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The study of the epitope specificity and interlinked function of responding T cells may help to improve the understanding of immunopathogenesis of nerve and skin lesions and may advance the search for immunodiagnostic and vaccine subunits. Considering the many antigens with structural homology among the pathogenic Mycobacterium leprae, Mycobacterium tuberculosis, and environmental mycobacteria (2) only a few have been studied by using synthetic peptides for the stimulation of cloned T cells (10) or blood lymphocytes (3, 12). Although most M. leprae antigens have close homologues in the M. tuberculosis complex (20), the 35-kDa antigen is an exception, with homologues in Mycobacterium avium only (21, 22). The 35-kDa antigen of M. leprae, a major membrane protein (8), has been cloned and sequenced (25). It contains an M. leprae-specific B-cell epitope of conformational nature (9), which is the target of serum antibodies (Ab), elevated in a very high proportion of multibacillary, but only few tuberculoid (TT/BT), leprosy patients (17, 19). The purified 35-kDa antigen of M. leprae has previously been found to be stimulatory for peripheral blood mononuclear cells (PBMCs) of TT/BT leprosy patients and Mycobacterium bovis BCG-vaccinated healthy subjects in California (15), and the purified recombinant protein was stimulatory of both proliferation and gamma interferon (IFN-) production in 65% of paucibacillary patients and 80% of healthy contacts in Nepal (21). These studies with the whole antigen indicated a strong immunogenicity but also its lack of M. leprae specificity, attributable to sensitization by the 88% homologous 35-kDa antigen of M. avium, since the gene encoding the 35-kDa protein is not found in the M. tuberculosis complex or BCG (21, 25). The purpose of this study was to identify immunogenic peptides and search for M. leprae-specific epitopes involving the 12% of residues that differ between M. leprae and M. avium. The mapping of T-cell epitopes is complicated in humans because of heterozygosity at three HLA class II loci (DR, DQ, and DP). Predictive motifs are only of partial value, and empirical mapping by PBMC proliferation is too elaborate for larger proteins. Therefore, for the analysis of the 35-kDa protein we opted for a strategy in which the initial screening of peptides was carried out by binding to purified HLA-DR molecules.

Affinity of peptide binding to purified HLA-DR molecules. On the basis of the 35-kDa protein sequence (25), a total of 58 15-mer peptides overlapping by five residues were synthesized and characterized by previously described methods (23). HLA-DR molecules of 10 different haplotypes were purified (5, 6), and the binding assay was performed with mixtures containing purified HLA-DR protein (0.5 µM), N-terminally biotinylated reference peptide (1.8 µM class II-associated invariant chain peptide [CLIP], 1.6 µM influenza hemagglutinin 307-319, or 1.6 µM HLA-A3 153-168), and competitor peptide (0, 3, 30, or 300 µM) (11). The 50% inhibitory concentrations (IC₅₀) for competitor peptides were calculated by regression analysis of the measured absorbencies.

Peptide-stimulated proliferation of PBMCs from leprosy patients and controls. All 55 Indian leprosy patients recruited from the Central Jalma Institute for Leprosy, Agra, India, and the Army Hospital, Lucknow, India, had received less than 4 weeks of World Health Organization multidrug chemotherapy, while healthy controls were recruited from staff working in the same institutes. Leprosy patients without microbiologically active disease were from the Hospital for Tropical Diseases in London, United Kingdom (UK). Healthy purified protein derivative-negative (4) and purified protein derivative-positive (18) controls, none of whom had been exposed to leprosy, were recruited from the tuberculosis contact clinic at Northwick Park Hospital, Harrow, England. The absence of any likely exposure to leprosy was ascertained by excluding those who had traveled to any area where leprosy was known to be endemic and by excluding any in a health service occupation. Proliferation and cytokine assays were performed as previously described (23). Although peptides were essentially lipopolysaccharide free, polymyxin B (10 µg/ml) was added to cultures as an additional precaution. The lymphocyte stimulation index (SI) was calculated as the average counts per minute in the presence of antigen/the average counts per minute in the absence of antigen. A positive response was defined by a lymphocyte SI of >2. A total of 4 of 105 subjects having values for background counts per minute of >3 standard deviations from the population mean were excluded from the analysis.

Response to pairs of homologous peptides with either the M. leprae or M. avium sequence. In view of the abundant exposure to environmental M. avium in areas where leprosy is endemic, we investigated if cross-sensitization could be distinguished with a pair of 19-mer peptides (206-224) (rather than the original 14-mer, 206-219) containing five residues differing between M. leprae and M. avium. Proliferation was tested in PBMC cultures of leprosy patients and controls from India and the UK. In the latter group (excluding any contact with M. leprae infection) all but one subject (a total of five) responded only to the M. avium peptide (P = 0.019), thus suggesting a specific recognition of M. avium 206-224. In contrast, T cells from the UK leprosy patients recognized 206-224 in about equal proportions either alone or in conjunction with M. avium 206-224. These data suggest that T cells can recognize each of these two peptides specifically, but a contribution from cross-reactive T cells cannot be excluded. This conclusion is corroborated by the absence of any response to the M. avium peptide in all three responders of the Indian lepromatous (LL/BL) group, hence suggesting the presence of M. leprae 206-224-specific T cells. One problem of interpretation remains the initial finding of 50% responsiveness of UK controls to peptide 206-219 (Table 1). Although the four N-terminal residues of the extended 19-mer peptide used in the comparison shown in Table 2 were all conserved between the two species, the addition of these residues ostensibly improved specificity. Therefore, detailed analysis of the epitope localization (i.e., window pepscan, establishment of minimal epitope length, the role of flanking regions, and characterization of the epitope core) is required. Based on the lack of response in UK controls who were not exposed to M. leprae infection we conclude, tentatively, that peptide 206-224 carries M. leprae specificity.

