Isolation of a proline-rich mycobacterial protein eliciting delayed-type hypersensitivity reactions only in guinea pigs immunized with living mycobacteria.

Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced only by a previous immunization with living attenuated mycobacteria, usually bacillus Calmette-Guérin (BCG). Living and killed bacteria share a number of common antigens. To identify and to purify molecules that are dominant antigens during immunization with living bacteria, a two-step selection procedure was used. Quantitative delayed-type hypersensitivity (DTH) reactions elicited in guinea pigs immunized either with living or with killed BCG were used to select or counterselect antigens present in BCG culture filtrates. Each major fraction eluted from a series of HPLC columns (gel filtration, DEAE, reverse-phase chromatography) was assayed and titrated on guinea pigs of each group. A protein with an unusual amino acid composition (40% proline, 12% threonine) was purified and N-terminally sequenced. To our knowledge, the sequence Thr-Pro-Pro-Xaa-Glu-Xaa-Pro-Pro-Pro-Pro-Gln-Xaa-Val-Xaa-Leu has not been previously reported. The protein was 100-fold more potent on guinea pigs immunized with living bacteria than on guinea pigs immunized with dead bacteria to elicit a DTH reaction.