Delayed-type hypersensitivity responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the guinea pig

Abstract

Two antigens from Mycobacterium tuberculosis, ESAT-6 and MPT64, elicited delayed-type hypersensitivity (DTH) skin responses in outbred guinea pigs infected with M. tuberculosis by the aerosol and intravenous routes but not those sensitized with M. bovis BCG or M. avium. The DTH epitope of ESAT-6 was mapped to the C terminus. Nonresponders to the individual antigens were found, but all animals responded to a combination of ESAT-6 and MPT64 or their respective minimal target peptides. Correspondingly, these molecules could form the basis of a new skin test for tuberculosis.

FIG. 1

ESAT-6 and MPT64 discriminate TB infection from BCG vaccination or exposure to environmental mycobacteria. Guinea pigs (n = 5 per group) were infected i.v. with M. tuberculosis H37Rv (M. tub.) or immunized with either BCG or M. avium for 4 weeks. They were then tested with 10 TU (0.2 μg) of PPD (○), 1 μg of rESAT-6 (◊), or 1 μg of rMPT64 (▿).

FIG. 2

Responses of two different strains of outbred guinea pigs infected with M. tuberculosis to overlapping peptides derived from ESAT-6 (P1, amino acids [aa] 1 to 20; P2, aa 12 to 35; P3, aa 22 to 45; P4, aa 32 to 55; P5, aa 42 to 65; P6, aa 52 to 75; P7, aa 62 to 85; P8, aa 72 to 95). Eight outbred Ssc:AL (○) and six Dunkin Hartley (◊) guinea pigs were infected by i.v. inoculation with M. tuberculosis H37Rv. Twenty-eight days later the animals were skin tested with 10 TU (0.2 μg) of PPD, 1 μg of rESAT-6, or 1 μg each of the ESAT-6 peptides P1 to P8.

FIG. 3

A combination of rESAT-6 and rMPT64 identifies all animals infected with M. tuberculosis. Twenty-eight days following i.v. infection, Ssc:AL guinea pigs (n = 18) were tested with 10 TU (0.2 μg) of PPD, 1 μg of rESAT-6, 1 μg of rMPT64, or the same quantities of rESAT-6 and rMPT64 in combination.

FIG. 4

Guinea pigs infected by aerosol recognize ESAT-6-MPT64 and the reactive peptides. Dunkin Hartley guinea pigs (n = 6) were infected by the respiratory route and tested for DTH responses to rESAT-6-rMPT64 and peptides from ESAT-6 and MPT64. The guinea pigs were skin tested 35 days following exposure to M. tuberculosis. At skin testing guinea pigs received 0.2 μg of PPD, 2 μg of an equal mixture (wt/wt) of ESAT-6 and MPT64, and 1 μg of the peptides ESAT P8, MPT64 P12, or the two peptides in combination. Reactions were read at 24 h after intradermal injection with the skin test reagents.