MTSA-10, the product of the Rv3874 gene of Mycobacterium tuberculosis, elicits tuberculosis-specific, delayed-type hypersensitivity in guinea pigs

Abstract

In a search for new skin test reagents specific for tuberculosis, we found that the antigen encoded by gene Rv3874 of Mycobacterium tuberculosis elicited delayed-type hypersensitivity in M. tuberculosis-infected guinea pigs but not in control animals immunized with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium avium. The antigen, which was named MTSA-10 (for M. tuberculosis-specific antigen 10), is a prime candidate for a component of a new tuberculin that will allow discrimination by a skin test of latent M. tuberculosis infection from vaccination with BCG or from sensitization with environmental, nontuberculous mycobacteria.

FIG. 1

The DTH response to MTSA-10 distinguishes TB infection from immunization with BCG or with M. avium. For skin test experiments, M. tuberculosis H₃₇Rv was grown to mid-log phase by shaking at 37°C in liquid glycerol-alanine-salts medium with 0.05% (vol/vol) Tween 80 in a biosafety level 3 laboratory. M. bovis BCG Japan and BCG Pasteur were grown in Sauton's medium with 0.025% (vol/vol) tyloxapol at 37°C. M. avium TMC 724 was grown by shaking at 37°C in Middlebrook 7H9 (Difco) liquid medium with 2% glycerol, 10% oleic acid-albumin-dextrose-catalase supplement, and 0.05% (vol/vol) Tween 80. PPD from M. tuberculosis was prepared at Colorado State University by using a standard protocol (25) and was applied to a Detoxi-gel column (Pierce, Rockford, Ill.) to remove the lipopolysaccharide. Purification of native MPT32 from M. tuberculosis H₃₇Rv was performed by the method of Dobos et al. (8). Purification of recombinant MPT64 from E. coli was described previously (6). Guinea pigs were aerosol infected with M. tuberculosis (◊) or were immunized with BCG (▵) or with M. avium (□). Six to eight weeks later, animals were tested intradermally with 1 μg of PPD and 2 μg of purified recombinant antigens. Each point represents 1 animal. Results are expressed as diameter of erythema measured 24 h after antigen injection. (A) In a first experiment, the group sizes were six animals for M. tuberculosis, eight for M. bovis BCG Japan, four for M. avium, and four for saline. Antigens were PPD, MPT64 (BCG Japan produces the M. bovis homolog MPB64; see text), and MTSA-10. (B) In a second experiment, the group sizes were six animals for M. tuberculosis, six for M. bovis BCG Pasteur, six for M. avium, and five for saline. Antigens were PPD, MPT32 (BCG Pasteur does not produce MPB64; see text), and MTSA-10.