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.     

Ribosomes of Acid-Fast Bacilli: Immunogenicity, Serology, and In Vitro Correlates of Delayed Hypersensitivity

Ribosomal fractions obtained from Mycobacterium bovis (BCG) and M. smegmatis (strain butyricum) were studied to determine their antigenicity, their ability to stimulate the production of soluble mediators of delayed hypersensitivity (in vitro correlates) by sensitized peritoneal exudate cells, and the antigenic relations of ribosomal antigens of BCG to BCG protoplasm and H37Rv culture filtrates. The crude ribosomes and the 50-30S ribosomal subunit pool obtained from each of the organisms induced both delayed and immediate hypersensitivity when injected in incomplete Freund adjuvant into rabbits, and skin reactions could be elicited in sensitized rabbits with those antigens. The crude ribosomes and 50-30S ribosomal subunit pool of M. smegmatis stimulated lymphocytes of guinea pigs sensitized with viable organisms to produce macrophage migration inhibition factor. Comparable ribosomal fractions from BCG bacilli caused lymphocytes of guinea pigs sensitized with viable M. bovis (BCG) to produce skin reactive factor. Immunoelectrophoretic studies showed that H37Rv culture filtrate, protoplasm, crude ribosomes, and 50-30S ribosomal subunits of BCG contain multiple precipitinogens and that many of these were shared between the different antigen systems. Comparative electrophoresis revealed that BCG protoplasm and H37Rv culture filtrate shared a major portion of their components with each other and relatively few with ribosomal systems. The ribosomal systems shared the major portion of their components with each other and relatively few with the other antigen systems.     

Immunotherapy of a guinea pig hepatoma with mycobacterial vaccines: comparison of BCG cell walls and cell wall skeletons.

BCG cell wall skeletons (SK) derived from BCG cell walls (CW) by treatment with proteolytic enzymes and organic solvents were tested for their potency to cause regression of a transplanted guinea pig hepatoma. On a weight basic, SK were as effective as CW in causing tumor regression, and they, as well as purified protein derivative of mycobacteria, provoked delayed cutaneous hypersensitivity reactions in animals immunized with CW or with SK. On a weight basis, CW were more active than SK in eliciting delayed cutaneous hypersensitivity in sensitized guinea pigs whether the animals were immunized with CW or with SK. In unimmunized animals the inflammatory response to intradermally administered CW was greater than that evoked by SK. CW and SK provoked delayed cutaneous hypersensitivity reactions of similar strength in animals immunized with living BCG. This study provided no compelling reasons for using SK instead of CW in clinical trials of cancer treatment by mycobacterial vaccines.     

Immunotherapy of guinea pigs with a transplanted hepatoma: comparison of intralesionally administered killed BCG cells and BCG cell walls.

Heat-killed whole BCG cells (KC) and BCG cell walls (CW) were each tested in emulsified form for their potency to cause regression of a transplanted guinea pig hepatoma. On a weight basis, KC were at least as effective as CW in causing tumor regression and elimination of microscopic lymph node metastasis, and they, as well as purified protein derivative of mycobacteria, provoked delayed cutaneous hypersensitivity reactions in animals immunized with CW or with KC. On a weight basis, KC were as active as CW in eliciting delayed cutaneous hypersensitivity in sensitized guinea pigs whether the animals were immunized with CW or with KC. In unimmunized animals the inflammatory response to intradermally administered KC was similar to that induced by CW. Because KC are easier to prepare than CW, it is suggested that whole killed BCG might be used instead of CW in clinical trials of cancer treatment requiring administration of nonliving mycobacteria.     

Isolation, characterization, and biological properties of a tuberculin-active peptidoglycan isolated from the culture filtrate of Mycobacterium tuberculosis.

