Characterization, sequence determination, and immunogenicity of a 64-kilodalton protein of Mycobacterium bovis BCG expressed in escherichia coli K-12.

We report the DNA sequence of a previously cloned Mycobacterium bovis BCG gene encoding an immunogenic 64-kilodalton protein. This protein, MbaA, was purified from overproducing Escherichia coli K-12 cells, and the presence of antibodies to MbaA in human sera was determined by an enzyme-linked immunosorbent assay. In about 80% of serum samples from tuberculosis patients and in about 60% of samples from BCG-vaccinated individuals, significant levels of anti-MbaA antibodies were found. Surprisingly, in about 30% of the control serum samples obtained from children, anti-MbaA antibodies were also observed. Guinea pigs sensitized with M. bovis BCG or MbaA showed a delayed-type hypersensitivity reaction after challenge with purified MbaA, supporting the previously observed strong reactivity of human T-cell clones with this, for mycobacteria, common antigen.     

重组卡介苗表达的MalE蛋白T细胞表位的鉴定

目的 鉴定重组卡介苗表达的MalE蛋白T细胞表位。方法 用抗原提呈试验、T细胞增殖试验、ELISPOT试验和表位作图测试重组MalE蛋白的T细胞表位。结果 重组BCG.MalE功能性表达了MalE蛋白的4种H-2^d限制性T细胞表位。结论 在4种表位中，p68-82是重组MalE蛋白的主要T细胞表位，而其余3个为次要表位。     

Cloning of Mycobacterium bovis BCG DNA and expression of antigens in Escherichia coli.

A gene bank of Mycobacterium bovis BCG DNA in Escherichia coli was constructed by cloning Sau3A-cleaved mycobacterium DNA fragments into the lambda vector EMBL3. The expression of mycobacterial antigens was analyzed by Western blotting with hyperimmune rabbit sera. Among 770 clones tested, several were found that produced various mycobacterial antigens in low amounts, with concentrations generally close to the detection limit. One particular clone was chosen for further investigation. This clone produced a 64-kilodalton (kDa) antigen. By placing the lambda promoter PL in front of the structural gene of this antigen, an overproducing E. coli strain was obtained. Rocket-line immunoelectrophoresis experiments showed that antigens cross-reacting with the 64-kDa protein are present in a wide variety of mycobacteria and also in so-called purified protein derivatives which are routinely used for skin tests. Preliminary experiments indicate the presence of antibodies against the 64-kDa antigen in sera from tuberculosis patients.     

Identification of a 25-kilodalton protein of Mycobacterium bovis BCG to distinguish BCG strains from Mycobacterium tuberculosis.

Mycobacterium bovis BCG vaccine strains were compared with Mycobacterium tuberculosis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 25-kDa protein observed in the BCG strains was absent in M. tuberculosis. Rabbit antibodies specific to the 25-kDa protein uniquely identified this protein in BCG strains but not in M. tuberculosis. It is suggested that the 25-kDa protein and polyclonal antibodies directed against this antigen can be exploited to distinguish BCG strains from M. tuberculosis.     

Genes for immunodominant protein antigens are highly homologous in Mycobacterium tuberculosis, Mycobacterium africanum, and the vaccine strain Mycobacterium bovis BCG.

The relatedness of immunodominant protein antigens in Mycobacterium tuberculosis, M. africanum, and M. bovis BCG was investigated by comparing the genes that encode major protein antigens in M. tuberculosis with their counterparts in the other two mycobacteria. Genes encoding homologs of M. tuberculosis major protein antigens were isolated from M. africanum and M. bovis BCG by constructing lambda gt11 recombinant DNA expression libraries and screening them with murine monoclonal antibodies and DNA probes. The antibodies were directed against four major protein antigens of M. tuberculosis with molecular masses of 71, 65, 19, and 14 kilodaltons. The isolated M. africanum and M. bovis BCG DNA clones were mapped with restriction endonucleases, and the maps of the mycobacterial genes were confirmed by Southern analysis of mycobacterial genomic DNA. The restriction maps of DNA containing the four genes in M. tuberculosis, M. africanum, and M. bovis BCG are identical, indicating that the immunodominant proteins that they encode are highly homologous in the three mycobacteria. Thus, the immunity against tuberculosis engendered by M. bovis BCG vaccination could be provided, at least in part, by the immune response to these homologous antigens.     

