This application focuses on the structure and antigenicity of proteins of Mycobacterium lapre that are releveant for the pathogenesis of leprosy, the diagnosis of infection with the leprosy bacillus, or for stimulation of effective cell-mediated immunity against the pathogen. Five protein molecules with subunit molecular masses of 65K, 36K, 28K, 18K and 12K daltons will be investigated initially. Each of thee proteins is known to contain at least on M. lepra species-specific epitope, is reconginzed by serum antibodies from humans with leprosy, and for each molcule there are available both recombinant DNA clones that express the protein and monoclonal antibodies that recognize the native molecule as well as the products of the recombinant DNA clones. Since the leprosy bacillus cannot be cultured in vitro, lysogenized Y1089 E. coli containing M. leprae gens or gene fragments which encode for proteins or peptides of interest, will be used to prepare bacterial lysates from which the M. leprae protein antigens will be purified. Monoclonal antibodies with known molecular and mycobacterial species specificity will be utilized for immunoaffinity purification and immunoassays of native and recombinant proteins adn peptides of interest. The structure of the epitopes recognized by monoclonal antibodies wil be identified by determining the nucleotide sequence of the smallest cubclone of the DNA clone which still expresses the epitope, or by directly determining the amino-acid sequence of the smallest antigenic fragment of the protein molecule obtained from the immnoaffinity column, or by enzymatic cleavage for the purified recombinant molecule followed by isolation and purification using HPLC, charge, sizing, hydrophobic interaction or immunoaffinity techniques. Purified M. leprae proteins and their fragments will be supplied from this grant to collaborating investigators and consultants who will separately investigate the T-cell stimulating capacities of each preparation. Peptide synthesis will be used to vary the amino- acid sequence of the linear structure domains shown to react with antibodies or T-cells to determine the precise structural requirements of each eitope. Since an effective cell-mediated immune response to the leprosy bacillus is required for resistance to leprosy, particular attention will be directed at alpha helical amphipathic regions of the protein sequence which are highly correlated with T-cell stimulating activity. These investigations should contribute to the body or knowledge elucidating structural differences between epitopes reactive with T-cells and antibodies, using a disease where cell-mediated immunity is fundamental to protection. Conceivably, they may identify the epitope(s) to which the T cells of individuals susceptible to leprosy are unable to respond, and suggest ways to modify or enhance this eitopes(s) to permit an effective immune response and the possibility of eventual immunotherapy and/or vaccine development.
| Pope, R M; Wallis, R S; Sailer, D et al. (1991) T cell activation by mycobacterial antigens in inflammatory synovitis. Cell Immunol 133:95-108 |
| Anderson, D C; Barry, M E; Buchanan, T M (1988) Exact definition of species-specific and cross-reactive epitopes of the 65-kilodalton protein of Mycobacterium leprae using synthetic peptides. J Immunol 141:607-13 |
| Anderson, D C; van Schooten, W C; Barry, M E et al. (1988) A Mycobacterium leprae-specific human T cell epitope cross-reactive with an HLA-DR2 peptide. Science 242:259-61 |
| Gaylord, H; Brennan, P J; Young, D B et al. (1987) Most Mycobacterium leprae carbohydrate-reactive monoclonal antibodies are directed to lipoarabinomannan. Infect Immun 55:2860-3 |
| Buchanan, T M; Nomaguchi, H; Anderson, D C et al. (1987) Characterization of antibody-reactive epitopes on the 65-kilodalton protein of Mycobacterium leprae. Infect Immun 55:1000-3 |
