Use of synthetic peptides corresponding to sequences of Mycobacterium leprae proteins to study delayed-type hypersensitivity response in sensitized guinea pigs.

In this work we report the synthesis of 10 peptides (P1-P10) corresponding to one or several segments of the amino acid sequence of proteins from Mycobacterium leprae: 65 kDa, 28 kDa, 18 kDa, and 28 kDa superoxide dismutase, recently renamed antigens 2L, 9L, 12L, and 14L, respectively. They were assayed in the guinea pig model for the induction of a delayed-type hypersensitivity response in M. leprae and BCG-sensitized animals. To sensitize the animals two schemes were used: either a single dose of 5 x 10(9) irradiated or autoclaved whole bacilli, or four weekly intramuscular injections each containing 500 micrograms of soluble extract of M. leprae (MLSE) in incomplete Freund's adjuvant. Because the second scheme used far too much antigen, we decided to use the first scheme for the experiments we report here. DTH reactions of sensitized animals were induced after 30 days with intradermal injections of 5 micrograms of MLSE and with each of the 10 peptides at three different concentrations: 250 micrograms, 100 micrograms, and 0.05 micrograms. All M. leprae-sensitized guinea pigs gave indurations of 10 mm or more with MLSE, which indicates that the animals were sensitized. None of them gave DTH indurations with 250 micrograms or 100 micrograms, but some of them had positive DTH reactions with the 0.05 micrograms doses of the synthetic peptides. This is most likely due to the fact that we have used an outbred strain of guinea pigs. The peptides were also tested at 0.05 micrograms in animals sensitized with BCG. P7 and P10 seem to be nonspecific peptides; the remaining peptides only induced DTH in the M. leprae-sensitized guinea pigs. P3 (segments 65-85 of the 65-kDa protein) induced a positive DTH in 58% of the tested animals. In other experiments, guinea pigs were sensitized with a single injection (500 micrograms) of each of the synthetic peptides. All animals, except those sensitized with P4 and P8, had positive DTH responses when the homologous peptide was used. Those sensitized with P2, P4, P5, P7, and P8 were able to produce indurations when MLSE was used for the induction of the DTH reaction.