Immunization of mice with recombinant protein CobB or AsnC confers protection against Brucella abortus infection

Abstract

Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ) assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB) or BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy.

Figure 1. SDS-PAGE of the recombinant proteins.

The expression of recombinant fusion proteins were analyzed by SDS-PAGE. M, molecular size protein markers; lane 1–12, the purified fusion proteins: BAB1_0063, BAB1_0116, BAB1_0381, BAB1_0512, BAB1_0553, BAB1_0560, BAB1_0597, BAB1_0722, BAB1_0812, BAB1_0917, BAB1_1108, BAB1_1124.

Figure 2. IFN-γ secretion by splenocytes stimulated with the recombinant B. abortus proteins.

Spleen cells from mice immunized with S19 or PBS were stimulated in vitro with recombinant proteins, ConA (positive control), or complete medium (negative control). Supernatants were collected and IFN-γ was determined by using an ELISA Quantikine Mouse kit (R&D Systems). All assays were performed in triplicate and the concentrations for IFN-γ in the culture supernatants were calculated by using the quantification formula. Significant differences between S19-immunized mice and PBS-immunized mice are indicated as follows: *, P<0.001.

Figure 3. Humoral immune response induced by CobB, AsnC, and Cu-Zn SOD.

BALB/c mice (n = 5) were inoculated intraperitoneally with purified recombinant proteins plus adjuvant. At 0, 15, 30, and 45 days post the immunization, serum samples were collected and IgG antibody titers were determined by ELISA. The values are mean titers ± SD of antibodies from the five mice.