Brucellosis 2003 proceedings - PHIDIAS

Short Oral Communications
Vaccines
VO10- DEVELOPMENT OF Brucella abortus STRAIN RB51 AS AN EXPRESSION
VECTOR FOR HETEROLOGOUS EUKARYOTIC AND VIRAL PROTEINS AND AS
A CARRIER FOR AIDS VACCINE.
Yakir Ophir 1 , Gerhardt Schurig 2 , Ramesh Vemulapalli 3 , George N. Pavlakis 4 , Barbara Felber 4 , A.T.M.
Shamsul Hoque 1 , Weila Wang 1 , Hana Golding 1 and Basil Golding 1 . (1) CBER, FDA, Bethesda, MD,
USA. (2) VA-MD Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA. (3)
Purdue University, West Lafayette, IN, USA. (4) NCI, NIH, Fredrick, MD, USA.
HIV-1 infection is associated with a loss in T-helper cell responses prior to
onset of AIDS. Therefore, therapeutic vaccines should be T-helper cell independent.
Earlier, we demonstrated that heat-killed Brucella abortus conjugated to a V3-loop
peptide from HIV-1 elicits neutralizing antibodies and CTL even in mice depleted of
CD4+ T-cells. Currently we are attempting to express heterologous eukaryotic and
viral genes in B. abortus RB51. It was previously reported that RB51 can express
heterologous bacterial proteins and mice vaccinated with these recombinants
developed Th1-like immunity against the expressed proteins. HIV-derived proteins
(gag and pol) and ovalbumin (OVA) were selected for expression in strain RB51.
These genes were cloned into three B. abortus expression vectors: pBBSODpro and
pBBgroE under the sodC and groE promoter sequences for constitutive expression;
and pNOF100 under the tightly regulated inducible tac promoter. The expression of
the cloned proteins was analyzed in whole cell extracts of B. abortus RB51 by
Western blotting. It was possible to obtain low level expression of pol in pBBSODpro
and improved expression in pNOF100 when grown in LB medium supplemented with
glycerol, but not in TSB medium. OVA was only expressed in pNOF100 when grown
in LB medium supplemented with glycerol. Optimal expression of eukaryotic genes in
Brucella will most likely require selective codon optimization of the foreign genes, and
possibly further modifications of expression vectors.
VO11- PROTECTION AGAINST Neospora caninum IN A GERBIL MODEL USING
Brucella abortus STRAIN RB51 EXPRESSING N. caninum PROTEINS.
S. Ramamoorthy, G. Schurig, D. Lindsay, R. Vemulapalli, S.M. Boyle, N. Srirangananthan. Virginia-
Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, USA.
N. caninum, the protozoan parasite, is of emerging importance as a cause of
abortions in cattle. The attenuated B. abortus strain RB51 has been used as an
effective vaccine for cattle brucellosis. Strain RB51 has also been used as a vector
for heterologous protein expression. It was hypothesized that putative virulence
factors of N. caninum could be expressed in strain RB51 to develop a combined
vaccine for neosporosis and brucellosis. The GRA7, SRS2 and MIC3 genes of N.
caninum were cloned separately into plasmid pBBR1MCS downstream of the
Brucella groE promoter and used to transform B. abortus strain RB51. The
recombinant RB51vaccine strains were inoculated individually and in combination
into groups of three gerbils. Each gerbil received intraperitoneally a primary dose of
6x10 8 CFU/ml followed by a booster dose of 2x10 6 CFU/ml administered four weeks
later. Five weeks post immunization, the gerbils were challenged with 2x10 6 N.
caninum NC-1 tachyzoites. Four out of five gerbils survived in the groups
administered GRA7 and SRS2 alone. Two out of three gerbils survived in the MIC3
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Brucellosis 2003 International Research Conference