Serology and leprosy: immunoassays comparing immunoglobulin G antibody responses to 28- and 30-kilodalton proteins purified from Mycobacterium bovis BCG.

Two major proteins from Mycobacterium bovis BCG culture filtrates with molecular masses of 28 kDa (P28) and 30 kDa (P30), identified as components of the BCG 85 complex, were purified and used in enzyme-linked immunosorbent assays (ELISAs) for the determination of specific immunoglobulin G (IgG) levels in patients with leprosy or tuberculosis or with exposure to these diseases. High reactivity to both antigens was observed with sera from lepromatous leprosy patients, whereas antibody levels in sera from paucibacillary leprosy patients were not significantly different from those in sera from healthy individuals from an area in which leprosy is endemic. High IgG responses were also found in some contacts of lepromatous leprosy patients. A comparison of the levels of anti-P28 and anti-P30 within the multibacillary leprosy patient group showed much higher IgG reactivity to P28 than to P30, suggesting that the antibody response of lepromatous patients is directed predominantly against the 28-kDa protein. A high degree of correlation in values of ELISAs based on P28 and on the phenolic glycolipid of Mycobacterium leprae was observed in all groups analyzed. The potential use of an assay based on the 28-kDa protein to selectively distinguish individuals destined to develop multibacillary leprosy is discussed, as also is the likelihood that the 28-kDa-30-kDa complex, part of the fibronectin-binding family, is an important component of M. leprae.     

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.     

Serological responses of patients with lepromatous and tuberculoid leprosy to 30-, 31-, and 32-kilodalton antigens of Mycobacterium tuberculosis.

Sera from patients with lepromatous and tuberculoid leprosy were examined in immunoblot assays for antibodies to Mycobacterium tuberculosis culture filtrate antigens. Antibodies to 30- and 31-kilodalton proteins were present in 88 and 81%, respectively, of 16 patients with lepromatous disease and absent in 16 patients with tuberculoid disease. Antibodies to a 32-kilodalton protein were found in 12 and 38% of lepromatous and tuberculoid patients, respectively. These reactivities may be useful for distinguishing lepromatous and tuberculoid leprosy.     

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.     

Immunological evaluation of a component isolated from Mycobacterium bovis BCG with a monoclonal antibody to M. bovis BCG.

A component of Mycobacterium bovis BCG referred to as BCG-a was isolated through the combined use of monoclonal antibody directed to BCG and affinity chromatography. Analysis of BCG-a by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single prominent band with a molecular weight of ca. 10,000. Structural characterization of BCG-a consisting of amino acid composition and amino-terminal sequence determination was carried out. The intact BCG-a antigen was bound by neither the lectin from common lentils nor concanavalin A, implying that BCG-a does not carry any asparagine-linked oligosaccharides. Immunoprecipitation of 125I-labeled BCG-a with polyclonal and monoclonal antibodies directed against BCG resulted in bands having the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as did free 125I-BCG-a. In radioimmunoassays 125I-BCG-a was bound by the monoclonal antibody and by polyclonal antibodies from rabbits that had been immunized to BCG and to Mycobacterium tuberculosis H37Rv. Antibodies to nontuberculous and to nonacid-fast bacteria bound BCG-a poorly or not at all. The binding of 125I-BCG-a by the monoclonal antibody was readily inhibited by extracts of BCG and H37Rv, but it was not as readily inhibited by extracts of nontuberculous mycobacteria and was not at all inhibited by extracts of nonacid-fast bacteria. Considerable inhibition was similarly observed by surface antigens of nonviable, intact BCG organisms. Delayed cutaneous hypersensitivity reactions to small concentrations of BCG-a were elicited in guinea pigs that had been immunized with BCG or H37Rv antigens, but such reactions were not elicited in unimmunized animals.     

Specific skin-reactive protein from culture filtrate of Mycobacterium bovis BCG.

A highly purified protein, named MPB70, was isolated from the culture filtrate of Mycobacterium bovis BCG. This protein accounted for more than 10% of the proteins secreted into the culture medium. MPB70 was purified by precipitation with ammonium sulfate, followed by treatment with diethylaminoethyl ion exchanger, with or without 3 M urea, and by gel filtration. The final MPB70 preparation was homogenous as judged by several analyses. The molecular weight was estimated to be 18,000 by electrophoresis or molecular sieve and 15,100 by sedimentation equilibrium. The preparation did not contain sugars. The amino acid composition did not include cysteine or tryptophan. In skin reaction, MPB70 was a strictly BCG-specific antigen and, among the guinea pigs sensitized with the heat-killed cells of the various species of mycobacteria--Mycobacterium tuberculosis strains H37Rv and Aoyama B, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium phlei, and BCG, it elicited a delayed cutaneous reaction only in the guinea pigs sensitized with BCG. The potency of MPB70 in the skin reaction was about one-twentieth of the standard purified protein derivative.     

The immune response to the cell wall of Mycobacterium bovis BCG.

