Waithaka MwangiDept. of Veterinary Pathobiology
College of Veterinary Medicine and Biomedical SciencesTexas A&M University
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
• ASFV is a highly contagious pathogen that causes devastatinghemorrhagic fever in pigs with ~100% case mortality rates.
• It causes major economic losses, threatens food security, andlimits pig production in affected countries.
Goal:Develop a vaccine capable of induction of ASFV-specific protectiveimmunity.
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 proteinvirion mass and are the majorcomponents of the core shell
• Indispensable for viral replication andproduction of viable virus
Three synthetic codon-optimized chimeric genes
generated: Designated saf1, saf2, and saf3.
• Replication-incompetent adenovirus (Ad5): - Systemic and mucosal immunization
• Bacillus subtilis: mucosal immunization
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.
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.
1 2 3 4 5 6 7 8 9 10 11Positive clone
Negative control
Test clone
Mwangi, W., et al., 2011
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 probedwith an isotype-matched AP-conjugated mAb.
Mwangi, W., et al 2011
Generation of rAdenovirus
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
0
50
100
150
200
250
300
350
400
450
PHA O1Campos FMDV1 10 ug FMDV1 30 ug PBS
# sp
ot/1
0E5
cells
603605
Filgueira, M.P., et al., 2011
IgA values: calculated as the S/P ratio = (sample – negative control)/(positive – negative control).
Hargis, B.M., et al 2011
Hydropathic profile of the SAF1 chimeric polypeptide
Mwangi, W., et al 2011
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.
• Recombinant SAFI-III proteins
• Recombinant Adenovirus-SAFI-III
• Recombinant B. subtilis-SAFI-III
Quality control analysis
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
Conduct immunization studies in pigs;
• Prime Evaluate SAFI-III-specific immune responses
• Boost
• Challenge
• Protective index: Survival
Mwangi, W., et al., 2011
DC-targeted Control
A 1 wk post-immunization 3 wks post-immunization
pCC98MSP1 pICMSP1 Vector0
100
200
300
400
500
IFN
-γ+
SFC
/106
CD
8- γδ
- PB
MC ∗p<0.001∗
pCC98MSP1 pICMSP1 Vector0
50
100
150
200
250
IFN
-γ+ S
FC/1
06 C
D8- γ
δ- P
BM
C ∗p<0.001∗
Mwangi, W., et al., 2011
A 19 wks post-immunization 1 wk post-Boost
∗ ∗∗p<0.001 ∗p<0.001
Mwangi, W., et al., 2011
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