Analysis of p206-224 specificity in HLA-DRB1*0101 transgenic and FVB/N (H2-A^q) control mice. The transgenic mice express both DRB1*0101 and indigenous H2-A^q but only murine CD4 (1). Mice were immunized subcutaneously in each hind footpad with 50 µg of synthetic peptides of either the M. leprae or M. avium sequence or with 25 µg of recombinant 35-kDa protein (a gift from Warwick Britton, Centenary Institute, Sydney, Australia) (21) (rpL35) or phosphate-buffered saline emulsified in a 1:1 (vol/vol) ratio with incomplete Freund's adjuvant (Difco, Detroit, Mich.). After 8 days, the draining popliteal lymph node (LN) cells in triplicate cultures of 4 × 10⁵ cells/well were incubated in the presence of the synthetic peptides (50 µg/ml) or rpL35 (10 µg/ml), and [³H]thymidine uptake (on day 3 after 6 h) was determined. Reciprocal priming and in vitro stimulation of LN cells showed that LN cells from M. leprae 206-224-primed mice were stimulated by the homologous peptide but not by the M. avium peptide (Fig. 1); this response was higher in the DR1 transgenic mice than in the control FVB mice. Stimulation with rpL35 was also pronounced in the DR1 transgenic mice but was much weaker in the control mice, thus suggesting the immunodominance of M. leprae 206-224 in the context of DR1. Moreover, this immunodominant determinant appeared to be M. leprae-specific, since LN cells from M. avium 206-224-primed mice responded only to the homologous peptide and not the peptide or the whole antigen from M. leprae.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Proliferative responses of LN cells from HLA-DRB1*01-transgenic mice (black bars) and control FVB mice (white bars). Mice (two males per group) were primed by footpad inoculation with 50 µg of peptide 206-224 of M. leprae (Ml) or M. avium (Ma) or 25 µg of rpL35 (rP) in incomplete Freund's adjuvant. [3H]thymidine uptake corrected for phosphate-buffered saline controls of pooled 8-day primed LN cells incubated in the presence of 50 µg of peptide or protein stimuli per ml was measured for transgenic and control mice. Anti-HLA-DR monoclonal Ab inhibited 53.3% of the M. leprae 206-224-specific response and not the rpL35 stimulation of DR1 transgenic LN cells (results not shown). *31000, counts per minute.

Cytokine production by peptide-stimulated PBMCs. PBMCs (10⁷ cells/ml) were cultured in 96F plates (Nunc, Roskilde, Denmark), and the supernatants were harvested at 48 h for enzyme-linked immunosorbent assay. Median values of nonparametric variables with the range were calculated. In cultures of 22 Indian controls and leprosy patients, concanavalin A (5 µg/ml) stimulated higher IFN- levels in the healthy controls (415.4 pg/ml; range, 409.1 to 642.9 pg/ml) in UK controls than in either the TT/BT group (344.9 pg/ml; range, 101.5 to 608.2 pg/ml) or the LL/BL group (281.8 pg/ml; range, 70.9 to 583.5 pg/ml) (P < 0.05 in both cases). Although M. leprae soluble extract, obtained from R. J. W. Rees, World Health Organization Bank, also stimulated higher IFN- levels in TT/BT patients (206.0 pg/ml; range, 33.8 to 415.0 pg/ml) than in healthy controls (48.3 pg/ml; range, 0 to 279.1 pg/ml), this difference was not statistically significant, while LL/BL patients produced, as expected, the lowest levels (9.1 pg/ml; range, 0 to 262.2 pg/ml; P < 0.05, by comparison with the TT/BT group). Most surprisingly, the IFN- responses of both healthy controls and TT/BT patients to the tested peptides were very low (results not shown). Considering the high sensitivity of the IFN- assay (1 pg/ml), the low background production (median value, 0 pg/ml; the highest value in the range was 11 pg/ml), and the high values in a previous analysis of tuberculosis patients (24), we are confident that the lack of significant peptide-stimulated IFN- production was not due to technical reasons. This is in contrast with the corresponding proliferation and IFN- responses to the recombinant whole 35-kDa protein in the Nepali study (21). It seems unlikely that immunogenic peptides per se are not stimulatory of IFN-, since at least one peptide (p-624), derived from the LSR/A15 antigen of M. leprae, was reported to be stimulatory in Indian leprosy patients and controls (13).