Immunoproteomic identification of human T cell antigens of Mycobacterium tuberculosis that differentiate healthy contacts from tuberculosis patients

Abstract

Identification of Mycobacterium tuberculosis antigens inducing cellular immune responses is required to improve the diagnosis of and vaccine development against tuberculosis. To identify the antigens of M. tuberculosis that differentiated between tuberculosis (TB) patients and healthy contacts based on T cell reactivity, the culture filtrate of in vitro grown M. tuberculosis was fractionated by two-dimensional liquid phase electrophoresis and tested for the ability to stimulate T cells in a whole blood assay. This approach separated the culture filtrate into 350 fractions with sufficient protein quantity (at least 200 microg of protein) for mass spectrometry and immunological analyses. High levels of interferon-gamma (IFN-gamma) secretion were induced by 105 fractions in healthy contacts compared with TB patients (p < 0.05). Most interesting was the identification of 10 fractions that specifically induced strong IFN-gamma production in the healthy contact population but not in TB patients. Other immunological measurements showed 42 fractions that induced significant lymphocyte proliferative responses in the healthy contact group compared with the TB patients. The tumor necrosis factor-alpha response for most of the fractions did not significantly differ in the tested groups, and the interleukin-4 response was below the detectable range for all fractions and both study groups. Proteomic characterization of the 105 fractions that induced a significant IFN-gamma response in the healthy contacts compared with the TB patients led to the identification of 59 proteins of which 24 represented potentially novel T cell antigens. Likewise, the protein identification in the 10 healthy "contact-specific fractions" revealed 16 proteins that are key candidates as vaccine or diagnostic targets.

Fig. 1.

IFN-γ response of 10 contact-specific fractions. “C” represents the response of healthy contacts, and “TB” represents the response of TB patients. Filled circles represent the Individual contact response for each fraction. Open circles represent the Individual patient response for each fraction. Horizontal bars represent the median value of the cytokine response (Patient or contact group).

Fig. 2.

LPA response of 10 contact-specific fractions. Statistical analysis was performed with the Mann-Whitney U test. *, significant value (p < 0.05); **, highly significant value (p < 0.005). This error bar represents median ± standard error.

Fig. 3.

Venn diagram of proteins identified in immunodominant fractions. Very Highly Significant represents the proteins identified in fractions that induced very highly significant levels of IFN-γ (p < 0. 0005) in the healthy contacts as compared with the TB patients. Highly Significant represents the proteins identified in fractions that induced highly significant levels of IFN-γ (p < 0.005) in the healthy contacts as compared with the TB patients. Significant represents the proteins identified in fractions that produced significant levels of IFN-γ (p < 0.05) in the healthy contacts as compared with the TB patients.