Wild boars are susceptible.
• Soft ticks of the genus Ornithodores are involved in
transmission of ASFV.
From “Encyclopedia of Virology”
Chap.: African swine fever” by
L.Dixon and D. Chapman. 2008

5. 5
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
XIII
RSA/1/03
RSA/4/95
RSA/1/98
SPEC/154
VII
XIV
NYA/12
TAN/2/03
TAN/1/03
XVI
XV
TAN/1/01
XIII
SUM/14/11
XII
MZI/1/92
XI
KAB/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
Neighbor-Joining tree depicting the p72 gene relationships
and geographical distribution of the major ASFV genotypes
p72 gene
Contribution: Livio Heath (ARC-OVI)
Genotype I
Genotype II,III, IV, V,
VI, VII, XIX, XX, XIII
Genotype IX, X
Genotype VIII, XI, XII, XIII,
XV, XVI, XIV

6. TOWARDS A LIVE ATTENUATED VACCINE FOR AFRICAN SWINE FEVER

7. 7
Vaccine Candidates

8. 8
Vaccine Candidates
• Efficacy
• High level of protection, 100 % in many
cases in various doses
• Under optimal timing, 4 weeks post
immunization
• Duration of immunity ?
• Safety
• Different dose studies for some vaccine
candidates.
• Very different clinical readout system, some use
clinical score systems with many parameters
(King 2011 and Galindo-Cardiel 2013), others
use single parameters, e.g., fever.
• Route of immunization
• Initially: intramuscularly
• Orally route became interesting because of wild boar
• Less viremia using orally route

9. 9
ILRI ASFV Vaccine Activities
• Live vaccine (CRISPR/Cas9 deletions) and synthetic approach
• Deletion of genes for attenuation
• Testing in established animal model
• Subunit vaccine – activities
• Screening of antigens
• Viral vectors as delivery

10. 10
Isolated Virus
• Kenya 1033 (genotype IX) isolated by ILRI and
DVS Kenya.
• Genotype IX and X are especially circulation in
Eastern Africa.
• Isolated from a zone with outbreaks.
• Used as the challenging virus in the animal
model
• Used as backbone for deletion of genes to
generate attenuated viruses.
Gallardo C et al. A.J. Biotech 2011
Onzere C. et al. Virus Genes 2018

11. 11
ASFV Kenya 1033 – Virus Batch for Challenge
• This virus is very similar to the other genotype IX and X viruses.
• Animal model was set up. Different doses were tested.
5 animal per group, intramuscular injection.
0 2 4 6 8 10
0
50
100
Survival - Groups
Day after challenge
Percent
survival
102
103
104
105
0 2 4 6 8
38
40
42
Body temperature - Groups
Day after challenge
Body
temperature
102
103
104
105
0 2 4 6 8 10
0
5
10
15
Clinical score - Groups
Day after challenge
Clinical
score
102
103
104
105
Scoring system: Galindo-Cardiel 2013

12. 12
Genomic Stability and Production Cell Lines
Problems with instability of genomes in cell lines Progress on production cell lines
ZMAC – pig macrophage cell line
MA-104 cell line (Green monkey kidney epithelial
cell line)

13. 13
Scoring system: King et al. 2011
Virus batch for challenge experiments
Virulence of WSL Adapted WT-Virus
0 2 4 6 8 10
0
50
100
Days post infection
Probability
of
Survival
0 1 2 3 4 5 6 7 8
0
5
10
15
20
25
Days post infection
Clinical
score
Challenge with wild type virus.
Open circles: WSL cell line grown
virus , Solid squares: Macrophage
grown virus
• WSL (from FLI) is a fetal wild boar lung cell line, not immortalized.
• ASFV Kenya 1033 was adapted to WSL (20+ passages)
• 102 TCID50 was chosen to test if the virus grown in WSL cells was still lethal

14. 14
Virus batch for challenge experiments
Titers of ASFV Ken-1033 in WSL
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
Macrophage grown ASFV - 4 days WSL grown ASFV - 4 days
MOI 0.1 MOI 0.25 MOI 1 MOI 2.5 MOI 5

15. 15
CRISPR-Cas Editing of African Swine Fever Virus
ASFV gene
RHA
LHA
LHA RHA
GFP in ASF
genome
Infect with
WT-virus
Transfection with guide RNA
and GFP donor DNA
Stable CAS transfected
cell line (WSL)
Check for GFP insert
with PCR over junction
Cloning of cells with
fluorescent virus
➢ Directly
modification on
replicating virus
inside cells
Constructed 7-10 different viruses

16. 16
Synthetic Construction of African Swine Fever Virus
+
Virus Genome
Validated Parts
Modified
Parts
Isolate genome
and rescue virus
+
➢ Capacity to efficiently perform genome-wide changes in the virus genome in
a combinatorial manner to understand virus biology.
➢ Capacity to produce clinically-relevant viruses without extensive passaging
in tissue culture.
➢ Streamlines process to generate various designer vaccine candidates and
oncolytic viruses.

