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Infection and Immunity, June 2000, p. 3290-3296, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Ramesh Vemulapalli,* Yongqun He, Stephen M. Boyle, Nammalwar Sriranganathan, and Gerhardt G. Schurig

Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0342

Received 9 December 1999/Returned for modification 28 February 2000/Accepted 22 March 2000

Brucella abortus strain 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, beta -galactosidase of Escherichia coli, and the 65-kDa heat shock protein (HSP65) of Mycobacterium bovis in strain RB51. We cloned the promoter sequences of Brucella sodC and groE genes in pBBR1MCS to generate plasmids pBBSODpro 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 pBBSODpro/lacZ, increased levels of beta -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 beta -galactosidase-expressing recombinant RB51 strains developed specific antibodies of predominantly the immunoglobulin G2a (IgG2a) isotype, and in vitro stimulation 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 vaccinated with strain RB51 containing pBBgroE/hsp65. Studies with mice indicated 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.

* Corresponding author. Mailing address: Center for Molecular Medicine and Infectious Diseases, 1410 Prices Fork Rd., Blacksburg, VA 24061. Phone: (540) 231-7757. Fax: (540) 231-3426. E-mail: rvemulap{at}vt.edu.

Infection and Immunity, June 2000, p. 3290-3296, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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