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Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans, and existing animal vaccines have limitations. To search the putative vaccine candidates, we studied the exoproteome of Brucella abortus NCTC 10093 using 2-DE-MS approach. Twenty-six proteins were identified using MALDI-TOF/TOF tandem mass spectrometry. Outer membrane protein 25, d-galactose periplasmic-binding protein, oligopeptide ABC transporter protein and isopropylmalate synthase were found to be the most abundant proteins. Most proteins (6, 23%) were predicted to be involved in amino acid transport and metabolism followed by carbohydrate transport and metabolism (4, 15%). Outer membrane protein 25, Omp2b porin and one hypothetical protein were predicted as outer membrane proteins. In addition, Omp28, Omp31 and one ribosomal protein (L9) were also identified. The ribosomal protein L9 was produced as a recombinant protein and was studied in mouse model for vaccine potential. It was found to be immunogenic in terms of generating serum antibody response and release of IFN-γ from mice spleen cells. Recombinant L9-immunized mice were protected against challenge with virulent B. abortus strain 544, suggesting usefulness of ribosomal protein L9 as a good vaccine candidate against brucellosis.

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Brucellae are Gram-negative intracellular bacteria that cause an important zoonotic disease called brucellosis. The animal vaccines are available but have disadvantage of causing abortions in a proportion of pregnant animals. The animal vaccines are also pathogenic to humans. Recent trend in vaccine design has shifted to epitope-based vaccines that are safe and specific. In this study, efforts were made to identify MHC-I- and MHC-II-restricted T cell epitopes of Brucella abortus and evaluate their vaccine potential in mice. The peptides were designed using online available immunoinformatics tools, and five MHC-I- and one MHC-II-restricted T cell peptides were selected on the basis of their ability to produce interferon gamma (IFN-γ) in in vivo studies. The selected peptides were co-administered with poly DL-lactide-co-glycolide (PLG) microparticles and evaluated for immunogenicity and protection in BALB/c mice. Mice immunized with peptides either entrapped in PLG microparticles (EPLG-Pep) or adsorbed on PLG particles (APLG-Pep) showed significantly higher splenocyte proliferation and IFN-γ generation to all selected peptides than the mice immunized with corresponding irrelevant peptides formulated PLG microparticles or phosphate-buffered saline (PBS). A significant protection compared to PBS control was also observed in EPLG-Pep and APLG-Pep groups. A plasmid DNA vaccine construct (pVaxPep) for peptides encoding DNA sequences was generated and injected to mice by in vivo electroporation. Significant protection was observed (1.66 protection units) when compared with PBS and empty vector control group animals. Overall, the MHC-I and MHC-II peptides identified in this study are immunogenic and protective in mouse model and support the feasibility of peptide-based vaccine for brucellosis.

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