Protection of BALB/c mice against Brucella abortus 544 challenge by vaccination with bacterioferritin or P39 recombinant proteins with CpG oligodeoxynucleotides as adjuvant

Abstract

The P39 and the bacterioferrin (BFR) antigens of Brucella melitensis 16M were previously identified as T dominant antigens able to induce both delayed-type hypersensivity in sensitized guinea pigs and in vitro gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells from infected cattle. Here, we analyzed the potential for these antigens to function as a subunitary vaccine against Brucella abortus infection in BALB/c mice, and we characterized the humoral and cellular immune responses induced. Mice were injected with each of the recombinant proteins alone or adjuvanted with either CpG oligodeoxynucleotides (CpG ODN) or non-CpG ODN. Mice immunized with the recombinant antigens with CpG ODN were the only group demonstrating both significant IFN-gamma production and T-cell proliferation in response to either Brucella extract or to the respective antigen. The same conclusion holds true for the antibody response, which was only demonstrated in mice immunized with recombinant antigens mixed with CpG ODN. The antibody titers (both immunoglobulin G1 [IgG1] and IgG2a) induced by P39 immunization were higher than the titers induced by BFR (only IgG2a). Using a B. abortus 544 challenge, the level of protection was analyzed and compared to the protection conferred by one immunization with the vaccine strain B19. Immunization with P39 and CpG ODN gave a level of protection comparable to the one conferred by B19 at 4 weeks postchallenge, and the mice were still significantly protected at 8 weeks postchallenge, although to a lesser extent than the B19-vaccinated group. Intriguingly, no protection was detected after BFR vaccination. All other groups did not demonstrate any protection.

FIG. 1

SDS-PAGE analysis, followed by Coomassie blue staining of whole-cell lysate of E. coli BL21(DE3) transformed by pET-15b-bfr before induction (lane 1) and after 2 h of induction (lane 2) and of the purified recombinant BFR (lane 3). Molecular mass standards (lane MM) are indicated on the right.

FIG. 2

Analysis of IgG1 (A) and IgG2a (B) antibody responses of BALB/c mice to the recombinant BFR and P39 antigens and to B. abortus 544 extract. Mice were immunized three times with BFR or P39 in different ODN combinations. Sera from four mice per group collected 3 weeks after the last immunization were assayed individually by ELISA. Antibody levels are expressed as mean titer.

FIG. 3

Proliferation of splenocytes from BALB/c mice immunized with the recombinant proteins with CpG or non-CpG ODN (A) or with P39, BFR or ODN alone (B). Splenocytes were stimulated in vitro with BFR, P39 (10 μg/ml), or bacterial lysate (30 μg/ml) or were not stimulated (RPMI). For each mouse (n = 4) the assays were set up in triplicate. The data represent the mean ± the standard deviation of the triplicate from four mice. ND, not done.