Brucella abortus strain RB51 as a vector for heterologous protein expression and induction of specific Th1 type immune responses

Brucella abortus strain RB51 is a stable, rough, attenuated mutant widely u sed as a live vaccine for bovine brucellosis. Our ultimate goal is to devel op strain RB51 as a preferential vector for the delivery of protective anti gens of other intracellular pathogens to which the induction of a strong Th 1 type of immune response is needed for effective protection, As a first st ep in that direction, we studied the expression of a foreign reporter prote in, beta-gatactosidase of Escherichia coli, and the 65-kDa heat shock prote in (HSP65) of Mycobacterium bovis in strain RB51, We cloned the promoter se quences of Brucella sodC and groE genes in pBBR1MCS to generate plasmids pB BSODpro and pBBgroE, respectively. The genes for beta-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 beta-galactosidase expression is higher under the groE promoter than under the sodC promoter. In strain RB51 containing pBBgroE/lacZ, but not pBBSODp ro/lacZ, increased levels of beta-galactosidase expression were observed af ter subjecting the bacteria to heat shock or following internalization into macrophage-like J774A.1 cells, Mice vaccinated with either of the beta-gal actosidase-expressing recombinant RB51 strains developed specific antibodie s of predominantly the immunoglobulin G2a (IgG2a) isotype, and in vitro sti mulation of their splenocytes with beta-galactosidase induced the secretion of gamma interferon (IFN-gamma), 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-gamma, but not IL-4, secretion by the specific-antigen stimulated splenocytes, was also detected in mice vac cinated with strain RB51 containing pBBgroE/hsp65. Studies with mice indica ted that expression of beta-galactosidase or HSP65 did not alter either the attenuation characteristics of strain RB51 or its vaccine efficacy against B. abortus 2308 challenge.