Antigenic and structural similarities between Mycobacterium tuberculosis 50- to 55-kilodalton and Mycobacterium bovis BCG 45- to 47-kilodalton antigens.

The relationship between Mycobacterium tuberculosis 50- to 55-kDa protein and Mycobacterium bovis BCG 45- to 47-kDa antigen was examined by using immunological and biochemical criteria. Reciprocal cross-reactivity with a rabbit polyclonal antiserum against the M. bovis BCG protein and with a monoclonal antibody raised against the M. tuberculosis antigen was observed. The epitope recognized by this antibody was apparently present only in proteins of M. tuberculosis and M. bovis BCG among the 11 mycobacterial species tested. The amino-terminal sequences and total amino acid contents of these proteins showed strong similarities. Both antigens are glycoproteins as assessed by binding of concanavalin A, labeling of carbohydrate moieties with biotin-hydrazide, and digestion of carbohydrates with jack bean alpha-D-mannosidase, which produced a reduction of the molecular weights of the proteins and totally eliminated concanavalin A binding. Both M. tuberculosis and M. bovis BCG proteins are secreted, since they were found mainly in the culture medium. Analysis of M. tuberculosis 50- to 55-kDa antigen by two-dimensional gel electrophoresis showed at least seven different components, as previously described for the M. bovis BCG antigen. Solid-phase immunoassays showed that the purified M. tuberculosis 50- to 55-kDa protein was recognized by serum specimens from 70% of individuals with pulmonary tuberculosis from a total of 77 Mexican patients examined.     

Characterization of antibody-reactive epitopes on the 65-kilodalton protein of Mycobacterium leprae.

Twenty-three monoclonal antibodies (MAbs) prepared in seven different laboratories were studied, all of which recognized the 65-kilodalton (kDa) protein of Mycobacterium leprae as determined by Western blotting or gel radioimmunoassay or both. Fourteen of the MAbs recognized different epitopes, as evaluated by cross-competition studies using radiolabeled MAb and unlabeled inhibitors; the species specificity of these epitopes was defined by nitrocellulose dot blot immunoassays with bacterial sonic extract antigen preparations from 23 species of mycobacteria. Each of the 14 distinct MAbs recognized a 65-kDa protein produced by a lysogenized Escherichia coli Y1089 host containing cloned rDNA which included the gene for the M. leprae 65-kDa protein. Of the 14 distinct MAbs, 1 recognized an epitope found only on M. leprae, and the others recognized epitopes present on as few as 8 or as many as all 23 of the mycobacterial species studied. Identification of these distinct 65-kDa protein epitopes and use of the MAbs which recognize them should assist future structural studies of this protein and characterization of the T-cell reactive and serodiagnostically useful portions of the molecule.     

Purification and characterization of two protein antigens from the heterogeneous BCG85 complex in Mycobacterium bovis BCG

The heterogeneous BCG85 complex is a major component of BCG culture fluid. BCG85A and BCG85B were purified by combining ammonium sulphate precipitation with chromatography on hydroxyapatite, DEAE-Sephacel and phenyl-Sepharose columns. Twenty percent of BCG85B was recovered. The chromatographic separation procedures were monitored by fused rocket immunoelectrophoresis. The BCG85 complex was found to consist of three antigens, which were heterogenous with regard to electrophoretic mobility, molecular weight (MW), hydrophobic and immunological properties. They were designated A, B and C in increasing order, according to their electrophoretic mobilities. Thus BCG85A had the lowest electrophoretic mobility, BCG85C the highest. The MW of BCG85A was found to be 31,000, while BCG85B had a slightly lower MW, 29,000, as determined by SDS-PAGE. The antigenic relationship between the components was evaluated by crossed immunoelectrophoresis and double diffusion, and reactions of partial identity between the antigens were found. The BCG85 complex occurs in far lower concentration in sonicates of BCG than in culture fluid.     

