Brucellosis 2003 proceedings - PHIDIAS

Poster Session
plasmid-based expression system, we have previously demonstrated that strain
RB51 can serve as a vector for the delivery of heterologous proteins to induce a
preferential, specific Th1 type of immune response. Because safety concerns may
preclude the field use of strain RB51-based recombinant live vaccines, we are
exploring strategies to inactivate recombinant RB51 strains without interfering with
the induction of Th1 type immune responses. In this study, we compared the ability of
heat-killed and gamma-irradiated recombinant RB51 strains to induce heterologous
antigen-specific immune responses in BALB/c mice. Our studies revealed that
exposure of strain RB51LacZ (a recombinant RB51 expressing β-galactosidase of E.
coli) to a minimum of 300 krad of gamma-radiation rendered the bacteria nonviable.
These bacteria, however, remained metabolically active as shown by their active
electron transport chain. A single intraperitoneal inoculation of mice with 10 9 CFUequivalent
gamma-irradiated RB51LacZ, but not heat-killed RB51LacZ, induced a β-
galactosidase-specific Th1 type immune response. On day 3 post-inoculation, mice
inoculated with gamma-irradiated, but not heat-killed, RB51LacZ had enlarged
spleens with detectable levels of nonviable strain RB51LacZ. Preliminary
experiments with dendritic cells indicated that both gamma-irradiated and heat-killed
RB51LacZ were equally efficient in activating dendritic cell maturation. However,
dendritic cells exposed to gamma-irradiated bacteria secreted more IL-12. These
results suggest that recombinant RB51 strains exposed to a low dose of gammaradiation
become nonviable, but remain metabolically active, and retain their ability to
stimulate a strong Th1 type immune response specific to the expressed heterologous
antigens.
91- DEVELOPMENT OF Brucella melitensis AS A VACCINE AGAINST
BIOTERRORISM AGENTS.
A. B. Bandara 1 , V. Dobrean 1 , S H. Poff 1 , D. L. Hoover 2 , M. P. Nikolich 2 , N. Sriranganathan 1 , G. G.
Schurig 1 , and S. M. Boyle 1 . (1) Virginia Polytechnic Institute & State University, Blacksburg, Virginia,
USA. (2) Walter Reed Army Institute of Research, Silver Spring, Maryland. USA.
The goal of this research is to develop a live, attenuated, single vaccine
against anthrax and brucellosis in humans. The 2.3-kb pag gene encoding the
Bacillus anthracis protective antigen (PA) was fused to Brucella GroE promoter in
broad host range plasmid pBBR4MCS to produce pBB4PA. A purEK deletion
Brucella melitensis strain WR201 was electroporated with plasmid pBB4PA to
produce strain WR201PA. The 2.1-kb Brucella wboA gene encoding a
mannosyltransferase involved in O-side chain synthesis, was cloned into pBB4PA to
produce pBB4PA/WboA. A purEK, wboA mutant B. melitensis strain WRRP1 was
electroporated with pBB4PA/WboA to produce strain WRSPA. Immunoblot analyses
using rabbit anti-PA polyclonal serum was performed to determine the expression of
PA by recombinant strains. Escherichia coli carrying pBB4PA and B. melitensis
strains WR201PA and WRSPA each produced an approximately 63-kDa protein
equivalent to the full length PA and a series of proteins between 4 to 30-kDa.
Immunoblot analyses using monoclonal rat antiserum to Brucella O-side chain
revealed that O-side chain synthesis was complemented in strain WRSPA. However,
crystal violet staining indicated that strain WRSPA exhibited a rough phenotype. One
week after inoculation, strain WRRP1 completely cleared from BALB/c mouse
spleens, whereas strain WRSPA cleared by 2.3 to 3.5 log 10 compared to B.
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