Evaluation of Brucella abortus phosphoglucomutase (pgm) mutant as a new live rough-phenotype vaccine

Abstract

Brucella abortus S19 is the vaccine most frequently used against bovine brucellosis. Although it induces good protection levels, it cannot be administered to pregnant cattle, revaccination is not advised due to interference in the discrimination between infected and vaccinated animals during immune-screening procedures, and the vaccine is virulent for humans. Due to these reasons, there is a continuous search for new bovine vaccine candidates that may confer protection levels comparable to those conferred by S19 but without its disadvantages. A previous study characterized the phenotype associated with the phosphoglucomutase (pgm) gene disruption in Brucella abortus S2308, as well as the possible role for the smooth lipopolysaccharide (LPS) in virulence and intracellular multiplication in HeLa cells (J. E. Ugalde, C. Czibener, M. F. Feldman, and R. A. Ugalde, Infect. Immun. 68:5716-5723, 2000). In this report, we analyze the protection, proliferative response, and cytokine production induced in BALB/c mice by a deltapgm deletion strain. We show that this strain synthesizes O antigen with a size of approximately 45 kDa but is rough. This is due to the fact that the deltapgm strain is unable to assemble the O side chain in the complete LPS. Vaccination with the deltapgm strain induced protection levels comparable to those induced by S19 and generated a proliferative splenocyte response and a cytokine profile typical of a Th1 response. On the other hand, we were unable to detect a specific anti-O-antigen antibody response by using the fluorescence polarization assay. In view of these results, the possibility that the deltapgm mutant could be used as a vaccination strain is discussed.

FIG. 1.

Western blot analysis of extracted LPS (A) and crude extracts (B) with nonoclonal anti-O-antigen antibodies. Lanes 1, wild type; lanes 2, Δpgm. Molecular mass markers in kilodaltons are shown on the left of panel A. SDS-PAGE and Western blot were carried out as described in Materials and Methods.

FIG. 2.

Virulence in mice. Mice were infected i.p. with 10⁷ CFU of the wild-type or Δpgm strain, and numbers of CFU recovered from spleens at different times postinfection were determined as described in Materials and Methods. Bars represent the means ± standard deviations (SD). *, P < 0.001.

FIG. 3.

Elicitation of anti-O-antigen antibodies. Groups of six mice were inoculated with 5 × 10⁵ CFU, and sera were collected at different times postinfection. Titers of specific anti-O-antigen antibodies were determined by FPA as described in Materials and Methods. Horizontal bars represent the average values for the group. *, P < 0.01; **, P < 0.001 compared with control S2308-inoculated mice.

FIG. 4.

Lymphocyte proliferation assay. Mice were inoculated with 10⁷ CFU of the Δpgm mutant or with PBS. Eight weeks after inoculation, spleen lymphocytes were recovered and stimulated with heat-inactivated B. abortus S2308, RPMI 1640, or ConA, and proliferation assays were performed as described in Materials and Methods. Bars represent the means ± SD of results for quadruplicate sets of cells. *, P < 0.001.