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.
Highlights
Identification of Mycobacterium tuberculosis antigens inducing cellular immune responses is required to improve the diagnosis of and vaccine development against tuberculosis
Molecular Characterization of Immunodominant 2D liquid phase electrophoresis (LPE) Fractions—Given that current in vitro T cell-based diagnostic assays for TB are based on the measurement of IFN-␥ responses [26] and the majority of anti-TB vaccine candidates were selected based on their ability to induce an antigen-specific IFN-␥ response [27], we focused protein identification efforts on those 2D LPE fractions that resulted in a significant IFN-␥ response in the healthy contact population
Only one protein (Rv2465c) was found in a 2D LPE fraction of each group, and a total of eight proteins was shared between two of the groups (Fig. 3). These data demonstrate that immunological responses are stratified with respect to different antigens, and it is this stratification that has allowed for the identification of the 10 contact-specific fractions and their corresponding proteins. This present study was developed to identify the antigens of M. tuberculosis that have a potential use in a TB vaccine or in diagnostic development
Summary
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 g of protein) for mass spectrometry and immunological analyses. Proteomic characterization of the 105 fractions that induced a significant IFN-␥ 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. There is evidence that proteins actively secreted by M. tuberculosis during growth induce cell-mediated immune responses by causing expansion of specific interferon-␥ (IFN-␥)-producing T lymphocytes that are capable of recognizing and exerting antimicrobial effects against infected macrophages [7].
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