Evaluation of protective effect of DNA vaccines encoding the BAB1_0263 and BAB1_0278 open reading frames of Brucella abortus in BALB/c mice
Highlights
► DNA vaccines pVF263 and pVF278, stimulate a Th1-cell-mediated immune responses. ► DNA vaccines pVF263 and pVF278, stimulate antibody production. ► DNA plasmid pVF278 induces a better protection in mice than pVF263. ► This study provides the first evidence with protective effect for BAB1_0278 antigen.
Introduction
Bacteria of the genus Brucella are facultative intracellular pathogens of human and animals, causing brucellosis. Brucellosis is a zoonotic disease, causing infertility, arthritis, endocarditis and meningitis in humans. In many animal species Brucella can cause abortion and infertility [1]. Brucellosis remains endemic in many developing countries where it undermines animal health and productivity, causing important economic loses [2]. Since Brucella is localized intracellularly, it has been suggested that cell-mediated immunity (CMI) of a Th1 immune response is crucial in the control of Brucella sp. infection [3]. The administration of live attenuated Brucella abortus S19 and RB51 strains is the most common strategy used to control brucellosis in cattle. These vaccine preparations, however, can be unsafe in humans [4]. This justifies the development of a new vaccine that is non-infectious to humans and effective in stimulating a broad protective immunity.
A powerful strategy for developing safe and efficacious vaccines is immunization with plasmid DNA encoding protective antigen. Because of the ability of DNA vaccines to induce a strong CMI response, they can be effective against intracellular bacteria [5]. DNA vaccines appear to offer the best approach to activate the CMI and humoral components of the immune response. DNA vaccines against Brucella contain genes including the ribosomal L7/L12 [6], bacterioferritin [7], lumazine synthase [8], Omp 31 [9], Cu–Zn superoxide dismutase [10] and a combination of L7L12/Omp16 [6]. Until now, 14 antigens have been verified experimentally to induce protective immunity against Brucella [11].
Bacteria possess the ability to gain large fragments of DNA by horizontal transfer from other bacteria permitting survival in new environments. Genomic islands are found within these DNA fragments [12]. Genomic island 3 (GI-3), present in Brucella melitensis and B. abortus, contains 29 genes, most with an unknown function [13]. It is therefore important to identify potential genes that may have a role in immune responses. We have previously demonstrated that the deletion of BAB1_0263 or BAB1_0278, two open reading frames (ORFs) of the GI-3 present in B. abortus, did not affect the process of cell invasion or the normal rate of growth in culture medium [14]. On the other hand, in contrast to the deletion in BAB1_0263, the deletion of BAB1_0278 affected the efficiency of bacterial growth within nonprofessional and professional phagocytes and its virulence in BALB/c mice [15].
In this study we report the cloning of two genes, BAB1_0263 and BAB1_0278, encoding a 15 kDa and 13 kDa protein respectively. The following factors may influence the immunogenicity of these proteins. First, BAB1_0263 and BAB1_0278 are located in the B. abortus GI-3 [16], and the role or contribution of the GI-3 in Brucella are currently unknown. Second, amino acid sequence analysis has demonstrated that the BAB1_0263 protein shares 42% sequence homology with the small protein Lsr2 in the Saccharopolyspora erythraea strain NRRL 23338. Recent studies suggest that Lsr2 is a regulatory protein involved in multiple cellular processes, including cell wall biosynthesis and resistance to antibiotics, and is also a DNA-binding protein [17], [18], [19]. BAB1_0278 shares 50% sequence homology with the GcrA protein in the alpha-proteobacterium Octadecabacter antarcticus. GcrA is a key regulator of the prokaryotic cell cycle and is present in many bacterial species [20]. Third, previous work in our laboratory found that mutants that have deletions of these proteins showed decreased virulence [15]. Therefore, we analyzed the immune response (humoral and cellular) against BAB1_0263 and BAB1_0278 of B. abortus using recombinant vectors and determined the level of protection against B. abortus virulent strain 2308 in a murine model.
Section snippets
Animals
Seven-to-eight-week-old female BALB/c mice (obtained from Instituto de Salud Pública, Santiago, Chile) were acclimated and randomly distributed into experimental groups. The mice were kept in conventional animal facilities and received water and food ad libitum. Mice were handled and disposed of according to the guidelines of Institutional Ethical Committee.
Bacterial strains and growth conditions
The virulent B. abortus strain 2308 and the attenuated strain RB51 were obtained from our culture collection. The bacterial cells were
Humoral immune response elicited by pVF263 and pVF278 immunization
To evaluate the humoral immune response, sera collected before of each immunization were analyzed to quantify rF263 or rF278 specific antibodies by indirect ELISA, using, as antigen, recombinant proteins rF263 or rF278, and CBP. As shown in Fig. 1A, 15 days after immunization the sera from mice immunized with pVF263 achieved a significantly higher level of anti-rF263 IgG [OD450 = 0.213 ± 0.04]. The levels of anti-rF263 IgG 30 and 45 days after decreased, but were still significantly higher than the
Discussion
In countries with a high incidence of brucellosis, vaccination is one of the main methods of controlling the spread of this disease. In order to overcome the significant disadvantages of the currently used live vaccines, the development of new generation vaccine systems to prevent brucellosis is essential. Among various immunization strategies, DNA vaccines offer many advantages associated with live vaccines. Notably, these include the processing and presentation of DNA-expressed antigens along
Acknowledgments
This work was supported by grant ADI 08/2006 from “Programa Bicentenario de Ciencia y Tecnología”, CONICYT, and grant 1100032 from “Fondo Nacional de Investigación Científica y Tecnológica” (FONDECYT), Chile.
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