Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4+ and CD8+ T cells - PubMed (original) (raw)

Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4+ and CD8+ T cells

Carola Muñoz-Montesino et al. Infect Immun. 2004 Apr.

Abstract

In the development of vaccines capable of providing immunity against brucellosis, Cu-Zn superoxide dismutase (SOD) has been demonstrated to be one of the protective immunogens of Brucella abortus. In an earlier study, we provided strong evidence that intramuscular injection with a plasmid DNA carrying the SOD gene (pcDNA-SOD) was able to induce a protective immune response. The present study was designed to characterize T-cell immune responses after an intraspleen (i.s.) vaccination of BALB/c mice with pcDNA-SOD. Animals vaccinated with pcDNA-SOD did not develop SOD-specific antibodies, at least until week 4 after immunization (the end of the experiment), and in vitro stimulation of their splenocytes with either recombinant Cu-Zn SOD or crude Brucella protein induced the secretion of gamma interferon (IFN-gamma), but not interleukin-4, and elicited the induction of cytotoxic-T-lymphocyte activity. Upon analyzing the SOD-specific T-cell responses, the pcDNA-SOD vaccination was found to be stimulating both CD4(+)- and CD8(+)-T-cell populations. However, only the CD4(+) population was able to produce IFN-gamma and only the CD8(+) population was able to induce cytotoxic activity. Nevertheless, although i.s. route vaccination induces a significant level of protection in BALB/c mice against challenge with the virulent B. abortus strain 2308, vaccination by the intramuscular route with a similar amount of plasmid DNA does not protect. Based on these results, we conclude that i.s. immunization with pcDNA-SOD vaccine efficiently induced a Th1 type of immune response and a protective response that could be related to IFN-gamma production and cytotoxic activity against infected cells by SOD-specific CD4(+) and CD8(+) T cells, respectively.

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Figures

FIG. 1.

Lymphocyte proliferation assay. BALB/c mice were immunized with pcDNA-SOD and the parental plasmid pcDNA3. The T-cell proliferative response was measured at week 4 after immunization. Splenocytes from each group (4 × 105 per well) were prepared and stimulated in vitro with CBPs (4 μg/ml) and purified rSOD (0.4 μg/ml) as antigens. Each value is the average number of counts per minute of triplicate cultures of cells (± standard deviation) obtained from a pool of five mice in each group. *, P < 0.005 compared with the value for pcDNA3-immunized control mice.

FIG. 2.

Quantitative ELISA analysis of IFN-γ secreted by lymphocytes upon stimulation with different antigens. Spleen cells (4 × 106/ml) from mice inoculated with pcDNA3 or pcDNA-SOD were stimulated with CBPs (4 μg/ml), rSOD (0.4 μg/ml), or RPMI 1640 (negative control) for 48 h. Each bar represents the geometric mean ± standard deviation (error bars) of the responses in spleen cells from five individual mice. *, statistically significant differences compared to RPMI 1640 (P ≤ 0.07).

FIG. 3.

Specific cytotoxic activity of total T cells. Four weeks after immunization, splenocytes from mice immunized with pcDNA-SOD or the parental plasmid pcDNA3 were stimulated for 5 days with J774.A1 macrophages (Mφ) infected with B. abortus RB51-SOD at a ratio of 1:100 (cells to RB51-SOD). These effector cells were incubated with J774.A1 cells alone or J744.A1 cells infected with RB51-SOD. A neutral red uptake assay was used to measure target lysis. The data are means for triplicate estimations, and standard deviations did not exceed 20% of the means. E, effector; T, target.

FIG. 4.

Lymphocyte proliferation assay for T-cell populations. BALB/c mice were immunized with pcDNA-SOD (solid bars) or the parental plasmid pcDNA3 (open bars). At week 4 after immunization, splenocytes from each mouse were separated by flow cytometry into CD4+ (A) and CD8+ (B) T cells and stimulated in vitro with CBPs (4 μg/ml) and purified rSOD (0.4 μg/ml) as antigens. Each value is the average number of counts per minute of triplicate cultures of cells plus standard deviation (error bars) obtained from a pool of five mice in each group. *, P ≤ 0.05 compared with the value for c-RPMI medium.

FIG. 5.

IFN-γ release in CD4+- and CD8+-T-cell cultures exposed to target cells. Four weeks after immunization, splenocytes from immunized mice were collected and cocultivated with stimulator cells for 5 days as described in Material and Methods. T cells were recovered and sorted into CD4+ and CD8+ T cells. The separated cells were cultivated with J744.A1 cells infected with RB51-SOD (target cells), and IFN-γ release was measured in the supernatants after 48 h. The solid bars represent pcDNA-SOD-immunized mice, and the open bars represent pcDNA3-immunized mice. *, statistically significant difference compared to CD4+ T cells obtained from mice immunized with pcDNA3 (P < 0.005). The error bars indicate standard deviations.

FIG. 6.

Specific cytotoxic activity of CD4+ or CD8+ T cells. Four weeks after immunization, splenocytes from mice immunized with pcDNA-SOD or the parental plasmid pcDNA3 were stimulated for 5 days with J774.A1 macrophages infected with B. abortus RB51-SOD. These effector cells were recovered and sorted into CD4+ and CD8+ T cells. The separated T-cell subsets were incubated with target cells (J774.A1 cells infected with RB51-SOD), and cytotoxic activity was measured 16 h later by neutral red assay. The data are means for triplicate estimations, and standard deviations did not exceed 20% of the means.

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Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4+ and CD8+ T cells - PubMed (original) (raw)