A DNA vaccine coding for the Brucella outer membrane protein 31 confers protection against B. melitensis and B. ovis infection by eliciting a specific cytotoxic response

Abstract

The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The outer membrane proteins (Omps) of Brucella spp. have been extensively characterized as potential immunogenic and protective antigens. This study was conducted to evaluate the immunogenicity and protective efficacy of the B. melitensis Omp31 gene cloned in the pCI plasmid (pCIOmp31). Immunization of BALB/c mice with pCIOmp31 conferred protection against B. ovis and B. melitensis infection. Mice vaccinated with pCIOmp31 developed a very weak humoral response, and in vitro stimulation of their splenocytes with recombinant Omp31 did not induced the secretion of gamma interferon. Splenocytes from Omp31-vaccinated animals induced a specific cytotoxic-T-lymphocyte activity, which leads to the in vitro lysis of Brucella-infected macrophages. pCIOmp31 immunization elicited mainly CD8(+) T cells, which mediate cytotoxicity via perforins, but also CD4(+) T cells, which mediate lysis via the Fas-FasL pathway. In vivo depletion of T-cell subsets showed that the pCIOmp31-induced protection against Brucella infection is mediated predominantly by CD8(+) T cells, although CD4(+)T cells also contribute. Our results demonstrate that the Omp31 DNA vaccine induces cytotoxic responses that have the potential to contribute to protection against Brucella infection. The protective response could be related to the induction of CD8(+) T cells that eliminate Brucella-infected cells via the perforin pathway.

FIG. 1.

A. Kinetics of the humoral response elicited after immunization with the Omp31 DNA vaccine. Mice were immunized with pCIOmp31, pCI, or H38 and bled retro-orbitally at the indicated days. IgG-specific antibodies against rOmp31 were evaluated by ELISA. Each symbol represents the mean ± SD for eight animals. The arrow indicates the time of B. ovis infection. B. Proliferative responses of spleen cells from mice immunized with pCIOmp31. Cells from immunized mice (2 × 10⁵ cells/well) were stimulated with rOmp31 (1 μg/ml) for 5 days, and incorporation of [³H] thymidine was measured. The S.I. corresponds to the counts per minute of stimulated cells divided by counts per minute of unstimulated cells. Results are expressed as the mean ± SD for five mice. ★, significantly different from PBS-immunized group (P < 0.05). Data are representative of three separate experiments. C. Determination of IFN-γ production in cells from pCI-, pCIOmp31-, PBS-, or H38-immunized mice. Spleen cells (4 × 10⁶/ml) from mice were stimulated with RPMI, rOmp31 (1 μg/ml), or ConA (2.5 μg/ml) for 48 h. IFN-γ in cell supernatants was quantified by antibody capture ELISA. Each value represents the mean ± SD of the responses of spleen cells from five individual mice. Data are representative of four separate experiments. D. Expression of IFN-γ versus cell surface markers in spleen cells from pCI- or pCIOmp31-immunized mice. Spleen cells from pCI- or pCIOmp31-immunized mice were cultured for 5 days with mitomycin C-treated A20JOmp31. On day 5 they were restimulated with mitomycin C-treated A20JOmp31 cells and rOmp31 (10 μg/ml) or, where indicated, with phorbol myristate acetate (20 ng/ml) plus ionomycin (750 ng/ml) and then incubated for 4 h with 10 μg/ml of BFA before analysis by flow cytometry. Numbers within quadrants represent the percentage of positive gated cells.

FIG. 2.

Induction of Omp31-specific CTLs in spleen cells from pCIOmp31-immunized mice. Cytotoxicity was detected in a standard 6-h ⁵¹Cr release assay. Target cells were A20JpCI (dashed lines) or A20JOmp31 (solid lines) cells labeled with ⁵¹Cr. Effector cells were the splenocytes from immunized mice previously cultured for 5 days with mitomycin C-treated A20JOmp31.The cytotoxicity was measured at the indicated effector/target cell ratios. Each value represents the mean ± SD of the responses of spleen cells from five individual mice. Data are representative of three separate experiments.

FIG. 3.

CD4⁺ T and CD8⁺ T cells are involved in the Omp31-specific cytotoxic activity elicited by pCIOmp31 or H38 immunization. 51Cr release CTL assay was undertaken as described for Fig. 2. Effector cells from pCIOmp31- or H38-immunized mice were previously incubated for 2 h with culture supernatants from anti-mouse CD4⁺ or anti-mouse CD8⁺ hybridomas or an IgG isotype control (-). The effector/target cell ratio was 100:1. Each value represents the mean ± SD of the responses of spleen cells from five individual mice. Data are representative of two separate experiments. ★, significantly different from isotype control-treated cells (P < 0.05).

FIG. 4.

pCIOmp31 elicits effector CD8⁺ T cells that mediate cytotoxicity via perforin and CD4⁺-specific T cells that use the Fas-FasL pathway. 51Cr release CTL assay was performed as described for Fig. 2. Target cells were A20JOmp31 cells. The effector/target cell ratio was 100:1. pCIOmp31-specific effector cells were depleted of CD4⁺ T cells (−CD4⁺) or CD8⁺ T cells (−CD8⁺) by using mouse CD4 (L3T4) Dynabeads or mouse CD8 (Lyt2) Dynabeads or were not depleted (total cells). Depleted and nondepleted effector cells were preincubated for 2 h with RPMI (-), brefeldin A (BFA), or concanamycin A (CCA). Each value represents the mean of triplicates ± SD of the response of a pool of spleen cells from five mice. Data are representative of two separate experiments.

FIG. 5.

Vaccination with pCIOmp31 elicit CD8⁺- and CD4⁺-specific T cells that lyse Brucella-infected macrophages in vitro. Cytotoxicity was detected in a standard 6-h ⁵¹Cr release assay. The effector/target cell ratio was 100:1. A. Target cells were A20J, A20JOmp31, J774, or J774 B. ovis. Effector cells were the splenocytes from pCIOmp31- or pCI-immunized mice previously cultured for 5 days with mitomycin C-treated A20JOmp31. Each value represents the mean ± SD of the responses of spleen cells from five individual mice. Data are representative of two separate experiments. ★, significantly different from pCI-immunized mice (P < 0.05). B. Target cells were J774 B. ovis. Effector cells from pCIOmp31-immunized mice were depleted of CD4⁺ T cells (−CD4⁺) or CD8⁺ T cells (−CD8⁺) or were not depleted (total cells) as indicated in Fig. 4. Depleted and nondepleted effector cells were preincubated for 2 h with RPMI (-), BFA, or CCA. Each value represents the mean of triplicates ± SD of the response of a pool of spleen cells from five mice. Data are representative of three separate experiments.