Mice were immunized with the cell wall of BCG suspended in an oil-in-saline emulsion, and examined against time for the emergence of T cell-mediated acquired immunity. Evidence is presented that shows that levels of acquired resistance expressed in these animals over the first month following inoculation, and which enabled them to substantially resist an intravenous challenge infection with Mycobacterium tuberculosis, were completely nonspecific in nature, in that they were equally well expressed in normal and T cell-deficient mice, and were present at a time when no protective T cell activity could be passively transferred from the inoculated host. Paradoxically, in contrast, weak but statistically significant protective immunity could be detected in the spleens of CW-immunized mice approximately 3 months after inoculation, at a time when the donor animals were devoid of resistance to rechallenge. Finally, evidence is presented that shows that the CW material, if given subcutaneously, is highly immunogenic for the generation of delayed-type hypersensitivity effector T cells; however, these cells do not themselves contribute to protective immunity.     

T cell response to purified filtrate antigen 85 from Mycobacterium bovis Bacilli Calmette-Guérin (BCG) in leprosy patients.

Precipitating autoantibodies to the URNP particles were used to select 80 patients, and were further characterized by immunoblotting and quantitative ELISA. These immunochemical results have been related to clinical diagnosis, the frequency of nephritis, and Raynaud's phenomenon. Autoantibodies to the 70-kD polypeptide of the U1RNP particle were present in 16 out of 19 patients with mixed connective tissue disease (MCTD) and in 27 out of 61 patients with systemic lupus erythematosus (SLE). The ratio of anti-U1RNP/Sm by ELISA and the frequency of antibody to 70-kD protein were directly related to the frequency of Raynaud's phenomenon and inversely related to the frequency of nephritis.     

The etiologic agents of leprosy and tuberculosis share an immunoreactive protein antigen with the vaccine strain Mycobacterium bovis BCG.

The amino acid sequences of the 65-kilodalton antigens of Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis BCG display greater than 95% homology.     

MPB59, a widely cross-reacting protein of Mycobacterium bovis BCG

The MPB59 protein of Mycobacterium bovis BCG was purified to homogeneity from culture fluid of BCG substrain Tokyo, and characterized by biochemical and immunological techniques. The molecular weight was 28,000, determined by SDS-polyacrylamide gel electrophoresis, and the pI value was 5.3. The N-terminal amino acid sequence was determined for 32 steps and showed no significant homology with MPB64, MPB70 or MPB80. By crossed immunoelectrophoresis, MPB59 was found to belong to the BCG antigen 85 complex and identified as corresponding to the 85B component of this complex. The protein cross-reacted extensively with other species of mycobacteria, and induced a marked humoral immune response in armadillos and monkeys during development of systemic mycobacterial infection after inoculation with Mycobacterium leprae.     

Lymphocyte response of leprosy patients to human-derived and purified armadillo-derived Mycobacterium leprae, BCG and PPD.

The lymphocyte transformation test was applied to compare in vitro lymphocyte responses of tuberculoid (high resistant) and lepromatous (low resistant) leprosy patients to purified Mycobacterium leprae derived from experimentally infected armadillos and crude M. leprae derived from man, as well as to bacille Calmette-Guérin (BCG) and purified protein derivative (PPD). It was found that the purification procedure using enzymic digestion did not affect the immunogenicity of armadillo-derived M. leprae as compared with the crude human-derived preparation, although 2.5-5-fold higher doses of the purified organisms were required to elicitate equivalent lymphocyte responses. The result indicated the suitability of purified armadillo-derived M. leprae as the standard antigen for lymphocytes transformation tests in leprosy. The cross-reactivity studies show a close relationship between PPD and BCG, but not between M. leprae and PPD or BCG.     

Immunological properties of ribosomal proteins from Mycobacterium bovis BCG.

Two proteins with molecular mass 65 kDa, a heat shock protein, and an S1-like protein were found in a 30S ribosomal subunit from Mycobacterium bovis BCG. The 17-kDa protein in the 30S subunit was homologous to alpha-crystallin heat shock protein, and the 16-kDa protein in the 50S subunit was homologous to the L7/L12 protein. The latter provoked a strong delayed-type hypersensitivity reaction in the sensitized guinea pigs. The GroES-like protein (12 kDa) loosely associated with ribosomes.     

Identification of a Mycobacterium bovis BCG 45/47-kilodalton antigen complex, an immunodominant target for antibody response after immunization with living bacteria.

