Identification of Mycoplasma mycoides subsp. mycoides small colony genes coding for T-cell antigens

Abstract

Genes of the Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) coding for proteins capable of eliciting protective T-cell memory responses have potential for incorporation into a recombinant subunit vaccine against contagious bovine pleuropneumonia (CBPP). Here we used lymphocytes from cattle that had completely recovered from infection to screen products of MmmSC genes for recognition by CD4(+) effector memory (Tem) and central memory (Tcm) T lymphocytes. Six MmmSC genes (abc, gapN, glpO, lppA, lppB, and ptsG) were expressed as histidine-tagged recombinant polypeptides, or synthetic overlapping peptides, before inclusion in proliferation and gamma interferon (IFN-gamma) assays. Only two MmmSC antigens, LppA and PtsG, consistently induced recall proliferation from immune CD4(+) T cells and IFN-gamma production in all animals tested. Moreover, LppA and PtsG were shown to possess epitopes recognized by both short-lived CD4(+) Tem and long-lived CD4(+) Tcm cells.

FIG. 1.

LppA-induced recall proliferation of lymphocytes from CBPP-immunized (a) and naïve (b) cattle. Cells were loaded with CFSE and incubated for 7 days with medium alone (no antigen [NoAg]), whole MmmSC antigen (MmmSC), the mitogen concanavalin A (ConA), and recombinant LppA and Apx proteins. Proliferation is associated with a decrease in CFSE fluorescence intensity (FL1) and is indicated on the plots as percentages of total cells calculated within the M1 interval (percent CFSE^low).

FIG. 2.

Recall proliferation of primed CD4⁺ T cells in response to individual MmmSC recombinant proteins. Cells were loaded with CFSE and incubated for 7 and 10 days with medium alone (NoAg), whole MmmSC antigen (MmmSC), and recombinant proteins (Abc, Apx, GapN, GlpO, LppA, LppB, and PtsG). Results are mean percentages (±standard deviations [SD]) of CFSE^low cells for four animals and two independent experiments. Asterisks indicate significant differences (P < 0.05) from medium-alone controls.

FIG. 3.

Recall proliferation of primed CD4⁺ T cells in response to synthetic PtsG peptides. Cells were loaded with CFSE and incubated for 10 days with medium alone (NoAg), whole MmmSC antigen (MmmSC), PtsG peptides soluble in PBS (pool A), and PtsG peptides soluble in DMSO (pool B). Results are mean percentages (±SD) of CFSE^low cells for three animals and two independent experiments.

FIG. 4.

Both CD62L-positive and CD62L-negative CD4⁺ T cells proliferate in response to LppA. Results of a typical three-color flow cytometric analysis are shown in density plots for one CBPP-immunized animal. Cells were loaded with CFSE and incubated for 10 days with medium alone (NoAg) or LppA. An R2 gate was set to analyze CD4⁺ CFSE^low cells for CD62L expression. The percentages of CD62L-positive and CD62L-negative cells within R2 are indicated on the plot in the upper and lower quadrants, respectively.

FIG. 5.

CD62L expression among CD4⁺ T cells proliferating (R2 gate, as in Fig. 4) in response to the Abc, LppA, and PtsG (pool A) peptides. Results are mean percentages (±SD) for four animals and two independent experiments.

FIG. 6.

Recall IFN-γ production by primed cells in response to individual MmmSC recombinant proteins. IFN-γ was measured by ELISA in 7- and 10-day-old supernatants of cultures supplemented with medium alone (NoAg), whole MmmSC antigen (MmmSC), and recombinant proteins (Abc, Apx, GapN, GlpO, LppA, LppB, and PtsG). Results are expressed as optical densities (OD) and are mean (±SD) values for four animals and two independent experiments. Asterisks indicate significant differences (P < 0.05) from medium-alone controls.

FIG. 7.

Recall IFN-γ production by primed cells in response to synthetic PtsG peptides. IFN-γ was measured by ELISA in 10-day-old supernatants of cultures supplemented with medium alone (NoAg), whole MmmSC antigen (MmmSC), PtsG peptides soluble in PBS (pool A), and PtsG peptides soluble in DMSO (pool B). Results are expressed as optical densities (OD) and are mean (±SD) values for three animals and two independent experiments. Asterisks indicate significant differences (P < 0.05) from medium-alone controls.