Identification of Early Secretory Antigen Target-6 Epitopes for the Immunodiagnosis of Active Tuberculosis

Abstract

The early secretory antigenic target (ESAT)-6 purified protein and peptides from Mycobacterium tuberculosis were evaluated as antigens for the immunodiagnosis of tuberculosis (TB). Because the control of TB requires improved diagnostic procedures, efforts have increased to identify Mycobacterium tuberculosis-specific epitopes for the immunodiagnosis of active TB and to discriminate between active and latent states of infection. Two multiepitopic peptides from ESAT-6 protein were selected by computational analysis. Patients with active TB (7 HIV + and 20 HIV-) and control patients (17 HIV + and 28 HIV-) were enrolled. Enzyme-linked immunospot assay analysis for interferon-γ expression by peripheral blood mononuclear cells was quantified after stimulation with selected ESAT-6 peptides, purified protein derivative, or the intact ESAT-6 protein. During active TB, 20 of 27 patients responded in vitro to ESAT-6 peptides and 23 of 27 patients to purified protein derivative. None of the controls without active TB, including individuals with latent TB infection, recognized ESAT-6 peptides. By contrast, latently infected individuals did respond in vitro to both intact ESAT-6 protein and purified protein derivative. Thus, high T-cell response frequencies to ESAT-6 peptides are present only during active TB and can be used to discriminate between active and latent forms of infection.

Use of a T Cell–Based Assay for Monitoring Efficacy of Antituberculosis Therapy

“…ESAT-6 is present in M. tuberculosis but absent from all strains of Mycobacterium bovis bacille Calmette-Guérin, as well as in almost all environmental mycobacteria [6,7], although a homologue has been identified in Mycobacterium leprae [8]. On the basis of this line of research, we recently reported an in vitro immune diagnostic ELISPOT assay for IFN-g whose novelty consists of use of ESAT-6 peptides, which are multiepitopic and are selected by computational analysis [9]. This assay's results are positive only for patients with active tuberculosis; it has a sensitivity of 74% and a specificity of 100%, compared with the standard tuberculosis culture method.…”

“…Two M. tuberculosis-specific, ESAT-6-derived peptides (ESAT-6 6-28 and ESAT-6 66-79 ) were selected for their putative HLA class II binding, as described elsewhere [9,11]. Together, the designed peptide epitopes were able to cover 190% of the HLA class II haplotypes present in different human populations (data not shown).…”

“…We studied 18 patients enrolled from a previous study [9]. There were 12 men and 6 women (mean age [‫ע‬SD], years).…”

“…Together, the designed peptide epitopes were able to cover 190% of the HLA class II haplotypes present in different human populations (data not shown). Peptides were synthesized as described elsewhere [9]. Lyophilized peptides were diluted in dimethyl sulfoxide.…”

Current concepts in the diagnosis of tuberculosis

“…Auch bei Verwendung von nur einem Antigen, ESAT-6, ist die Spezifität mit 91 -100% sehr hoch (4,16,21,22,25,29,33,41,43 …”