Increased protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by a previous immunization with living avirulent mycobacteria, usually Mycobacterium bovis BCG. Only a transient and marginal protection is obtained after immunization with bacterial extracts or dead bacteria. Both living and heat-killed bacteria share a number of common antigens. In order to identify mycobacterial molecules which are dominant antigens during immunization with living bacteria, a two-step selection method was used. Two groups of guinea pigs were immunized either with living or with heat-killed BCG. Sera were then collected and used to select and counterselect antigens present in BCG culture filtrates. Each major fraction eluted from a series of high-pressure liquid chromatography columns (gel filtration, DEAE, and reverse-phase chromatography) was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on polyvinylidene difluoride sheets. The molecules present on twin immunoblots were stained with antibodies raised in guinea pigs immunized either with living or with heat-killed BCG. Cross-reactive antigens stained in twin immunoblots were eliminated. Major antigens interacting with antibodies raised after immunization only with living bacteria were further purified. A complex of 45- and 47-kDa major molecules (45/47-kDa complex) was thus identified and further purified. The complex was found to interact only with antibodies present in sera of guinea pigs immunized with living bacteria and not at all with antibodies raised after immunization with dead bacteria. The 45/47-kDa antigen complex molecules were resolved on two-dimensional electrophoresis in three major and seven minor proteins detected with silver staining. All the molecules interacted with the antibodies present in sera of guinea pigs immunized with living BCG. The three major proteins (two at 47 kDa and one at 45 kDa) were amino-terminal sequenced. The sequence A-P-E-P-A-P-P-V-P-P-A-A-A-A-P-P-A, which was not previously reported, was the same for these three molecules. By using a competitive enzyme-linked immunosorbent assay, the concentrations of the 45/47-kDa antigen complex were measured in BCG culture filtrates, freeze-dried BCG, and dried heat-killed BCG; they were, respectively, 2, 0.01, and 0.001% of the total mass. The low or very low values compared with the high antibody concentration emphasized the ability of the 45/47-kDa complex delivered through live BCG to trigger an antibody response.     

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.     

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.     

Humoral responses against the 85A and 85B antigens of Mycobacterium bovis BCG in patients with leprosy and tuberculosis.

Immunoglobulin G antibodies against the 85A and 85B components of the Mycobacterium bovis BCG antigen 85 complex separated by isoelectric focusing were investigated in serum samples from 129 patients representing the major forms of leprosy, 111 tuberculous patients, and 153 healthy subjects. For both of the antigens, a higher degree of staining was observed for lepromatous leprosy patients and patients with active tuberculosis than for the other groups. Because sera from some healthy subjects recognized the 85A antigen, we suggest that antigen 85B is the most useful component of the antigen 85 complex for the serodiagnosis of the multibacillary forms of leprosy or of the active forms of tuberculosis.     

Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids.

Profile analysis of mycolic acid ester patterns of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis bacillus Calmette-Gúerin (BCG) using high-performance liquid chromatography indicated that separation of BCG from M. tuberculosis and M. bovis by elution and relative retention times is possible. Mycolic acid patterns of BCG eluted from the column 0.5 min before M. tuberculosis or M. bovis, resulting in relative retention times for two peaks not seen in the pattern of M. tuberculosis or M. bovis. Identification was confirmed by phage typing, which has been the standard procedure for confirmation of BCG strains. These results showed that high-performance liquid chromatographic analysis of mycolic acid esters can be used in the mycobacterial reference laboratory for separation of BCG from M. tuberculosis and M. bovis.     

Preparation and properties of antigen 60 from Mycobacterium bovis BCG.

Antigen 60 (A60) is the main thermostable immunogen of both 'old tuberculin' (OT) and 'purified protein derivative' (PPD), known reagents for cutaneous tests in tuberculosis. It is recognized by bidimensional immunoelectrophoresis with anti-BCG antiserum, where it appears as the less mobile component. A60 was prepared from the cytoplasm of Mycobacterium bovis BCG, and purified by exclusion gel chromatography and lectin affinity chromatography. Labelled A60 was obtained by radioiodination and used for a radioimmunoassay. Composition of A60 was explored by use of organic solvents, chemicals and enzymes. It contained two fractions of free and bound lipids, as well as protein and polysaccharide moieties. After removal of both free and bound lipid fractions, the core still retained the ability to form immunoprecipitinogen lines with anti-BCG antiserum. The lipopolysaccharide and lipo-protein moieties of A60, as well as the free lipid fraction, were also complexed by antibodies. It is concluded that A60 is a lipopolysaccharide-protein complex of 10(6) to 10(7) daltons, which is a major immunogenic component of mycobacterial cytoplasm. The detailed structure of this antigen, its immunological properties, and its use for an ELISA type immunoassay for tuberculosis are described in two other publications.     

Granulomagenic activity of serologically active glycolipids from Mycobacterium bovis BCG.

The granulomagenic properties of serologically active glycolipids A1, B2, B3, and C isolated from Mycobacterium bovis BCG were studied. Glycolipid A1, dissolved in olive and injected intradermally in guinea pigs, was able to elicit a granulomatous response that seemed to be of the nonallergic type. This granulomagenic activity was quite striking since only 2 mug was necessary to elicit the reaction. The B and C glycolipids were milder granulomagenic agents. Glycolipid A1, dissolved in olive oil and injected intraperitoneally, was toxic for mice. Mice lost weight after the injection of as little as 10 mug of A1, although not even a dose of 100 mug was lethal. Glycolipid A1 failed to immunize mice against aerogenic infection with virulent tubercle bacilli.     

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.