A water-soluble tuberculin-active peptidoglycan (TAPG) with a molecular weight of ca. 28,000 to 30,000 was isolated from the culture filtrate of Mycobacterium tuberculosis. TAPG was approximately four to five times more potent than tuberculin purified protein derivative S in guinea pigs sensitized with M. tuberculosis or M. bovis (freeze-dried BCG). It showed little or no cross-reactivity at a dose of 0.1 to 0.4 microgram in guinea pigs sensitized with M. kansasii, M. scrofulaceum, M. intracellulare, or M. avium. TAPG did not show any adjuvant activity when injected in guinea pigs in a water-in-oil emulsion containing ovalbumin. TAPG, in Freund incomplete adjuvant, proved to be an effective immunogen for inducing delayed hypersensitivity in guinea pigs. Chemical analysis of TAPG showed that it contains proline, glutamic acid, alanine, diaminopimelic acid, tyrosine, threonine, glucosamine, and the reducing sugars, arabinose and galactose. In immunoelectrophoretic studies with reference M. tuberculosis H37Rv antiserum, TAPG did not show any precipitin bands.     

Modulation of expression of delayed hypersensitivity by mycobacterial antigen 85 fibronectin-binding proteins.

Although demonstration of delayed hypersensitivity to purified protein derivative of tuberculin (PPD) is an important element in the diagnosis of infection with Mycobacterium tuberculosis, many patients with tuberculosis are anergic. Several possible mechanisms for this specific lack of response have been described. We have now uncovered an additional one. T-cell fibronectin (FN), a lymphokine secreted by activated T cells, is closely associated with the initiation of delayed hypersensitivity reactions. Mycobacterial antigen 85 (Ag85) proteins have been shown to bind to plasma FN. The ability of Ag85 to bind to T-cell FN and modulate expression of delayed hypersensitivity was therefore studied. Purified Ag85 proteins from M. tuberculosis, Mycobacterium bovis BCG, or Mycobacterium kansasii bound to T-cell FN, fibroblast FN, and plasma FN in vitro. Purified 65-kDa heat shock protein (hsp65) from M. bovis BCG did not bind to any FN. Ag85, but not hsp65, inhibited the ability of T-cell FN to agglutinate monocytes in vitro in a dose-dependent manner. In vivo, mixtures of PPD or dinitrophenyl-ovalbumin and purified M. tuberculosis or M. bovis BCG Ag85 proteins elicited significantly smaller delayed hypersensitivity inflammatory reactions in sensitized guinea pigs than did PPD or dinitrophenyl-ovalbumin alone. Purified hsp65 did not inhibit expression of delayed hypersensitivity to PPD or dinitrophenyl-ovalbumin. We suggest that Ag85 proteins could inhibit in vivo expression of delayed hypersensitivity during mycobacterial infections because of their interaction with T-cell FN.     

Effects of diet and genetics on Mycobacterium bovis BCG vaccine efficacy in inbred guinea pigs.

Strain 2 and strain 13 guinea pigs were vaccinated with Mycobacterium bovis BCG and placed on low-protein or protein-adequate diets. Five weeks later all animals were infected by the respiratory route with virulent Mycobacterium tuberculosis H37Rv organisms. Four weeks postchallenge, guinea pigs were skin tested with purified protein derivative and sacrificed. Protein deficiency resulted in significant reductions in body weight and thymus weight and in an impairment in the ability to control the M. bovis BCG vaccine organisms and to mount delayed hypersensitivity reactions. Protein deficiency also adversely affected the efficacy of the BCG vaccine as demonstrated by the numbers of virulent organisms recovered in spleens and lungs. Strain differences were observed in the number of leukocytes, thymus weight, and the responsiveness of blood lymphocytes to purified protein derivative stimulation. In general, strain 13 guinea pigs responded more dramatically to dietary insult than did their strain 2 counterparts. Protein deprivation completely abolished BCG vaccine protection in the lungs and spleens of strain 13 animals and significantly reduced the protection afforded to strain 2 animals. In both strains, the BCG vaccine protected normally nourished guinea pigs. There was no significant difference between strains with respect to susceptibility to pulmonary infection with virulent mycobacteria. Thus, diet and genetic pedigree each had a significant influence on BCG vaccine efficacy.     

