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. 2010 Jan 13;5(1):e8644.
doi: 10.1371/journal.pone.0008644.

Vaccination of mice with salmonella expressing VapA: mucosal and systemic Th1 responses provide protection against Rhodococcus equi infection

Aline F Oliveira  1 , Luciana P Ruas, Silvia A Cardoso, Sandro G Soares, Maria-Cristina Roque-Barreira
Affiliations

Vaccination of mice with salmonella expressing VapA: mucosal and systemic Th1 responses provide protection against Rhodococcus equi infection

Aline F Oliveira et al. PLoS One. .

Abstract

Conventional vaccines to prevent the pneumonia caused by Rhodococcus equi have not been successful. We have recently demonstrated that immunization with Salmonella enterica Typhimurium expressing the VapA antigen protects mice against R. equi infection. We now report that oral vaccination of mice with this recombinant strain results in high and persistent fecal levels of antigen-specific IgA, and specific proliferation of the spleen cells of immunized mice in response to the in vitro stimulation with R. equi antigen. After in vitro stimulation, spleen cells of immunized mice produce high levels of Th1 cytokines and show a prominent mRNA expression of the Th1 transcription factor T-bet, in detriment of the Th2 transcription factor GATA-3. Following R. equi challenge, a high H2O2, NO, IL-12, and IFN-gamma content is detected in the organs of immunized mice. On the other hand, TNF-alpha and IL-4 levels are markedly lower in the organs of vaccinated mice, compared with the non-vaccinated ones. The IL-10 content and the mRNA transcription level of TGF-beta are also higher in the organs of immunized mice. A greater incidence of CD4+ and CD8+ T cells and B lymphocytes is verified in vaccinated mice. However, there is no difference between vaccinated and non-vaccinated mice in terms of the frequency of CD4+CD25+Foxp3+ T cells. Finally, we show that the vaccination confers a long-term protection against R. equi infection. Altogether, these data indicate that the oral vaccination of mice with S. enterica Typhimurium expressing VapA induces specific and long-lasting humoral and cellular responses against the pathogen, which are appropriately regulated and allow tissue integrity after challenge.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Humoral and cellular immune responses elicited by vaccination with Salmonella expressing the VapA protein.
(A) Fecal IgA antibody response. Groups of BALB/c mice were orally immunized on days 0 and 14 with S. enterica Typhimurium χ3987-pYA3137vapA (closed triangles/dashed line), or inoculated with either S. enterica Typhimurium χ3987-pYA3137 (closed squares) or PBS (open circles). Fecal extracts were obtained on days 7, 14, 21, 28, 35, and 42 after the second immunization, for measurement of specific IgA by ELISA. The R. equi surface antigen (APTX) was used as the coating antigen (1 µg/mL). Results are expressed as the mean of OD values of five mice per group and are a representative experiment of three assays. ***p<0.001 compared to control groups. (B) Lymphocyte proliferative response. Spleen cells were harvested from immunized (pYA3137vapA) and control mice (pYA3137 or PBS) on day 14 after the last immunization and cultured in the presence of medium alone, APTX (5 µg/mL), or Concanavalin A (2 µg/mL) for 72 h. Cell proliferation was measured by [3H]-thymidine incorporation assay. Each bar represents the average of five mice per group ± SD and is representative of four independent experiments. ***p<0.001 compared to control groups. (C–F) Cytokines production. Whole spleen cells were collected from immunized (pYA3137vapA) and control mice (pYA3137 or PBS) on day 14 after the last immunization and cultured in the presence of medium alone, APTX (5 µg/mL), or LPS (1 µg/mL) plus IFN-γ (1 ng/mL) for 48 h. The concentration of IL-12 (C), IFN-γ (D), TNF-α (E), IL-10 (F), and IL-4 (not shown) cytokines in the supernatants of the cell cultures was measured by ELISA. Each bar represents the mean ± SD of triplicate samples and is a representative experiment of three assays. ***p<0.001 compared to the two control groups; +++p<0.001 compared to PBS-inoculated mice.
Figure 2
Figure 2. Transcription levels of T-bet and GATA-3 mRNA in spleen cells of mice vaccinated with Salmonella expressing the VapA protein.