Expression of Treponema pallidum antigens in Escherichia coli K-12

A colony bank of recombinant plasmids harboring Treponema pallidum DNA inserts has been established in Escherichia coli K-12. By using an in situ immunoassay, we identified four E. coli clones that expressed T. pallidum antigens. Thus, recombinant DNA technology may provide powerful new tools for studying the pathogenesis of T. pallidum infection.     

Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes.

Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.     

Identification of murine B-cell and T-cell epitopes of Escherichia coli outer membrane protein F with synthetic polypeptides

The major pore-forming outer membrane proteins (Omps) of gram-negative bacteria demonstrate numerous immunomodulating properties and are involved in the virulence of pathogenic strains. Because Escherichia coli OmpF is the best-characterized porin in terms of structural and functional characteristics, in vitro B-cell and T-cell responses to this porin in six different strains of mice were analyzed. Mice were immunized with purified OmpF trimers or overlapping synthetic polypeptides (20-mers) spanning the entire 340-amino-acid sequence of the OmpF monomer. T-cell proliferative responses and immunoglobulin G antibody responses to native OmpF and the peptide analogues were determined. For each strain, patterns of T-cell proliferation were similar regardless of whether native OmpF or synthetic peptides were inoculated, although all strains recognized one or more cryptic determinants. Mice exhibited several haplotype-specific responses, but genetically permissive epitopes were also identified. Four peptides (75-94, 265-284, 295-314, and 305-324) elicited strong T-cell proliferative responses from all strains of mice when mice were presensitized with native OmpF or a homologous peptide. In general, 10 or fewer peptides were recognized by sera from mice immunized with native OmpF or synthetic peptides, and most sera from peptide-immunized mice reacted poorly with the native protein. Four peptides spanning amino acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from peptide-immunized mice. Peptides 245-264 and 305-324 were universally recognized by sera from peptide-immunized mice, but these sera reacted weakly or were negative when tested against the native protein. Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response. T-cell and B-cell responses have been investigated based on the assumption that differences in epitope specificity could influence protective or pathologic host reactions. Because of the high level of structural homology of OmpF to porins isolated from other enteric pathogens, the identification of T- and B-cell-stimulatory determinants of E. coli OmpF may have broader application.     

The Mycobacterium tuberculosis 65-kilodalton antigen is a heat shock protein which corresponds to common antigen and to the Escherichia coli GroEL protein.

Monoclonal hybridoma antibodies directed against a 65-kilodalton (kDa) mycobacterial protein could detect similarly sized antigens in many other bacterial species. In Pseudomonas aeruginosa, the cross-reacting protein corresponded to a 62-kDa antigen that has been called Common Antigen. The mycobacterial 65-kDa antigen and Common Antigen are similar in that both (i) are highly immunoreactive molecules, (ii) contain species-specific and genus-specific epitopes in addition to the broadly cross-reactive epitopes, (iii) can be isolated as homomultimers of greater than 240 kDa, and (iv) have similar amino acid compositions. In Escherichia coli, the cross-reactive protein corresponded to the GroEL protein. Both the GroEL protein and the mycobacterial 65-kDa protein are expressed as heat shock proteins.     

Characterization of the secreted antigens of Mycobacterium bovis BCG: comparison of the 46-kilodalton dimeric protein with proteins MPB64 and MPB70.

Western blot analysis showed that the 46-kilodalton (kDa) dimeric protein antigen secreted in large amounts by some daughter strains of Mycobacterium bovis BCG corresponded to protein MPB70 present in long-term culture filtrates of the Japanese substrain. The 46/23-kDa antigen is the most abundant protein in supernatant from a 5-day culture but is masked by leaked products in old culture supernatants. No similarities were found between the 46-kDa protein and MPB64, a protein with the same strain distribution, or with the antigen of similar molecular mass recognized by monoclonal antibody SA1.D2D.     

