Leprosy, a chronic infectious disease afflicting between 10 and 15 million people, is caused by the obligate intracellular parasite Mycobacterium leprae. Although M. leprae was the first identified bacterial pathogen of man, basic biochemical, immunological, diagnostic and therapeutic investigations have been severely limited because it remains one of the few human pathogens that have not been cultured in vitro. An M. leprae recombinant DNA expression library was constructed to provide a source of genes encoding proteins relevant for such studies. Monoclonal antibodies directed against M. leprae specific antigens have been used to isolate the genes encoding the five most immunogenic protein antigens of the leprosy bacillus. We report here that M. leprae specific epitopes recognized by all of 13 monoclonal antibodies tested were produced by recombinant phage in Escherichia coli.

A recombinant DNA expression strategy has been used to deduce the amino acid sequences of six different antigenic determinants in a single protein of Mycobacterium leprae, the etiologic agent of leprosy. The gene encoding the M. leprae 65-kDa antigen was sequenced and a Xgtll gene sublibrary was constructed with fragments of the gene. Recombinant DNA clones producing specific antigenic determinants were isolated by screening with monoclonal antibodies, and the sequences of their insert DNAs were determined with a rapid primer-extension method. The amino acid sequence of each determinant was deduced from the minimum overlap of insert DNAs from multiple antibody-positive DNA clones. Amino acid sequences for six different epitopes were elucidated. A peptide containing sequences for one of these epitopes was synthesized and shown to bind the appropriate monoclonal antibody; this antigenic determinant is unique to M. leprae. The approach described here can be used to rapidly elucidate protein epitopes that are recognized by antibodies or T cells.Antigenic determinants, or epitopes, are the specific segments of antigens that are recognized by antibodies or T cells. Various approaches have been used to determine the precise location and/or amino acid sequence of protein epitopes (reviewed in refs. 1 and 2). The most commonly used approaches include sequence analysis of viral variants that are resistant to neutralization by antibodies, screening purified proteolytic fragments, and screening collections of overlapping synthetic peptides.We have designed an efficient recombinant DNA strategy to deduce the amino acid sequences that comprise specific antigenic determinants in a protein. The strategy involves isolating a DNA clone that encodes the entire antigen of interest and determining its nucleotide sequence. A sublibrary containing fragments of the gene with random endpoints is constructed in the bacteriophage expression vector Xgtll (3). The expression of epitope coding sequences by individual recombinant bacteriophage is detected with monoclonal antibody probes and the appropriate DNA clones are isolated. The precise nucleotide sequences of the cloned DNA fragments are determined by using primer-directed DNA sequence analysis. The DNA sequence encoding the epitope is attributed to sequences that are shared by multiple antibody-positive recombinant clones.We have used this approach to define epitopes encoded within the gene for the 65-kDa protein antigen of Mycobacterium leprae. There are several reasons for choosing this antigen for detailed study. It is one of the major immunologically relevant proteins in a variety of medically important mycobacteria, including M. leprae, M. tuberculosis, and M.bovis BCG (4, 5). Antibodies and T cells that recognize the 65-kDa antigen can be detected in patients with leprosy or tuberculosis (refs. 6 and 7 and unpublished data). In addition, the antigen contains at least six different epitopes that can be distinguished with monoclonal antibodies in competitive inhibition rad...

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. As with other intracellular parasites, protective immunity is dependent on T cells and cell-mediated immunity. In animal models, immunization with killed armadillo-derived M. leprae elicits strong T-cell responses, delayed-type hypersensitivity and protection against viable challenge. We have recently shown that killed M. leprae can induce delayed-type hypersensitivity in healthy human volunteers. Identification of the M. leprae antigens that are recognized by T cells and may be involved in protection has been hampered by the inability to cultivate the organism in vitro and by difficulties in antigen purification from limited quantities of armadillo-derived bacillus. Because genes for the major protein antigens of M. leprae as seen by mouse monoclonal antibodies have been isolated, it has become possible to test whether these individual antigens are recognized by T cells. We screened crude lambda gtll phage lysates of Escherichia coli containing individual M. leprae antigens using M. leprae-specific T-cell clones isolated from M. leprae-vaccinated volunteers. Using this method, we find that nearly half of the M. leprae-specific T-cell clones are stimulated to proliferate by lysates containing an epitope of a M. leprae protein of relative molecular mass 18,000 (18K).