Staphylococcus aureus- Cure-Associated Antigens Elicit Type 3 Immune Memory T Cells

Abstract

Background: Staphylococcus aureus is one of the most frequently major mastitis pathogens that cause clinical and subclinical mastitis worldwide. Current antimicrobial treatments are usually ineffective, and the commercially available vaccines lack proven effectiveness. The immunological response elicited by the recombinant S. aureus-cure-associated proteins phosphoglycerate kinase (PGK), enolase (ENO), and elongation factor-G (EF-G) in combination with the granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA vaccination was studied in this work.

Methods: Here, twenty-three C57BL/6 mice were divided into four groups and vaccinated with: G1: none (control); G2: GM-CSF DNA plasmid DNA vaccine; G3: the combination of EF-G+ENO+PGK; and G4: the combinations of EF-G+ENO+PGK proteins plus GM-CSF plasmid DNA vaccine. After 44 days, spleen cells were collected for immunophenotyping and lymphocyte proliferation evaluation by flow cytometry upon S. aureus stimulus.

Results: Immunization with the three S. aureus recombinant proteins alone resulted in a higher percentage of IL-17A⁺ cells among CD8⁺ T central memory cells, as well as the highest intensity of IL-17A production by overall lymphocytes indicating that the contribution of the combined lymphocyte populations is crucial to sustaining a type 3 cell immunity environment.

Conclusion: The immunization with three S. aureus-cure-associated recombinant proteins triggered type 3 immunity, which is a highly interesting path to pursue an effective bovine S. aureus mastitis vaccine.

Keywords: IL-17A; Staphylococcus aureus; T cell response; dairy cow; intramammary infection; mastitis; recombinant antigens; vaccine.

Figure 1

Scheme of the timeline of immunization (A) and experimental groups (B). EF-G: elongation factor g recombinant S. aureus protein; PGK: transmembrane protein phosphoglycerate kinase recombinant S. aureus protein; ENO: enolase recombinant S. aureus protein; GM-CSF: pCI-granulocyte and macrophage colony-stimulating factor plasmid DNA. The unvaccinated group was merely administrated saponin adjuvant to liposome.

Figure 2

Percentage of IL-17A⁺ T cells in mice vaccinated with three S. aureus-cure-associated recombinant proteins. T cell responses are normalized by dividing the percentage of IL-17A⁺ cells upon S. aureus stimulation per the percentage of IL-17A⁺ cells under unstimulated control a stimulation index. (I) Representative gating of IL-17A production by different lymphocyte populations. (II) Stimulation index of (A) T CD8⁺ CD44⁺ CD27⁺ (central memory) cells, (B) overall γδ TCR⁺ cells, (C) γδ TCR⁺ CD44⁺ CD27⁺ (central memory) cells, and (D) TCRVγ4⁺ Ki67⁺ cells. Granulocyte-macrophage colony-stimulating factor; EF-G: elongation factor-G, ENO: enolase; and PGK: phosphoglycerate kinase. * indicate p ≤ 0.05, ** indicate p ≤ 0.01. (One-way ANOVA followed by Tukey test).

Figure 3

Geometric mean fluorescence intensity (GMFI) of IL-17A⁺ production by T cells in mice vaccinated with three-antigens S. aureus-cure-associated recombinant proteins. T cell responses are normalized by dividing the GMFI of IL-17A⁺ cells upon S. aureus stimulation per the percentage of IL-17A⁺ cells under unstimulated control condition, thereby creating a stimulation index. Here, we show the GMFI of stimulation index of (A) overall lymphocytes, (B) T CD4⁺, (C) TCRVγ4⁺ and (D) T CD8⁺ CD44⁺ CD27⁺ (central memory) and (E) T CD44⁺ lymphocytes. Granulocyte-macrophage colony-stimulating factor; EF-G: elongation factor-G, ENO: enolase; and PGK: phosphoglycerate kinase. * indicate p ≤ 0.05, ** indicate p ≤ 0.01, *** indicate p ≤ 0.001 (One-way ANOVA followed by Tukey test).

Figure 4

Percentage of T CD4⁺ cells in immunized animals, and the antidromic trend of the percentage TCRVγ4⁺ effector and central memory cells in mice vaccinated with GM-CSF DNA vaccine. (A) Overall T CD4⁺, (B) TCRVγ4⁺ CD44⁺ CD27⁻ (effector memory cells), and (C) TCRVγ4⁺ CD44⁺ CD27⁺ (central memory cells). GM-CSF: Granulocyte-macrophage colony-stimulating factor; EF-G: elongation factor-G, ENO: enolase; and PGK: phosphoglycerate kinase. * Indicate p ≤ 0.05, ** indicate p ≤ 0.01. (One-way ANOVA followed by Tukey test).