Human T-cell clones to the 70-kilodalton heat shock protein of Mycobacterium leprae define mycobacterium-specific epitopes rather than shared epitopes.

The mycobacterial 70-kDa heat shock protein (Hsp70) is a dominant antigen during the human T-cell response to mycobacterial infection despite the conserved sequence with the human homolog. To determine whether this response is pathogen specific, CD4+ T-cell clones were isolated from Mycobacterium leprae Hsp70-reactive individuals. The cytokine profile of the clones was mixed, with all of the clones releasing interferon gamma and half releasing interleukin-4 on stimulation, while six demonstrated cytolytic activity. Five clones reacted with the N-terminal half of the molecule, and the epitopes identified were mycobacterium specific. Residues 241 to 260 were identified by three clones, one of which was restricted by HLA-DR7 (DR7), while a DR1-restricted clone identified residues 71 to 90 and residues 261 to 280 were recognized in the context of DR3. The remaining five T-cell clones reacted with the C-terminal half of the molecule, and the precise position of these epitopes was mapped with 12-mer peptides overlapping by 11 residues. Two of these clones identified overlapping epitopes from residues 411 to 425 and 412 to 428, the latter restricted by DR3. Further epitopes were mapped to residues 298 to 313 restricted by DRw53, residues 388 to 406 restricted by DRw52 or DQ2, and residues 471 to 486 restricted by DR1. The sequences of three epitopes, residues 411 to 425, 412 to 428, and 471 to 486, showed significant identity with the equivalent regions of the prototype human Hsp70. However, when amino acid substitutions that made the sequence more like the human sequence were introduced, the changes were tolerated poorly as measured by proliferation, cytokine production, and cytotoxic potential. Therefore, T-cell recognition of the M. leprae Hsp70 antigen occurs in the context of multiple HLA-DR phenotypes and is exquisitely species specific.     

Analysis of human antibody epitopes on the 65-kilodalton protein of Mycobacterium leprae by using synthetic peptides.

In order to study antibody reactivity to the Mycobacterium leprae 65-kilodalton (kDa) antigen, peptides representing overlapping sequences of the 65-kDa protein were synthesized, and a recombinant protein expression system for r65-kDa was constructed. Mouse monoclonal antibodies and leprosy patient seroreactivity to peptides and r65-kDa were tested by an enzyme-linked immunosorbent assay. All seven of the monoclonal antibodies used in this study reacted with their previously defined epitopes when tested against peptides. All monoclonal antibodies also reacted with r65-kDa. Leprosy patient seroreactivity to peptides and r65-kDa was seen in about one-third of active multibacillary cases. Specimens from patients positive for antibodies to peptides were seen to recognize different epitopes than did mouse monoclonal antibodies used in this study. It is concluded that substantial differences exist between mouse monoclonal antibodies and human leprosy patient reactivity to the 65-kDa antigen and that human seroreactivity to the 65-kDa antigen is indicative of a highly elevated bacillary load.     

Cosmid cloning of Rickettsia prowazekii antigens in Escherichia coli K-12.

Rickettsia prowazekii DNA was partially digested with Sau3A or HindIII, ligated with the cosmid vector pHC79, packaged in vitro, and transduced into Escherichia coli HB101. Cosmid cloning of Sau3A-digested rickettsial DNA yielded 1,288 ampicillin-resistant colonies; 798 cosmid clones resulted with HindIII-digested rickettsial DNA. Chimeric cosmid DNA was extracted from the latter gene bank, digested to completion with HindIII, and compared by agarose gel electrophoresis with a HindIII digest of rickettsial genomic DNA. The two digestion profiles were quite similar in their overall banding patterns, indicating that the clone bank was significantly representative of the rickettsial genome. When both clone banks were screened for expression of rickettsial antigens by enzyme-linked immunosorbent assay with goat anti-R. prowazekii serum, ca. 20% of the clones reacted positively. Two clones were randomly selected for more detailed analysis. Each contained a large chimeric plasmid (40.2 and 38.1 kilobases) which apparently yielded smaller deletion derivatives (13.6 and 12.6 kilobases) when transformed into an E. coli minicell strain. Each recombinant plasmid directed the synthesis of new protein species not observed in control minicells. One of the clones produced a 51,000-dalton protein in minicells, which comigrated with a protein reactive with anti-R. prowazekii serum. This protein was not present in negative controls. When antibodies to this protein were incubated with a Western blot of rickettsial total protein, they bound to a 52,000-dalton polypeptide. Hence, the cloned rickettsial gene product in E. coli corresponds to a protein of similar size in R. prowazekii. This study demonstrates the feasibility of cosmid cloning of rickettsial antigens in E. coli.     

