Presented by Waithaka Mwangi at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011

1. ASFV Diagnostics, Surveillance, Epidemiology and Control:
Identification of Researchable Issues Targeted to the Endemic Areas
within sub-Saharan Africa
Hosted by BecA-ILRI and sponsored by CSIRO-AusAID
Waithaka Mwangi
Dept. of Veterinary Pathobiology
College of Veterinary Medicine and Biomedical Sciences
Texas A&M University

2. • ASFV is a highly contagious pathogen that causes devastating
hemorrhagic fever in pigs with ~100% case mortality rates.
• It causes major economic losses, threatens food security, and
limits pig production in affected countries.
Goal:
Develop a vaccine capable of induction of ASFV-specific protective
immunity.

4. p32: immunogenic, implicated in virus
internalization, antibody target
p54: Transmembrane, involved in virus
particle maturation, antibody target
p72: Main component of the viral
capsid, antibody and
CTL target
Pp220 and pp62 polyproteins:
• Produce structural proteins that
account for ~32% of the total protein
virion mass and are the major
components of the core shell
• Indispensable for viral replication and
production of viable virus

5. Three synthetic codon-optimized chimeric genes
generated: Designated saf1, saf2, and saf3.

6. • Replication-incompetent adenovirus (Ad5):
- Systemic and mucosal immunization
• Bacillus subtilis: mucosal immunization

7. Evaluate immunogenicity and protective efficacy of the
lead vaccine candidates following intradermal or mucosal
immunization with recombinant adenovirus (rAd) or Bacillus (rBa),
respectively, expressing saf1, saf2, and saf3.

8. Immunization of pigs with adenovirus- or Bacillus-vectored ASFV
chimeric antigens will confer systemic and/or mucosal immunity
against ASFV
Specific Aims:
• Test whether intradermal or mucosal immunization of pigs with
rAdSAF1-3 will confer protection against ASFV challenge.
• Test whether mucosal immunization of pigs with rBaSAF1-3 will
confer protection against mucosal ASFV challenge.

9. Positive clone 1 2 3 4 5 6 7 8 9 10 11
Test clone
Negative control
Mwangi, W., et al., 2011

10. Generation of rAdenovirus
A) B) C)
Immunocytometric analysis of 293A cells infected with;
A and B) rAdFMD virus; and C) control adenovirus.
A and C) were probed with anti-FLAG AP-conjugated mAb, B was probed
with an isotype-matched AP-conjugated mAb.
Mwangi, W., et al 2011

11. A) B)
Immunization of calves with a single dose of the rAdFMD vaccine primed significant;
A) FMD1-specific IFN-γ-secreting T cell responses; and B) FMD1-specific T cell
proliferation, detectable in seven days.
Mwangi, W., et al 2011

12. 450
400 603
605
350
# spot/10E5 cells
300
250
200
150
100
50
0
PHA O1Campos FMDV1 10 ug FMDV1 30 ug PBS
Filgueira, M.P., et al., 2011

13. IgA values: calculated as the S/P ratio = (sample – negative control)/(positive – negative control).
Hargis, B.M., et al 2011

14. Hydropathic profile of the SAF1 chimeric polypeptide

15. A) B) C) D)
Immunocytometric analysis of 293A cells transfected with:
A) SAFI; B) SAFII; C) SAFIII expression constructs; and D) vector control
The cells were probed with anti-FLAG AP-conjugated mAb.
Mwangi, W., et al 2011

16. • Recombinant SAFI-III proteins
• Recombinant Adenovirus-SAFI-III
• Recombinant B. subtilis-SAFI-III
Quality control analysis

17. Conduct dose-escalation immunization studies in pigs
• Evaluate SAFI-III-specific immune responses
• Evaluate recall responses upon boost
- test sera for recognition of native ASFV antigens
- test T cells for reactivity against ASF virus
• identify dose required to induce optimal immune responses

18. Conduct immunization studies in pigs;
• Prime Evaluate SAFI-III-specific immune responses
• Boost
• Challenge
• Protective index: Survival

19. DC-targeted Control
Mwangi, W., et al., 2011

20. A 1 wk post-immunization 3 wks post-immunization
250 ∗ ∗p<0.001 500
∗ ∗p<0.001
IFN- γ + SFC/106 CD8-γ δ -PBMC
IFN- γ + SFC/106 CD8-γ δ -PBMC
200 400
150 300
100 200
50 100
0 0
pCC98MSP1 pICMSP1 Vector pCC98MSP1 pICMSP1 Vector
Mwangi, W., et al., 2011

21. A 19 wks post-immunization 1 wk post-Boost
∗ ∗p<0.001 ∗ ∗p<0.001
Mwangi, W., et al., 2011

23. Mwangi Lab: Jocelyn Bray, Shehnaz Lokhandwala,
Ann-Marrie
Surya Waghela Texas A&M University
Luc Berghman, Texas A&M University
Mariano Pérez- Filgueira, Instituto de Virología, CICVyA, INTA-
Castelar, Argentina
Billy M. Hargis University of Arkansas
Richard Bishop ILRI in collaboration with DVS, Kenya
and CINA-INIA, Valdeolmos, Spain