Mapping of the delayed-type hypersensitivity-inducing epitope of secreted protein MPT64 from Mycobacterium tuberculosis.

The gene encoding the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was expressed in Escherichia coli K-12 and purified as a recombinant protein. The purified recombinant MPT64 elicited delayed-type hypersensitivity (DTH) in outbred guinea pigs sensitized with Mycobacterium bovis BCG Tokyo. The skin reactions were comparable to those obtained with native MPT64. No skin reactions were observed when either recombinant MPT64 or native MPT64 was used in guinea pigs sensitized with M. bovis BCG Danish 1331. Amino- and carboxy-terminal deletion mutants of MPT64 were purified as fusion proteins for the mapping of DTH-inducing epitopes on recombinant MPT64 by use of the guinea pig skin test model. The part of the molecule responsible for the biological activity was located at the carboxy-terminal end. Further studies with overlapping synthetic peptides have pinpointed the biological activity at a single DTH-inducing epitope consisting of 15 residues between amino acids Gly-173 and Ala-187. Screening by PCR of 56 clinical isolates of M. tuberculosis from Danish and Tanzanian patients demonstrated the presence of mpt64 in all of the strains. These results point to MPT64 as a possible candidate for a skin test reagent specific for diagnosis of human tuberculosis.     

Delayed Hypersensitivity Reactions Provoked by Ribosomes from Acid-Fast Bacilli I. Ribosomal Isolation, Characterization, Delayed Hypersensitivity, and Specificity

Ribosomes and ribosomal subunits of Mycobacterium bovis (strain BCG) and M. smegmatis have been isolated and employed as skin test antigens in guinea pigs sensitized with homologous or heterologous organisms. Ribosomes and ribosomal subunits were found to be potent antigens for skin test purposes, and the 30S subunits were found to be more specific and active than the 50S subunits.     

Comparative studies with various substrains of Mycobacterium bovis BCG on the production of an antigenic protein, MPB70.

A protein, isolated and purified from the unheated culture filtrate of Mycobacterium bovis BCG (substrain Tokyo 172) and designated MPB70, elicited a delayed skin reaction in guinea pigs sensitized with viable cells of BCG but not in those sensitized with heat-killed cells. The skin reaction reached the maximum 4 to 8 weeks after the inoculation of the BCG and then decreased gradually, resulting in conversion to negative after 20 weeks, whereas the skin reaction to purified protein derivative (PPD) continued to be positive. Guinea pigs immunized with viable cells of various substrains of BCG were skin tested with MPB70 and PPD. Guinea pigs immunized with the BCG substrain Tokyo 172 and the substrain Moreau (Brazil) showed strong delayed skin reactions to both MPB70 and PPD. On the other hand, guinea pigs immunized with the Pasteur substrain 1173P2, the Glaxo substrain 1077, the Copenhagen substrain 1331, the Tice substrain, or the Beijing substrain 64-42 showed negative skin reactions to MPB70, whereas they were strongly positive to PPD. In a two-dimensional acrylamide gel electrophoretic analysis of proteins from the culture filtrates of the BCG substrains, the culture filtrates of the Tokyo and Moreau substrains showed the spot of MPB70 on the gel slabs, whereas those of the other BCG substrains did not.     

