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