17. 17
First Viruses: Experimental Setup
Day -21 0 31 51
Quarantine Clinical scoring Clinical scoring
Immunisation (1 injection) Challenge
104 ASF1033_∆CD2v (9x) 102 ASF1033 (8x)
104 ASF1033_∆CD2v∆A238L (9x) 102 ASF1033 (8x)
PBS (9x) 102 ASF1033 (8x)
CD2v
Immunomodulatory molecule promoting apoptosis of
lymphocytes.
A238L
Mimic NFκB subunit, inhibits NFκB activity, which is
crucial in the pro-inflammatory response.

18. 18
Clinical Scores After Immunization
0 10 20 30
0
5
10
15
20
ASF1033_CD2v
DPI
Clincal
sore
0 10 20 30
0
5
10
15
20
ASF1033_CD2vA238L
DPI
Clinical
score
0 10 20 30
0
5
10
15
20
PBS
DPI
Clincal
score
0 10 20 30
0
5
10
15
Post-immunisation
DPI
Clinical
score
ASF1033_CD2v
ASF1033_CD2vA238L
PBS
1 2 3 4
-2.5
0.0
2.5
5.0
7.5
Weight gain
Week
Kg
ASF1033_CD2v
ASF1033_CD2vA238L
PBS
ASF1033_∆CD2v ASF1033_∆CD2v∆A238L PBS

19. 19
Clinical Scores After Challenge
0 7 14 21
0
5
10
15
20
ASF1033_CD2v
DPC
Clincal
score
0 7 14 21
0
5
10
15
20
ASF1033_CD2vA238L
DPC
Clinical
score
0 7 14 21
0
5
10
15
20
PBS
DPC
Clinical
score
0 7 14 21
0
5
10
15
20
Clinical scores Challenge
DPC
Clincal
score
ASF1033_CD2v
ASF1033_CD2vA238L
PBS
0 7 14 21
0
5
10
15
20
Challenge
DPC
Clinical
score
ASF1033_CD2v
ASF1033_CD2vA238L
PBS
ASF1033_∆CD2v ASF1033_∆CD2v∆A238L PBS

20. 20
Survival Plot
0 7 14 21
0
50
100
Survival proportions
DPC
Probability
of
Survival
ASF1033_CD2v
ASF1033_CD2vA238L
PBS
medium
WT
medium
WT
medium
WT
0
200
400
600
800
1000
SFU/
million
cells
ASF1033_CD2v
ASF1033_CD2vA238L
PBS
IFNγ-ELISpot D28

21. 21
• ∆CD2v is more efficient than the double knockout but less attenuated.
87.5% protection versus 50%.
• ∆A238L seems to add to the attenuation, but with a loss in ability to
protect.
Conclusion
0
2
4
6
8
10
12
14
16
18
0 5 10 15 20 25
Clinical
score
DPI
ΔA238L-only
Controls

22. 22
Second viruses: Experimental Setup
Day -21 0 31 51
Quarantine Clinical scoring Clinical scoring
Immunisation (1 injection) Challenge
103 ASF1033_∆X (7x) 102 ASF1033 _∆X (6x)
103 ASF1033_∆Y (7x) 102 ASF1033 _∆Y (6x)
PBS (7x) 102 ASF1033 (6x)
28

23. 23
New Gene-Deleted Viruses
G ro u p 1
C
lin
ic
a
l
s
c
o
r
e
(
a
v
e
r
a
g
e
)
0 10 20 30 40 50
0
5
10
15
20
25 Remaining data:
Viremia data
Antibody titers
PM data
G r o u p 2
C
lin
ic
a
l
s
c
o
r
e
(
a
v
)
0 1 0 2 0 3 0 4 0 5 0
0
5
1 0
1 5
2 0
2 5
A ll g ro u p s
D a y s p o s t in fe c tio n
C
lin
ic
a
l
s
c
o
r
e
s
(m
e
a
n
)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
2
0
2
1
2
2
2
3
2
4
2
5
2
6
2
7
2
8
2
9
3
0
3
1
3
2
3
3
3
4
3
5
3
6
3
7
3
8
3
9
4
0
4
1
4
2
4
3
4
4
0
5
1 0
1 5
2 0
2 5
Group1
Group2
Group3
E lis p o t D 2 8 _ 2 x 1 0 ^ 5
S
F
U
/1
0
^
6
P
B
M
C
M
E
D
I
A
A
S
F
V
W
T
M
O
I
_
0
.
1
C
o
n
_
A
-2 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
G ro u p 1
G ro u p 2
G ro u p 3
Challenge
Immunization
Immunization Challenge Immunization Challenge
Days post infection Days post infection
IFNγ-ELISpot D28

24. 24
ILRI
Hussein Abkallo
Hanneke Hemmink
Nicholas Svitek
Jeremiah Khayumba
Anna Lacasta
Elias Awino
Rosemary Saya
Bernard Odour
Emanuel Khazalwa
Lucilla Steinaa
Collaborators
Sanjay Vashee,
J. Craig Venter Institute
Walter Fuchs,
Friedrich Loeffler Institute
Acknowledgements
∆CD2v virus / WT-virus
Friedrich Loeffler Institute
Gunther Keil
Raquel Portugal
Sandra Blome
ILRI
Richard Bishop, now WSU
Edward Okoth

25. THANK YOU