Biological Activity in Sensitized Guinea Pigs of MPB 70, a Protein Specific for Some Strains of Mycobacterium bovis BCG

MPB 70 is a protein found in large quantities in the culture filtrate (CF) of the Tokyo and some other strains of Mycobacterium bovis BCG, and it has a remarkable degree of specificity for these strains. We estimated the molecular weight of MPB 70 to 22,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and immunoblotting showed that MPB 70 was present in high quantities in CF from BCG Tokyo, that it could be also demonstrated in BCG Copenhagen, and that it was absent in CF from M. tuberculosis H37Rv. When the purified MPB 70 preparation used in the present study was run in SDS-PAGE, blotted and stained with a polyclonal rabbit or a monoclonal mouse anti-MPB 70 antibody, several bands in addition to the main 22 kDa band were seen, indicating a tendency of the MPB 70 molecules and/or fragments thereof to form very stable aggregates with themselves. The biological activity of MPB 70 was studied in groups of guinea pigs sensitized with live BCG of the Tokyo and Copenhagen strains. Guinea pigs from both groups developed reactivity to tuberculin PPD as assessed by skin tests and lymphocyte stimulation tests with peripheral blood or lymph node lymphocytes. In addition, a strong and persistent reactivity to MPB 70 was demonstrated in the BCG Tokyo group with both methods. Guinea pigs sensitized with the Copenhagen strain were only weakly reactive to MPB 70. Skin reactions in guinea pigs that had been repeatedly tested with MPB 70 and tuberculin were compared with reactions in animals tested only once. Reactions to MPB 70 in BCG Tokyo sensitized guinea pigs were suppressed by repeated tests, whereas tuberculin reactions were boosted by the interim tests. The levels of specific anti-MPB 70 antibodies were higher in BCG Tokyo- than in BCG Copenhagen-sensitized guinea pigs. MPB 70 has a high degree of specificity and is a strongly immunogenic protein, which may prove useful in studies of mycobacterial immunology.     

Cell-mediated immunity in malnourished guinea pigs after Mycobacterium bovis BCG vaccination.

Specific-pathogen-free guinea pigs were vaccinated with viable Mycobacterium bovis BCG and maintained on purified, isocaloric diets containing either 30% or 7.5% casein, or commercial chow. At intervals of 4, 5, 6, and 8 weeks postvaccination, groups of guinea pigs from each diet treatment were skin tested with purified protein derivative and killed. Protein-deficient animals exhibited progressive reductions in total serum proteins and albumin. Significantly greater numbers of viable M. bovis BCG were recovered from the vaccination site and inguinal lymph nodes of protein-deficient guinea pigs at all intervals. In contrast, the development of delayed hypersensitivity was markedly retarded in the 7.5% casein group and was also reduced somewhat in the 30% casein group as compared to chow control. Peripheral blood lymphocytes from protein-deficient animals did not respond normally in vitro to a polyclonal T cell mitogen, phytohemagglutinin. These results demonstrate that protein-calorie malnutrition in this model impairs the development of cell-mediated immunity as evidenced by skin test anergy, lymphocyte hyporesponsiveness, and failure to control levels of viable M. bovis BCG after vaccination.     

Immunobiology and species distribution of Mycobacterium tuberculosis antigen 5.

The immunobiology and mycobacterial species distribution of immunoabsorbent affinity chromatography-purified Mycobacterium tuberculosis antigen 5 have been studied. In delayed hypersensitivity skin tests, antigen 5 was nearly equipotent with tuberculin-purified protein derivative in sensitized guinea pigs. In vitro, antigen 5 was capable of stimulating the production of migration inhibitory factor by cultured lymphocytes from sensitized guinea pigs and humans. Antigen 5 stimulated thymidine incorporation by cultured guinea pig lymphocytes but did not stimulate thymidine incorporation by cultured human lymphocytes. Although erythrocytes were readily sensitized with antigen 5 for passive hemagglutination, their use did not offer any advantage over previous hemagglutination techniques for the serodiagnosis or evaluation of patients with tuberculosis. By immunoelectrophoresis and immunodiffusion, antigen 5 was readily identified in culture filtrates of 10 strains of M. tuberculosis and M. bovis but not in those of 30 strains of 12 other myobacterial species.     