(A–B) Groups of BALB/c mice were orally immunized on days 0 and 14 with S. enterica Typhimurium χ3987-pYA3137vapA, or inoculated with either S. enterica Typhimurium χ3987-pYA3137 or PBS. On day 14 after the second immunization, spleen cells were harvested and the total RNA was extracted and assessed by real-time PCR. (C–D) Immunized (pYA3137vapA) and control (pYA3137 or PBS) mice were intravenously challenged with 4×106 CFU of virulent R. equi strain 14 days after the last immunization. Spleen cells were collected 8 days post-infection, and the total RNA was extracted and assessed by real-time PCR. The cDNA contents were normalized on the basis of predetermined β-actin levels. Data are mean ± SD of triplicate samples in one of the three similar experiments. *p<0.05 compared to the two control groups; ++p<0.01 compared to the PBS control group.
Figure 3
Figure 3. Cytokine levels in the organs of mice after R. equi infection.
BALB/c mice were orally immunized on days 0 and 14 with S. enterica Typhimurium χ3987-pYA3137vapA (closed triangles/dashed line), or inoculated with either S. enterica Typhimurium χ3987-pYA3137 (closed squares) or PBS (open circles). All mice were intravenously challenged with 4×106 CFU of R. equi ATCC33701 14 days after the last immunization. The lung (A–E), liver (F–J), and spleen (K–O) were harvested 2, 4, 8, and 10 days post-infection and homogenized for IL-12 (A, F, and K), IFN-γ (B, G, and L), TNF-α (C, H, and M), IL-10 (D, I, and N), and IL-4 (E, J, and O) detection. Results are expressed as means of five mice per group ± SD and are representative of three experiments. *p<0.05, **p<0.01, and ***p<0.001 compared to the two control groups; and +p<0.05, ++p<0.01, and +++p<0.001 compared to the PBS control group.
Figure 4
Figure 4. Nitrite and hydrogen peroxide production in the organs of mice following R. equi infection.
BALB/c mice were orally immunized on days 0 and 14 with S. enterica Typhimurium χ3987-pYA3137vapA (closed triangles/dashed line), or inoculated with either S. enterica Typhimurium χ3987-pYA3137 (closed squares) or PBS (open circles). All mice were intravenously challenged with 4×106 CFU of R. equi ATCC33701 14 days after the last immunization. On days 2, 4, 8, and 10 after infection, the lung, liver, and spleen were harvested and homogenized for NO (A, C, and E) and H2O2 (B, D, and F) detection. The endogenous H2O2 release was measured by the horseradish peroxidase-dependent phenol red oxidation methods, whereas the nitrite levels were measured by Griess reaction. Data are expressed as means of five mice per group ± SD and are representative of three separate experiments. *p<0.05, **p<0.01, and ***p<0.001 compared to the two control groups.
Figure 5
Figure 5. Alteration of lymphocyte subpopulation in the spleen of mice.
BALB/c mice orally administered with S. enterica Typhimurium χ3987-pYA3137vapA, S. enterica Typhimurium χ3987-pYA3137, or PBS, on days 0 and 14, were intravenously challenged with 4×106 CFU of virulent R. equi strain 14 days after the last immunization. Spleen cells were harvested 8 days post-infection and analyzed by flow cytometry. The cells were stained with specific anti-CD19 (A), anti-CD3 plus anti-CD4 (B), and anti-CD3 plus anti-CD8 (C) monoclonal antibodies. (D) CD4+ spleen T cells stained with specific anti-CD25 plus anti-Foxp-3 monoclonal antibodies. The average of the percentage of spleen cells positive for the respective CD markers in five samples is shown. Similar results were obtained in three independent experiments. (E) Total RNA was extracted from spleen cells harvested 8 days post-infection and assessed by real-time PCR for detection of TGF-β mRNA. The cDNA contents were normalized on the basis of predetermined β-actin levels. Data are mean ± SD of triplicate samples in one of the three similar experiments. *p<0.05, **p<0.01, and ***p<0.001 compared to the two control groups.
Figure 6
Figure 6. Recall immune response induced by immunization with Salmonella expressing the VapA protein.
BALB/c mice were orally immunized with S. enterica Typhimurium χ3987-pYA3137vapA, or inoculated with either S. enterica Typhimurium χ3987-pYA3137 or PBS on days 0 and 14. Five months after the last immunization, all mice were intravenously challenged with 4×106 CFU of virulent R. equi. On day 8 after infection, the spleen (A and B) and liver (not shown) were harvested and homogenized for bacterial burdens and NO quantification. Results are expressed as the mean of five mice per group ± SD. The experiment was performed twice with similar results. *p<0.05, **p<0.01, and ***p<0.001 compared to the control groups.
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