Brucella abortusStrain RB51 as a Vector for Heterologous Protein Expression and Induction of Specific Th1 Type Immune Responses

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Abstract
Brucella abortusstrain RB51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Our ultimate goal is to develop strain RB51 as a preferential vector for the delivery of protective antigens of other intracellular pathogens to which the induction of a strong Th1 type of immune response is needed for effective protection. As a first step in that direction, we studied the expression of a foreign reporter protein, β-galactosidase ofEscherichia coli, and the 65-kDa heat shock protein (HSP65) ofMycobacterium bovisin strain RB51. We cloned the promoter sequences ofBrucella sodCandgroEgenes in pBBR1MCS to generate plasmids pBBSODpro and pBBgroE, respectively. The genes for β-galactosidase (lacZ) and HSP65 were cloned in these plasmids and used to transform strain RB51. An enzyme assay in the recombinant RB51 strains indicated that the level of β-galactosidase expression is higher under thegroEpromoter than under thesodCpromoter. In strain RB51 containing pBBgroE/lacZ, but not pBBSODpro/lacZ, increased levels of β-galactosidase expression were observed after subjecting the bacteria to heat shock or following internalization into macrophage-like J774A.1 cells. Mice vaccinated with either of the β-galactosidase-expressing recombinant RB51 strains developed specific antibodies of predominantly the immunoglobulin G2a (IgG2a) isotype, and in vitro stimulation of their splenocytes with β-galactosidase induced the secretion of gamma interferon (IFN-γ), but not interleukin-4 (IL-4). A Th1 type of immune response to HSP65, as indicated by the presence of specific serum IgG2a, but not IgG1, antibodies, and IFN-γ, but not IL-4, secretion by the specific-antigen-stimulated splenocytes, was also detected in mice vaccinated with strain RB51 containing pBBgroE/hsp65. Studies with mice indicated that expression of β-galactosidase or HSP65 did not alter either the attenuation characteristics of strain RB51 or its vaccine efficacy againstB. abortus2308 challenge.