application of novel delivery systems for asfv antigens
DESCRIPTION
Presented by Waithaka Mwangi at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011TRANSCRIPT
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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
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• 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.
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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
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Three synthetic codon-optimized chimeric genes
generated: Designated saf1, saf2, and saf3.
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• Replication-incompetent adenovirus (Ad5): - Systemic and mucosal immunization
• Bacillus subtilis: mucosal immunization
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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.
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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.
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1 2 3 4 5 6 7 8 9 10 11Positive clone
Negative control
Test clone
Mwangi, W., et al., 2011
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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
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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
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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
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IgA values: calculated as the S/P ratio = (sample – negative control)/(positive – negative control).
Hargis, B.M., et al 2011
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Hydropathic profile of the SAF1 chimeric polypeptide
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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.
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• Recombinant SAFI-III proteins
• Recombinant Adenovirus-SAFI-III
• Recombinant B. subtilis-SAFI-III
Quality control analysis
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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
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Conduct immunization studies in pigs;
• Prime Evaluate SAFI-III-specific immune responses
• Boost
• Challenge
• Protective index: Survival
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Mwangi, W., et al., 2011
DC-targeted Control
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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
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A 19 wks post-immunization 1 wk post-Boost
∗ ∗∗p<0.001 ∗p<0.001
Mwangi, W., et al., 2011
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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