MAPPING OF THE DELAYED-TYPE HYPERSENSITIVITY-INDUCING EPITOPE OF SECRETED PROTEIN MPT64 FROM MYCOBACTERIUM-TUBERCULOSIS

The gene encoding the immunogenic protein MPT64 found in culture filtr ates of Mycobacterium tuberculosis H37Rv was expressed in Escherichia coli K-12 and purified as a recombinant protein. The purified recombin ant MPT64 elicited delayed-type hypersensitivity (DTH) in outbred guin ea pigs sensitized with Mycobacterium bovis BCG Tokyo. The skin reacti ons were comparable to those obtained with native MPT64. No skin react ions were observed when either recombinant MPT64 or native MPT64 was u sed in guinea pigs sensitized with M. bovis BCG Danish 1331. Amino- an d carboxy-terminal deletion mutants of MPT64 were purified as fusion p roteins for the mapping of DTH-inducing epitopes on recombinant MPT64 by use of the guinea pig skin test model. The part of the molecule res ponsible for the biological activity was located at the carboxy-termin al end. Further studies with overlapping synthetic peptides have pinpo inted the biological activity at a single DTH-inducing epitope consist ing of 15 residues between amino acids Gly-173 and Ala-187. Screening by PCR of 56 clinical isolates of M. tuberculosis from Danish and Tanz anian patients demonstrated the presence of mpt64 in all of the strain s. These results point to MPT64 as a possible candidate for a skin tes t reagent specific for diagnosis of human tuberculosis.