Delayed hypersensitivity to hapten-skin protein conjugates in guinea pigs sensitized to benzo(a)pyrene

Guinea pigs were sensitized to 3,4-benzo(a)pyrene, by epicutaneous application or by footbad injection in Freund's complete adjuvant. Conjugates of benzo(a)pyrene and guinea pig skin protein formed by ultraviolet radiation could elicit cutaneous delayed hypersensitivity and could inhibit the migration of macrophages obtained from guinea pigs sensitized to the carcinogen. Extracts of benzo(a)pyrene-treated guinea pig skin and conjugates formed in vitro with benzo(a)pyrene isocyanate were unable to consistently elicit delayed hypersensitivity reactions in vivo or in vitro. The results indicate a high degree of hapten-carrier specificity to contact sensitivity to benzo(a)pyrene.     

Purification, characterization and identification of a 32 kDa protein antigen of Mycobacterium bovis BCG

An immunogenic protein called P32 has been purified from Sauton zinc deficient culture filtrate of Mycobacterium bovis BCG using successively hydrophobic chromatography on Phenyl-Sepharose, ion exchange on DEAE-Sephacel and molecular sieving on Sephadex G-100. The final preparation was found to be homogeneous as based on several analyses. This P32 protein was a constituent of BCG cells grown in normal conditions. It represented about 3% of the soluble fraction of a cellular extract, and appeared as the major protein released in normal Sauton culture filtrate. This protein was found to have a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis and in molecular sieving. Its amino acid composition showed an abundance of acidic amino acids (or their amides). The NH2-terminal amino acid sequence (6 amino acids) was determined. Purified P32 was tested by various crossed immunoelectrophoresis techniques, and was shown to belong to the antigen 85 complex in the reference system for BCG antigens. It was more precisely identified as antigen 85A. The protein antigen elicited a weak delayed hypersensitivity reaction in guinea pigs sensitized with heat-killed or living BCG. No delayed hypersensitivity reaction was observed in living BCG sensitized mice, however, it induced significant amounts of gamma interferon in cultured spleen cells from BCG-sensitized mice. Moreover, P32 either pure or as part of BCG soluble extract promoted substantial antibody levels when injected in rabbits.     

Mycobacterium bovis BCG vaccine fails to protect protein-deficient guinea pigs against respiratory challenge with virulent Mycobacterium tuberculosis.

Specific-pathogen-free Hartley guinea pigs were maintained on isocaloric-purified diets either adequate (30%) or moderately deficient (10%) in protein. Half of each diet group was vaccinated with viable Mycobacterium bovis BCG. Six weeks later, all animals were challenged by the respiratory route with virulent Mycobacterium tuberculosis H37Rv. At intervals of 1, 2, and 3 weeks postchallenge, guinea pigs from each diet and vaccination group were skin tested with tuberculin and sacrificed. Protein deficiency resulted in loss of tuberculin hypersensitivity. Vaccination with M. bovis BCG protected control animals, as determined by significant reductions in the number of M. tuberculosis H37Rv organisms recovered from lungs, spleen, and bronchotracheal lymph nodes 2 and 3 weeks postchallenge. Based upon the same criteria, the degree of protection afforded protein-deficient animals by M. bovis BCG vaccine ranged from partial (spleen and lymph nodes) to none at all (lungs). Approximately the same numbers of tubercle bacilli were recovered from nonvaccinated guinea pigs in both diet groups. Protein deficiency appears to impair M. bovis BCG-induced immunity while not affecting primary pulmonary infection with virulent M. tuberculosis.     

A 35-kilodalton protein is a major target of the human immune response to Mycobacterium leprae.

