Immunological activity of a 14-kilodalton recombinant protein of Mycobacterium tuberculosis H37Rv.

A 14-kilodalton peptide antigen from Mycobacterium tuberculosis was isolated from an Escherichia coli lambda gt 11 recombinant DNA clone and was identified by Western blotting (immunoblotting) with monoclonal antibody TB68. Immunization of mice and guinea pigs with the recombinant peptide (rTB68) induced in vitro lymphoproliferative responses in draining lymph node lymphocyte cultures as well as in vivo delayed-type hypersensitivity reactions. Moreover, rTB68 was found both to induce and to cross-react with Mycobacterium leprae immune lymphocytes, but did not generate protective effects against live M. leprae challenge in mice. These findings showed that a 14-kilodalton peptide which has been characterized as specific for M. tuberculosis on the basis of B-cell recognition was capable of generating cell-mediated immune responses and moreover contained T-cell epitopes which were cross-reactive with M. leprae antigens.     

Comparison of the Immunological Activity of Five Defined Antigens from Mycobacterium tuberculosis in Seven Inbred Guinea Pig Strains. The 38-kDa Antigen is Immunodominant

We have examined the immunological activity of five affinity-purified protein antigens from Mycobacterium tuberculosis in seven inbred and one outbred guinea pig strains. The test systems were measurements of delayed-type hypersensitivity (Dth) responses, lymphocyte stimulation assays (LS), and antibody response measurements. The results showed significant differences in the immunogenicity of the single-protein antigens and, when the antigens were considered separately, highly significant guinea pig strain differences. The outbred guinea pig strain behaved as a Dth high responder to all antigens studied. The order of magnitude of the Dth responses was not usually correlated with that of the corresponding antibody responses for the individual guinea pig strain-antigen combinations. In particular, when compared with the other strains, strain 2 guinea pigs generally gave the lowest Dth, but the highest antibody responses. A 38,000 molecular weight protein, possessing M. tuberculosis complex-specific B-cell determinants, appeared immunodominant in 5 out of 7 strains. Our Dth data in the inbred strains further suggest the presence of an M. tuberculosis-specific T-cell epitope. A T-cell line, 11D9, derived from the high-responder guinea pig strain 13 reactive to this protein, was shown to be able to confer a tuberculin-like skin reaction in vivo. LS assays with recombinant 38-kDa protein and truncated versions of the protein mapped the 11D9-defined T-cell epitope to the middle part of the molecule.     

Strain variations in the murine cellular immune response to the phenolic glycolipid I antigen of Mycobacterium leprae.

The cellular immune response to the Mycobacterium leprae-specific phenolic glycolipid I was examined in inbred mice immunized with M. leprae by in vivo delayed cutaneous hypersensitivity and in vitro lymphocyte proliferation. Whereas all mouse strains responded to M.leprae-induced delayed-type hypersensitivity and lymphocyte proliferation, only BALB.K was responsive in both assays to the glycolipid. Responsiveness was determined in part by non-H-2 genes, while the influence of H-2 genes was not apparent. Among congenic BALB/c mice differing only at Igh-C allotype loci, variations in responsiveness were found in both delayed-type hypersensitivity and lymphocytes proliferation assays, indicating a possible role for Igh-C loci-linked genes. Unresponsiveness in the lymphocyte proliferation assay to the glycolipid was inherited as a dominant trait in one set of responder X nonresponder F1 progeny. We conclude that after immunization with M. leprae organisms, the cell-mediated responses to the glycolipid, endowed with a single carbohydrate epitope, are under polygenic control, predominantly non-H-2-linked genes.     

Adoptive cell transfer of resistance to Mycobacterium leprae infections in mice.

Cells were transferred from mice intradermally vaccinated with killed Mycobacterium leprae to sublethally irradiated recipients. Unseparated cells from lymph nodes or spleens of M. leprae vaccinated mice were found to cause significant inhibition of the growth of a subsequent M. leprae challenge in mouse footpads for up to 26 weeks after vaccination. Vaccination with live BCG and cells transferred from BCG-vaccinated mice caused no significant inhibition of M. leprae growth in mouse footpads. Cell separation into fractions containing predominantly B and T lymphocytes showed that the inhibition of growth was due to M. leprae-sensitized T lymphocytes. M. leprae vaccinated mice were also skin tested with soluble M. leprae antigen and showed maximum delayed hypersensitivity responses 4 weeks after vaccination.     

Immunotherapy of experimental cancer by intralesional injection of emulsified nonliving mycobacteria: comparison of Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis.

Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis were each tested in emulsified form for their potency to cause regression of transplants of a syngeneic murine fibrosarcoma and of a syngeneic guinea pig hepatoma. On a weight basis, M. phlei and M. smegmatis were as effective as BCG in causing tumor regression. M. phlei and M. smegmatis were comparable to BCG in provoking delayed cutaneous hypersensitivity reactions in guinea pigs sensitized to M. phlei or M. smegmatis. In BCG-sensitized guinea pigs, M. phlei and M. smegmatis provoked weaker delayed cutaneous hypersensitivity reactions than did BCG. Purified protein derivative of M. tuberculosis was more active in eliciting delayed cutaneous hypersensitivity in BCG-sensitized guinea pigs than in animals sensitized with M. phlei or M. smegmatis.     

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.     

Vaccination of mice against Mycobacterium leprae infection.

Intradermal immunization with killed Mycobacterium leprae renders mice immune to infection with viable M. leprae. This protection is long lasting and systemic in that immunization in the left flank results in protection in both the left and right footpads. Immunization with Mycobacterium vaccae was ineffective in protecting mice against M. leprae infection, while Mycobacterium bovis BCG provided partial protection. Mycobacterium habana TMC 5135 (now known as Mycobacterium simiae) was found to be as effective as M. leprae in protecting mice against footpad infection.     

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.     

Two-Dimensional Electrophoresis for Analysis of Mycobacterium tuberculosis Culture Filtrate and Purification and Characterization of Six Novel Proteins

Culture filtrate from Mycobacterium tuberculosis contains molecules which promote high levels of protective immunity in animal models of subunit vaccination against tuberculosis. We have used two-dimensional electrophoresis for analysis and purification of six novel M. tuberculosis culture filtrate proteins (CFPs): CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28. The proteins were tested for recognition by M. tuberculosis-reactive memory cells from different strains of inbred mice and for their capacity to induce a skin test response in M. tuberculosis-infected guinea pigs. CFP17, CFP20, CFP21 and CFP25 induced both a high gamma interferon release and a strong delayed-type hypersensitivity response, and CFP21 was broadly recognized by different strains of inbred mice. N-terminal sequences were obtained for the six proteins, and the corresponding genes were identified in the Sanger M. tuberculosis genome database. In parallel we established a two-dimensional electrophoresis reference map of short-term culture filtrate components and mapped novel proteins as well as already-known CFP.     

Nerve damage following intraneural injection of Mycobacterium leprae into rabbits pre-sensitized to mycobacteria.

Nerve damage is a common feature of leprosy although the mechanism responsible for the damage is not clearly understood. In the tuberculoid end of the leprosy spectrum where both intraneural Mycobacterium leprae or their antigens and cell-mediated hypersensitivity to M. leprae co-exist, acute neuritis affecting major nerve trunks can occur during reversal reactions. These reactions are known to be associated with increased hypersensitivity to M. leprae antigens. The nerve involvement is therefore thought to be a direct consequence of the patient's hypersensitivity to M. leprae. So far the only indirect evidence based on in vitro studies have been produced to support such a contention. We sensitized rabbits with M. leprae and then injected M. leprae sonicate into the sciatic nerves at the peak of hypersensitivity. Seventy-two hours later, the nerves were dissected out and studied histologically. Our results show that cellular infiltration and axonal degeneration can occur as a direct consequence of hypersensitivity to intraneural M. leprae antigens. This study, therefore, offers direct evidence for the involvement of specific cell-mediated hypersensitivity to M. leprae antigens in the pathogenesis of major nerve trunk damage in the tuberculoid end of the leprosy spectrum especially during acute reversal reactions.     

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.     

Lipids from Mycobacterium leprae cell wall suppress T-cell activation in vivo and in vitro.

The influence of Mycobacterium leprae cell wall lipids on lymphocyte functions has been investigated in vivo (delayed-type hypersensitivity) and in vitro. The inflammatory response has been earlier evaluated by the mouse footpad oedema model and the delipidated mycobacteria evoked a mild but significant inflammatory response. Herein a higher level of hypersensitivity reaction was observed with delipidated bacilli than with the intact mycobacteria. The lipids obtained from the extract of M. leprae external cell wall were used to prepare liposomes, which have not been shown to be toxic to lymphocytes. The method of lipidic extraction and the sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the lipid fraction did not reveal any trace of proteins. Thin-layer chromatography of this extract detected four different bands with an apolar character, suggestive of mycolic and fatty acids. These same M. leprae liposomes potently suppressed lymph node cells, as well CD4+ and CD8+ T-cell lines, and an antigen-specific T-cell clone (T 4-9) proliferation, even under potent stimulus. Cholesterol-choline liposomes, unrelated to M. leprae liposomes, used as a control in the biological assays showed no significant effect on lymphoblastic activity, which points to the specificity of M. leprae lipids. These data demonstrated that M. leprae cell wall lipids induce immune suppression in mice without causing any membrane alteration in T cells as assessed throughout kinetic studies in vitro. This fact is closely related to the down-regulating effect induced by M. leprae lipids which we have previously observed in macrophage functions in vivo and in vitro. Although this lipidic fraction showed a suppressive action on T lymphocytes in vitro (proliferation) and in vivo (delayed-type hypersensitivity), its possible significance in the establishment of a specific immune response to M. leprae must be further investigated.     

