Overexpression of protective antigen as a novel approach to enhance vaccine efficacy of Brucella abortus strain RB51

Abstract

Brucella abortus strain RB51 is an attenuated rough strain that is currently being used as the official live vaccine for bovine brucellosis in the United States and several other countries. We reasoned that overexpression of a protective antigen(s) of B. abortus in strain RB51 should enhance its vaccine efficacy. To test this hypothesis, we overexpressed Cu/Zn superoxide dismutase (SOD) protein of B. abortus in strain RB51. This was accomplished by transforming strain RB51 with a broad-host-range plasmid, pBBR1MCS, containing the sodC gene along with its promoter. Strain RB51 overexpressing SOD (RB51SOD) was tested in BALB/c mice for its ability to protect against challenge infection with virulent strain 2308. Mice vaccinated with RB51SOD, but not RB51, developed antibodies and cell-mediated immune responses to Cu/Zn SOD. Strain RB51SOD vaccinated mice developed significantly (P < 0.05) more resistance to challenge than those vaccinated with strain RB51 alone. The presence of the plasmid alone in strain RB51 did not alter its vaccine efficacy. Also, overexpression of SOD did not alter the attenuation characteristic of strain RB51.

FIG. 1

Demonstration of overexpression of Cu/Zn SOD in strain RB51SOD by SDS-PAGE (A) and Western blot analysis (B). (A) Lane 1, molecular weight marker; lanes 2 to 4, contain antigens of strains RB51, RB51pBB, and RB51SOD, respectively. The gel was stained with Coomassie brilliant blue G. (B) Lanes 1 and 2, the antigens of strains RB51pBB and RB51SOD, respectively. The sera indicated below each blot were used as the primary antibodies for reacting with the antigens. Asterisks indicate the Cu/Zn SOD protein; numbers at the left indicate approximate molecular masses in kilodaltons.

FIG. 2

Resistance to B. abortus 2308 challenge infection in mice vaccinated with strains RB51SOD, RB51pBB, and RB51. Two weeks after challenge infection, the number of strain 2308 CFU in the spleen was determined. The mean value in each group is indicated by a solid horizontal line. The horizontal broken line above the x axis indicates the lower detection limit (<20 CFU/spleen). The P value indicated in the RB51SOD group is in comparison with the RB51 group. In all three experiments, strain RB51-vaccinated mice contained significantly lower numbers of CFUs in comparison with the saline-inoculated group (P ≤ 0.001). In experiment 1, there was no significant difference in the number of CFU between RB51- and RB51pBB-vaccinated groups (P > 0.05).