Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

Abstract

Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate. The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested in cultures of peripheral blood mononuclear cells from human tuberculosis (TB) patients, Mycobacterium bovis BCG-vaccinated donors, and nonvaccinated donors. The two ESAT-6 family members, TB10.4 and CFP10, were very strongly recognized and induced gamma interferon release at the same level (CFP10) as or at an even higher level (TB10.4) than ESAT-6. The non-ESAT-6 family member, TB7.3, for comparison, was recognized at a much lower level. CFP10 was found to distinguish TB patients from BCG-vaccinated donors and is, together with ESAT-6, an interesting candidate for the diagnosis of TB. The striking immunodominance of antigens within the ESAT-6 family is discussed, and hypotheses are presented to explain this targeting of the immune response during TB infection.

FIG. 1

Alignment of the protein sequence of ESAT-6 with the two ESAT-6 family proteins TB10.4 and CFP10. Identical residues are marked by vertical lines.

FIG. 2

2-DE map of novel and previously identified M. tuberculosis proteins in ST-CF. (A) Silver-stained two-dimensional SDS-PAGE of total ST-CF. Previously identified proteins have been indicated. (B) Silver-stained two-dimensional SDS-PAGE of the low-molecular-mass region of ST-CF. The novel molecules have all been mapped by Western blotting of duplicate gels, and their positions (indicated by rings) have been transferred to the silver-stained reference gels. The white ring indicates CFP10. TB7.3 is present in only very low concentrations in culture filtrate and is not detectable by silver staining. Previously characterized proteins are shown for comparison. Molecular masses are given to the left in kilodaltons. The pH scale is shown below.

FIG. 3

Human lymphocyte responses to rTB7.3, rTB10.4, and rCFP10. The IFN-γ response resulting from stimulation of PBMC from two human TB patients (circles) and two healthy BCG-vaccinated human donors (upside-down triangles) with increasing concentrations of rTB7.3 (A), rTB10.4 (B), and rCFP10 (C). The data depicted are the means of duplicate analysis.

FIG. 4

IFN-γ responses to low-mass antigens from M. tuberculosis in different groups of donors. Seven healthy nonvaccinated donors, 7 healthy BCG-vaccinated donors, and 17 TB patients were stimulated with 5 μg of ST-CF or recombinant antigens. Individual antigen-specific responses are shown as delta values (IFN-γ release in the antigen-stimulated well minus IFN-γ release in the unstimulated well). Positive responses are defined as delta values above 200 pg of IFN-γ/ml. IFN-γ release in unstimulated wells was generally below 100 pg of IFN-γ/ml. Three TB patients induced an IFN-γ release against ST-CF that was higher than 10,000 pg/ml. These results are not included in the figure.