Induction of a type 1 immune response to a recombinant antigen from Mycobacterium tuberculosis expressed in Mycobacterium vaccae.

A 19-kDa lipoprotein from Mycobacterium tuberculosis was expressed as a recombinant antigen in the nonpathogenic mycobacterial host strain M. vaccae. Immunization of mice with the recombinant M. vaccae resulted in induction of a strong type 1 immune response to the 19-kDa antigen, characterized by immunoglobulin G2a (IgG2a) antibodies and gamma interferon (IFN-gamma) production by splenocytes. Immunization with the same antigen in incomplete Freund's adjuvant induced a strong IgG1 response with only low levels of IFN-gamma. Subsequent intravenous and aerosol challenges of immunized mice with virulent M. tuberculosis demonstrated no evidence of protection associated with the response to the 19-kDa antigen; in fact, the presence of the recombinant 19-kDa antigen abrogated the limited protection conferred by M. vaccae (vector control). The recombinant M. vaccae system is a convenient approach to induction of type 1 responses to M. tuberculosis antigens. However, the unexpected reduction in protective efficacy of M. vaccae expressing the 19-kDa antigen highlights the complexity of testing recombinant subunit vaccines and the need for a better understanding of the immune mechanisms required for effective vaccination against tuberculosis.     

Persistence and Protective Efficacy of a Mycobacterium tuberculosis Auxotroph Vaccine

New vaccines against tuberculosis are urgently required because of the impressive incidence of this disease worldwide and the highly variable protective efficacy of the current vaccine. The possibility of creating new live vaccines by the rational attenuation of strains from the Mycobacterium tuberculosis complex was investigated. Two auxotrophic mutants of M. tuberculosis and M. bovis BCG were constructed by disruption of one of their purine biosynthetic genes. These mutants appeared unable to multiply in vitro within mouse bone-marrow derived macrophages. They were also attenuated in vivo in the mouse and guinea pig animal models. In guinea pigs, the two mutants induced strong delayed-type hypersensitivity response to purified protein derivative. In a preliminary experiment, the two mutants were compared to the BCG vaccine for their protective efficacy in a challenge against aerosolized virulent M. tuberculosis in the guinea pig model. Both mutants conferred some level of protection. These experiments demonstrate that the rational attenuation of M. tuberculosis could lead to the design of new candidate live vaccines against tuberculosis.     

Phenolic glycolipid 1 of Mycobacterium leprae causes nonspecific inflammation but has no effect on cell-mediated responses in mice

The involvement of the phenolic glycolipid from Mycobacterium leprae in cell-mediated immunity has been investigated in this study. The phenolic glycolipid itself does not appear to stimulate cell-mediated immunity directly, as shown by its failure to elicit a classical delayed-type hypersensitivity response in mice immunized with M. leprae or to stimulate M. leprae-immune lymph node cells in a lymphoproliferative assay. Intradermal vaccination with the phenolic glycolipid failed to influence the growth of M. leprae in mouse footpads. A nonspecific inflammatory response to the sonicated glycolipid was observed in mice vaccinated with whole M. leprae and in control animals. No evidence was obtained for any adjuvant or suppressive effect on cell-mediated immunity by the phenolic glycolipid either to M. leprae or an unrelated antigen (sheep erythrocytes); neither sensitization nor elicitation to either antigen was affected.     

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.     

MTC28, a novel 28-kilodalton proline-rich secreted antigen specific for the Mycobacterium tuberculosis complex.

Proteins that are actively secreted by Mycobacterium tuberculosis serve as major targets of immune responses in the infected host. To identify and purify novel proteins in the filtrates of M. tuberculosis cultures, a bacteriophage lambda library of M. tuberculosis H37Rv DNA was immunoscreened by using an anti-culture filtrate rabbit antiserum. Of 20 positive clones isolated, 6 were analyzed and found to express the genes for two known components of the early culture filtrate, the secreted 45/47-kDa antigen complex and the KatG protein, and two novel genes. Here we report the molecular cloning and nucleotide sequence of one of the new genes encoding a culture filtrate protein of 310 amino acid (aa) residues. We called this gene mtc28. The deduced polypeptide sequence contained an NH2-terminal, highly hydrophobic 32-aa region having properties of a secretion signal peptide. The putative 278-aa mature MTC28 protein was characterized at its NH2 and COOH termini by a high content of proline and alanine residues organized in an (AP)n motif. Thus, MTC28 is a new member of a group of proline-rich antigens found in M. tuberculosis and Mycobacterium leprae. As shown by DNA hybridization experiments, the mtc28 gene was present only in species of the M. tuberculosis complex. Purified recombinant MTC28 antigen evoked strong delayed-type hypersensitivity and antibody responses in guinea pigs immunized with Mycobacterium bovis BCG, but not in guinea pigs immunized with Mycobacterium avium. The strong immunological activity of MTC28 and the absence of B- and T-cell epitopes cross-reactive with a common environmental mycobacterial species, such as M. avium, make this novel antigen an attractive reagent for immunodiagnosis of tuberculosis.     

