Presentation by Lucilla Steinaa at a Global African Swine Fever Research Alliance (GARA)/International Alliance for Biological Standardization (IABS) webinar on current efforts in African swine fever vaccines, 6 May 2021

1. Better lives through livestock
Recent advances in African swine fever vaccine development at
the International Livestock Research Institute
Lucilla Steinaa
Principal Scientist
Animal and Human Health Program, ILRI
Global African Swine Fever Research Alliance (GARA)/International Alliance for Biological Standardization (IABS) webinar:
Current efforts in African swine fever vaccines
6 May 2021

2. 2
African swine fever in Africa
• All 24 genotypes are present in Africa.
• ASFV present in about 26 African
countries.
• There is a wildlife reservoir: warthogs and
bush pigs.
• Soft ticks of the genus Ornithodoros are
involved in transmission of ASFV.

3. 3
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

4. 4
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 backbone for deletion of genes to
generate attenuated viruses.
• Used as the challenging virus in the animal
model
Gallardo C et al. A.J. Biotech 2011
Onzere C. et al. Virus Genes 2018

5. TOWARDS A LIVE ATTENUATED VACCINE FOR AFRICAN SWINE FEVER

6. 6
CRISPR-Cas9 modification of African swine fever
Non Homologous End-
Joining Pathway
One gRNA approach in stable
Cas9 transfected WSL cell line
Indels detected by Genome
cleavage detection Kit, verified
by sequencing
Generated 1 mutant
Many in-frame-indels
Homology Directed
Recombination Pathway
gRNA + linearized donor DNA
(fluorochrome) in stable Cas9
transfected WSL cell line
Modification detected by
fluorescent foci.
Generated 7-10 mutants

7. 7
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

8. 8
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
• 102 TCID50 was chosen to test if the virus grown in
WSL cells was still lethal (20+ passages).

9. 9
First viruses to be tested
ASFV-1033_∆CD2v
ASFV-1033_∆CD2v∆A238L
CD2v
Immunomodulatory molecule promoting
apoptosis of lymphocytes.
A238L
Mimic NFκB subunit, inhibits NFκB activity, which
is crucial in the pro-inflammatory response.

10. 10
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)

11. 11
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

12. 12
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

13. 13
Survival plot
0 7 14 21
0
50
100
Survival proportions
DPC
Probability
of
Survival ASF1033_CD2v
ASF1033_CD2vA238L
PBS

14. 14
Remaining data
• Viremia, blood, nasal
• Autopsy, macroscopic pathology
• Immunological data, ELISpot and antibodies

15. 15
• Both viruses are attenuated but not sufficiently. All pigs
survived the immunization with 104 TCID50.
• ∆CD2v seemed to be 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.
• Possibility to do 2 injections with the double knock-out.
• Next step – to test other mutations
Conclusion

16. TOWARDS A SUBUNIT VACCINE FOR AFRICAN SWINE FEVER

17. 17
• 10 European breed animals
• 10 local breed animals
Immunization using ASF1033 ΔCD2v

18. 18
Clinical scoring/temperature after immunization
using ASF 1033ΔCD2v

19. 19
• 217 gene pools in all
• 42 pools recognized in
all 3 screenings.
• 62 pools not recognized
Gene
pool
Recognition of overlapping peptides spanning the entire
proteome by PBMC

20. 20
CD8 cells
PBMC
Peptide pools recognized in ELISpot using
full proteome library

21. 21
PBMC
Best recognition
of single Ag:
17/22 animals
CD8 cells
Best
recognition of
single Ag:
6/22 animals
Single antigen recognition by number of animals

22. 22
• 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
Production of viral vectors with ASF genes

23. 23
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
∆CD2 virus / WT-virus
Friedrich Loeffler Institute
Gunther Keil
Raquel Portugal
Sandra Blome
ILRI
Richard Bishop, now WSU
Edward Okoth

24. THANK YOU