Immunogenicity and Protective Response Induced by Recombinant Plasmids Based on the BAB1_0267 and BAB1_0270 Open Reading Frames of Brucella abortus 2308 in BALB/c Mice

Abstract

Immunogenicity induced by recombinant plasmids based on the BAB1_0267 and BAB1_0270 open reading frames (ORFs) of Brucella abortus 2308 was evaluated. Bioinformatics analyses indicate that the BAB1_0267 and BAB1_0270 ORFs encode a protein with a SH3 domain and a Zn-dependent metalloproteinase, respectively. Both ORFs have important effects on intracellular survival and replication of B. abortus 2308, mediated via professional and non-professional phagocytic cells. Our results show that immunization with the recombinant plasmid based on the BAB1_0267 ORF significantly increases the production of IgG1, levels of IFN-γ and the lymphoproliferative response of splenocytes. However, BAB1_0267 did not provide significant levels of protection. The plasmid based on the BAB1_0270 significantly increased IgG2a production, levels of IFN-γ and TNF-α, and the lymphoproliferative response of splenocytes. These results demonstrate that immunization with the BAB1_0270 derived recombinant plasmid induce a Th1-type immune response, correlated with a heightened resistance to B. abortus 2308 infection in mice. It is concluded that the Th1-type immune response against bacterial Zn-dependent metalloproteinase induces a protective response in mice, and that pV270 recombinant plasmid is an effective candidate microbicide against brucellosis.

Keywords: DNA vaccines; Src homology 3 domain; brucellosis; interferon gamma; zinc-dependent metalloproteinase.

Figure 1

Electrophoresis of SH3-267 (A) and ZM270 (B) recombinant proteins using 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). Lane 1: molecular weight marker (kDa), lane 2: recombinant proteins.

Figure 2

Serum IgG (A), IgG1 (B), and IgG2a (C) titers measured by ELISA at day 0, 15, 30, and 45 post-immunization (n = 5). Results were expressed as mean ± standard deviation (SD) of the last serum dilution yielding a specific optical density higher than the cutoff value. ^**P < 0.01 and ^***P < 0.001.

Figure 3

Expression of IFN-γ (A), TNF-α (B), and IL-4 (C) by spleen cell cultures from mice stimulated with 0.8 and 4 μg/ml of recombinant SH3-267 and ZM270 proteins. Results were expressed as mean ± SD (n = 5). ^*P < 0.05 and ^***P < 0.001.

Figure 4

Lymphocyte proliferation after in vitro stimulation with (A) 0.8 or 4 μg/ml recombinant proteins and (B) 1.2 or 6 μg/ml B. abortus 2308 total proteins. Results are shown as mean ± SD of ³H-thymidine incorporation from mice splenocytes (n = 5). ^**P < 0.01 and ^***P < 0.001.