Mapping of the delayed-type hypersensitivity-inducing epitope of secreted protein MPT64 from Mycobacterium tuberculosis

Abstract

The gene encoding the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was expressed in Escherichia coli K-12 and purified as a recombinant protein. The purified recombinant MPT64 elicited delayed-type hypersensitivity (DTH) in outbred guinea pigs sensitized with Mycobacterium bovis BCG Tokyo. The skin reactions were comparable to those obtained with native MPT64. No skin reactions were observed when either recombinant MPT64 or native MPT64 was used in guinea pigs sensitized with M. bovis BCG Danish 1331. Amino-and carboxy-terminal deletion mutants of MPT64 were purified as fusion proteins for the mapping of DTH-inducing epitopes on recombinant MPT64 by use of the guinea pig skin test model. The part of the molecule responsible for the biological activity was located at the carboxy-terminal end. Further studies with overlapping synthetic peptides have pinpointed the biological activity at a single DTH-inducing epitope consisting of 15 residues between amino acids Gly-173 and Ala-187. Screening by PCR of 56 clinical isolates of M. tuberculosis from Danish and Tanzanian patients demonstrated the presence of mpt64 in all of the strains. These results point to MPT64 as a possible candidate for a skin test reagent specific for diagnosis of human tuberculosis.

Comparison of Antigen‐Specific T‐Cell Responses of Tuberculosis Patients using Complex or Single Antigens of Mycobacterium tuberculosis

“…The present study extends these data to include humans and shows that ESAT-6 is also an important antigen during natural infection with M. tuberculosis. Similar data showing that MPT59, MPT64 and MPB70 are important antigens in animal models already exist [11,12,43,[46][47][48][49]. Our present data extend these findings and indicate that these antigens are important in human tuberculosis [50][51][52].…”

T‐Cell Recognition of Mycobacterial Proteins MPB70 and MPB64 in Cattle Immunized with Antigen and Infected with Mycobacterium bovis

“…In guinea pigs the epitope involved in delayed-type hypersensitivity responses to MPB64 has been characterized and shown to have partial overlap with the epitope in peptide no. 19 predicted in this study [34]. The conserved nature of T-cell recognition of MPB64 epitopes across species may further suggest broad recognition within species with obvious benefits in diagnosis.…”

Human T‐ and B‐Cell Reactivity to the 16 kDa α‐Crystallin Protein of Mycobacterium tuberculosis

“…There must be doubt, however, concerning its ability to induce DTH, given the lack of skin reaction in outbred guinea pigs. The antigens MPT64 and 38 kDa of M. tuberculosis elicited skin reactions at the same or lower doses in identical experiments [14,24]. The chemical form or high purity (less adjuvant effect of impurities) of the preparation used may be important.…”