Differential effects of prior exposure to environmental mycobacteria on vaccination with Mycobacterium bovis BCG or a recombinant BCG strain expressing RD1 antigens

In silico analysis reveals that most protective antigens expressed by the antituberculous vaccine Mycobacterium bovis BCG (BCG) are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates cross-reactive immune responses blocking BCG activity. We investigated the impact of sensitization with M. avium, M. scrofulaceum, or M. vaccae on the protective efficacy of a recombinant BCG strain expressing RD1 antigens (BCG::RD1), using a mouse model of experimental tuberculosis (TB). No evidence that the RD1-encoded antigens ESAT-6, CFP-10, and PPE68 were expressed by these environmental strains could be demonstrated by Western blot analysis. Mice sensitized with each of these strains did not prime cellular immune responses cross-reacting with the immunodominant ESAT-6. Importantly, clearance of BCG::RD1 from the lungs and spleens of mice exposed to each of the environmental strains before vaccination was minimal compared to that of BCG. In mice sensitized with M. avium, increased persistence of BCG::RD1 correlated with stronger antimycobacterial gamma interferon responses and enhanced protection against aerosol infection with M. tuberculosis, compared to BCG. In contrast, animals exposed to M. scrofulaceum or M. vaccae prior to vaccination with BCG or BCG::RD1 were better protected against TB than were the unsensitized controls. Our results suggest that the inhibitory effect of environmental mycobacteria on the protective efficacy of BCG depends critically on the extent of cross-recognition of antigens shared with the vaccine. In hosts sensitized with M. avium, potent immunogenicity of ESAT-6 and increased persistence of BCG::RD1 may allow this recombinant vaccine to overcome preexisting antimycobacterial responses.     

T- and B-cell epitopes in the secreted Mycobacterium bovis antigen MPB70 in mice

MPB70 is a soluble secreted protein highly expressed in Mycobacterium bovis and strains of bacille Calmette-Guérin (BCG); as such, it is a candidate for subunit and DNA vaccines against tuberculosis. MPB70 was screened for T-cell epitopes in four different inbred mouse strains. Major histocompatibility complex (MHC) H-2b-expressing mice (C57BL/6) secreted interferon-gamma (IFN-gamma) after stimulation with peptides from the regions 1-20, 41-50, 81-110, 121-150 and 161-193 of the MPB70 sequence. H-2db mouse (B6D2) splenocytes secreted IFN-gamma after stimulation with some of the same peptides, whereas H-2d mice (BALB/c and DBA/2) did not secrete IFN-gamma upon stimulation with the peptides. Sera from H-2db mice immunized with native MPB70 in incomplete Freund's adjuvant (IFA), mpb70 DNA or live BCG Moreau were found to contain antibodies against the native MPB70 antigen. H-2db mice immunized with native MPB70 in IFA exhibited high titres of peptide-reactive immunoglobulin G1 (IgG1) antibodies, whereas DNA-immunized mice reacted with IgG2a antibodies against some of the same peptides. As some of the epitopes recognized by mouse T and B cells have previously been found to stimulate immune responses in humans, cattle and rabbits, we conclude that these epitopes may be good general epitopes for the stimulation of T- and B-cell responses and candidates for a DNA vaccine with a broad applicability.     