T cell epitopes on the 36K and 65K Mycobacterium leprae antigens defined by human T cell clones

To identify the molecular localization and specificity of Mycobacterium leprae antigenic determinants inducing T cell activation, we studied the reactivity of M. leprae-reactive T cell clones from two tuberculoid leprosy patients towards a battery of different mycobacterial strains and purified mycobacterial antigens. Of the 38 T cell clones tested 8 appeared to be M. leprae specific (specificity A), another 8 were cross-reactive with at least one of the three mycobacterial strains, M. lepraemurium, M. vaccae and M. scrofulaceum (specificity B), 5 were reactive with most but not all strains (specificity C) and the remaining 18 were reactive with all 17 mycobacterial strains tested (specificity D), but not with nonmycobacterial antigens. All T cell clones were tested with the 36K and 65K antigen isolated from M. leprae, and with the M. leprae and M. bovis BCG 65K proteins produced in E. coli by recombinant DNA. Four T cell clones appeared to recognize epitopes on the 36K antigen, nine T cell clones recognized the 65K antigen. These 2 M. leprae antigens, 36K and 65K, thus seem to contain major T cell epitopes. At least 3 different epitopes could be defined on the 36K antigen of which one is M. leprae specific, one of specificity B and one of specificity C. Two distinct epitopes were discerned on the 65K antigen of which one is M. leprae specific and one of specificity D. The M. leprae-specific epitopes on the 36K and 65K antigen may help in the development of a specific serodiagnostic and skin test.     

Induction of cell-mediated immunity to Mycobacterium leprae in guinea pigs.

Guinea pigs immunized with intact or disrupted armadillo-grown human Mycobacterium leprae administered in aqueous or oil vehicles were tested with various dilutions of M. leprae suspended in saline, water-soluble M. leprae extract, purified protein derivative, and a water-soluble extract of normal armadillo tissue. The results demonstrated the following. (i) Under no conditions was any skin test reactivity found to normal armadillo tissue extract. (ii) Positive sensitization to both M. leprae and its water-soluble extract was achieved by sensitizing guinea pigs with M. leprae suspended in Hanks solution or saline. Autoclaved M. leprae in Hanks solution or saline inoculated intradermally was an effective immunogen. Oil suspensions or emulsions were effective at sensitization, but appeared to be no better and, in general, slightly weaker, than simple inoculation in aqueous suspension. (iii) Living BCG failed to reveal a significant adjuvant effect on sensitization to M. leprae. However, cord factor appeared to potentiate slightly the sensitization to M. leprae in aqueous suspension. (iv) The minimum dose required for sensitization with M. leprae in aqueous suspension was 55 micrograms of purified bacilli. (v) Animals inoculated with M. leprae in saline or with M. leprae together with BCG showed positive skin test reactivity to the first skin test application made fully 1 year after the initial sensitization. The efficacy of autoclaved, irradiated M. leprae in aqueous, oil-free medium suggests a relatively safe approach to human vaccination studies.     

微卡对哮喘豚鼠肺组织嗜酸粒细胞凋亡及Bcl-2蛋白表达的影响

目的：研究微卡对哮喘豚鼠肺组织嗜酸粒细胞凋亡及Bcl-2蛋白表达的影响。 方法： 30只豚鼠随机分为生理盐水组、哮喘组及微卡组，每组10只。微卡组每只豚鼠在OVA致敏前10 d肌注22.5 μg微卡。应用TUNEL法检测肺组织嗜酸粒细胞的凋亡，免疫组化法检测肺组织Bcl-2蛋白的表达。 结果： 微卡组豚鼠肺组织嗜酸粒细胞凋亡指数（23.78±5.42）%显著高于哮喘组（4.56±0.68）%(P<0.01)；肺组织Bcl-2蛋白平均积分吸光度值（1 556.3±492.4）显著低于哮喘组（2 321.9±751.2）（P<0.05）。 结论： 微卡能诱导哮喘豚鼠肺组织嗜酸粒细胞凋亡，可能与其抑制Bcl-2蛋白在哮喘豚鼠肺组织的表达有关。