The control of leprosy will be facilitated by the identification of major Mycobacterium leprae-specific antigens which mirror the immune response to the organism across the leprosy spectrum. We have investigated the host response to a 35-kDa protein of M. leprae. Recombinant 35-kDa protein purified from Mycobacterium smegmatis resembled the native antigen in the formation of multimeric complexes and binding by monoclonal antibodies and sera from leprosy patients. These properties were not shared by two forms of 35-kDa protein purified from Escherichia coli. The M. smegmatis-derived 35-kDa protein stimulated a gamma interferon-secreting T-cell proliferative response in the majority of paucibacillary leprosy patients and healthy contacts of leprosy patients tested. Cellular responses to the protein in patients with multibacillary leprosy were weak or absent, consistent with hyporesponsiveness to M. leprae characteristic of this form of the disease. Almost all leprosy patients and contacts recognized the 35-kDa protein by either a T-cell proliferative or an immunoglobulin G antibody response, whereas few tuberculosis patients recognized the antigen. This specificity was confirmed in guinea pigs, with the 35-kDa protein eliciting strong delayed-type hypersensitivity in M. leprae-sensitized animals but not in those sensitized with Mycobacterium tuberculosis or Mycobacterium bovis BCG. Therefore, the M. leprae 35-kDa protein appears to be a major and relatively specific target of the human immune response to M. leprae and is a potential component of a diagnostic test to detect exposure to leprosy or a vaccine to combat the disease.     

Recombinant early secreted antigen target 6 protein as a skin test antigen for the specific detection of Mycobacterium tuberculosis infection

Although the delayed-type hypersensitivity skin test reaction to tuberculin purified protein derivative (PPD) is used worldwide for tuberculosis (TB) detection, it is incapable of distinguishing Mycobacterium tuberculosis (MTB) infection from bacille Calmette-Guérin (BCG) vaccination or infection with non-tuberculous Mycobacteria. As a result, there is an urgent need for a more specific diagnostic tool for TB. This study reports the skin reactions of guinea pigs and human volunteers to recombinant early secreted antigen target 6 (rESAT6), a secretory protein found only in MTB, M. bovis and few other mycobacterial species. These volunteers had varying histories of BCG vaccination and exposure to MTB, allowing us to determine the specificity of their response to TB exposure. Our results show that 1.0 microg of the purified MTB rESAT6 antigen elicited a positive skin response in both animals and humans exposed to MTB, as well as in animals exposed to M. bovis and M. marinum, all species of Mycobacteria that contain the gene for early secreted antigen target 6 (ESAT6). ESAT6 appears to be more specific to MTB infection than PPD, as demonstrated by the fact that we saw no skin responses in the BCG-vaccinated volunteers, nor in the guinea pigs sensitized with BCG vaccine, or with Mycobacteria that do not contain the gene encoding ESAT6. We believe that this is the first report of the use of a rESAT6 protein in a skin test in human volunteers, and that these data support its use in the specific detection of MTB infection.     

Delayed-Type Hypersensitivity Responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the Guinea Pig

Two antigens from Mycobacterium tuberculosis, ESAT-6 and MPT64, elicited delayed-type hypersensitivity (DTH) skin responses in outbred guinea pigs infected with M. tuberculosis by the aerosol and intravenous routes but not those sensitized with M. bovis BCG or M. avium. The DTH epitope of ESAT-6 was mapped to the C terminus. Nonresponders to the individual antigens were found, but all animals responded to a combination of ESAT-6 and MPT64 or their respective minimal target peptides. Correspondingly, these molecules could form the basis of a new skin test for tuberculosis.     

Preparation of various fractions from Mycobacterium smegmatis, their arthritogenicity and their preventive effect on adjuvant disease.

Cell walls of Mycobacterium smegmatis were able to produce much more severe arthritis in rats than the delipidated cells, whereas cell envelope and cell membrane fractions were unable to produce the disease. The lysozyme-solubilized product was able to produce mild disease with only 30% of incidence with an optimum dose, whereas the higher and the lower doses did not produce the disease. The rats immunized with cell envelope, cell membrane fraction and nonarthritogenic doses of lysozyme-solubilized product were protected against the subsequent homologous and heterologous challenge of delipidated cells. It was discussed that this preventative effect can be the result of antigenic competition between the arthritogenic and nonarthritogenic components of M. smegmatis. On the other hand, all the fractions separated here were able to serve as an immunoadjuvant in terms of inducing delayed hypersensitivity to ovalbumin in guinea pigs.