Analysis of B-cell epitopes in the variable C-terminal region of the Mycobacterium leprae 70-kilodalton heat shock protein.

The C-terminal region of the Mycobacterium leprae 70-kDa heat-shock protein is the major target for the humoral immune response to this protein and contains M. leprae-specific sequences. To examine B-cell responses to this region more closely, we constructed and expressed a recombinant fragment of the M. leprae P70 gene that encodes the C-terminal 142 residues (C-142) and synthesized a series of 10 overlapping peptides to encompass this region. The affinities of three monoclonal antibodies (MAbs) reactive with this region of P70 were measured, and the binding site of the highest-affinity MAb was determined to lie between residues 498 and 515. This reactivity was confirmed by a fluid-phase inhibition enzyme-linked immunosorbent assay. By contrast, sera from leprosy patients which were strongly reactive with the C-142 fragment failed to bind directly to the conjugated or unconjugated peptides. To determine whether the M. leprae-specific C-terminal 70 residues could stimulate B-cell responses, the reactivity of hyperimmune anti-M. leprae P70 antisera with the peptides was examined. Rabbit polyclonal anti-M. leprae P70 antisera recognized epitopes between residues 498 and 515 and in the M. leprae-specific region between residues 567 and 591. The latter, in turn, when coupled to ovalbumin, was able to generate a strong anti-P70 response specific for mycobacterial, but not human, HSP70. Three strains of mice immunized with either C-142 or P70 recognized epitopes in the region between residues 487 and 532, but the response varied with the strain and immunogen. These data demonstrate that two regions in the C-terminal portion of M. leprae P70 contain linear B-cell epitopes recognized by MAbs or hyperimmune serum. Sera from leprosy patients, however, react predominantly with conformational determinants in the immunodominant C-terminal part of the protein.     

Homology of the 70-kilodalton antigens from Mycobacterium leprae and Mycobacterium bovis with the Mycobacterium tuberculosis 71-kilodalton antigen and with the conserved heat shock protein 70 of eucaryotes.

Two lambda gt11 recombinant clones, JKL2 and JKL15, each containing an insert coding for part of the highly immunogenic 70-kilodalton (kDa) protein antigen, were isolated from a Mycobacterium leprae genomic library by immunoscreening with the monoclonal antibody L7. Clone JKL2 contained the largest insert, 2.3 kilobase pairs. Nonoverlapping fragments of this insert were used as probes and showed strong hybridization to a number of Mycobacterium tuberculosis-lambda gt11 recombinants producing proteins recognized by an anti-M. tuberculosis 71-kDa monoclonal antibody, IT11. One clone from a recombinant Mycobacterium bovis library was also characterized by using L7, and the insert from this clone, B5bt, hybridized strongly to the M. leprae probes as well. The nucleotide sequence of the 1,037-base-pair coding region of the JKL2 M. leprae clone which encodes the carboxy-terminal half of the 70-kDa protein had extensive homology with genes from a number of species. In all cases, these genes, including the recently described Ag63 and Ag361 of Plasmodium falciparum, were found to be members of the heat shock protein 70 (hsp 70) family of genes. At the amino acid level, homology was maximal between amino acids 83 through 107 and 159 through 184, which showed extreme conservation (92 and 85% identity) with Escherichia coli DnaK amino acids 386 through 409 and 460 through 485, respectively, and was 51% homologous over the entire coding region (amino acids 1 through 344 of JKL2). In contrast, amino acids 129 through 158 had maximal homology with the phylogenetically more distant Xenopus laevis hsp70. Homology declined substantially in the carboxy-terminal 34 amino acids. The predicted ATP-binding functional activity of the 70-kDa antigen from M. bovis was confirmed with affinity purification of the antigen by binding to ATP-agarose and elution with ATP. In view of the conservation of sequences between these mycobacterial antigens and mammalian endogenous cellular enzymes, further evaluation of these molecules in vivo may aid in understanding tolerance to self-antigens as well as provide potentially useful immunodiagnostic reagents.