Peripheral Blood and Pleural Fluid Mononuclear Cell Responses to Low-Molecular-Mass Secretory Polypeptides ofMycobacterium tuberculosisin Human Models of Immunity to Tuberculosis

Abstract

A total of 104 polypeptides were purified from the low-molecular-mass secretory proteome of Mycobacterium tuberculosis H 37 Rv using a combination of anion exchange column chromatography and high resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution. The goal of this study was to identify polypeptides from a low-molecular-mass secretory proteome recognized by human subjects infected with M. tuberculosis and to ascertain the differences in specificity of antigen recognition by the peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) of these individuals. The study identified CFP-8 (Rv0496), CFP-11 (Rv2433c), CFP-14.5 (Rv2445c), and CFP-31 (Rv0831c) as novel T-cell antigens apart from previously characterized ESAT-6, TB10.4, CFP10, GroES, MTSP14, MTSP17, CFP21, MPT64, Ag85A, and Ag85B on the basis of recognition by PBMCs of tuberculosis contacts and treated tuberculosis patients. Further, polypeptides prominently recognized by PFMCs of tuberculous pleurisy patients were the same as those recognized by PBMCs of healthy contacts and treated tuberculosis patients. The results of our study indicate the homogeneity of antigenic target recognition by lymphocytes at the site of infection and at the periphery in the human subjects studied and the need to evaluate these antigenic targets as components of future antituberculous vaccines.

Immunoproteomic Identification of Human T Cell Antigens of Mycobacterium tuberculosis That Differentiate Healthy Contacts from Tuberculosis Patients

“…In the present study, only 14.8% of the immunodominant T cell antigens were represented by antigens with a predicted mass of less than 15 kDa. A stronger immune response to the proteins below 25 kDa was observed in the present study (48.6%) in agreement with previous reports (16,63). However, a sizable number of recognized proteins (ϳ31%) were also found to be greater than 40 kDa.…”

“…Like many previous T cell antigen discovery efforts (15,16,28), we focused on the secreted proteins of M. tuberculosis. However, our current efforts differ from earlier studies, which used experimental animals (15), whereas our study aims at immunological responses to human TB.…”

“…It is generally believed that low molecular mass proteins of M. tuberculosis are predominantly recognized by human T cells (61,62), and most of the studies on human T cell antigens have focused on low molecular mass or selective pooling of fractions (16,28) and reported that antigens Ͻ15 kDa were highly immunogenic. In the present study, only 14.8% of the immunodominant T cell antigens were represented by antigens with a predicted mass of less than 15 kDa.…”

“…Later, twodimensional separation methods were used that involved protein separation by either IEF (15) or chromatography (16) in the first dimension and preparative SDS-PAGE followed by whole gel elution (17) in the second dimension. Mouse T cell antigens of M. tuberculosis were identified using this method (15).…”

“…Mouse T cell antigens of M. tuberculosis were identified using this method (15). Mycobacterial antigens that induce an immune response in healthy household contacts and treated TB patients were also mapped using this approach (16).…”