Mycobacterium Tuberculosis Culture Filtrate Research Articles

Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex

BALB/c and C57BL/6 mice were injected intramuscularly with plasmid DNA encoding the three components of the immunodominant 30–32 kDa antigen 85 complex (Ag85A, Ag85B, and Ag85C) from Mycobacterium tuberculosis culture filtrate, in order to investigate the utility of nucleic acid vaccination for induction of immune responses against mycobacterial antigens. Ag85A and Ag85B encoding plasmids induced a robust Th1-like response towards native Ag85, characterized by elevated levels of interleukin (IL)-2, interferon-γ, and TNF-a. Levels of IL-4, IL-6, and IL-10 were low or undetectable. Plasmid encoding Ag85C was not effective. Cytotoxic T cell activity was also generated in in vitro restimulated splenocyte cultures from Ag85A and Ag85B DNA vaccinated mice. Finally, Ag85A and Ag85B DNA vaccination conferred significant protection against mycobacterial replication in lungs from B6 mice, subsequently challenged. Therefore, this technique may be useful for the definition of protective antigens of M. tuberculosis and the development of a more effective tuberculosis vaccine.

Immunobiological properties of a 30 kDa secretory protein of Mycobacterium tuberculosis H 37Ra

Six different secretory proteins of molecular weights (15, 26, 30, 41, 55 and 70 kDa) were isolated from 8-day-old culture filtrate of Mycobacterium tuberculosis H 37Ra using different column chromatography techniques. These proteins were further examined for their ability to induce cell mediated (T-cell proliferation assay) and humoral immune response (ELISA) in mice immunized with total culture filtrate proteins. Out of six proteins, three proteins showed good reactivity. However, the activity was at a maximum with 30 kDa antigen. The immune response induced by 30 kDa antigen emulsified in Freund's incomplete adjuvant (FIA) was investigated and was found to be dose dependent. The T-cell response induced by this protein was skewed towards T-helper (Th1) cells as determined by the pronounced secretion of interleukin-2 (IL-2) and γ-interferon (IFN-γ). The protective activity of the 30 kDa protein was also evaluated and compared with reference to Bacillus Calmette Guerin (BCG) vaccine in the mice challenged with virulent M. tuberculosis H 37Rv. The degree of protection afforded by the 30 kDa antigen on the basis of mortality and the significant decrease in c.f.u.'s recovered from different organs (lung, liver, spleen) after 30 days of challenge with LD 50 of M. tuberculosis H 37Rv was significantly higher in comparison to BCG vaccinated animals. However, the degree of immunity induced by this antigen decreased with time (when challenged 8 and 12 weeks post-immunization) but it was still comparable with BCG. These findings suggest that 30 kDa secretory protein of M. tuberculosis is the key immunoprotective antigen and may be a suitable candidate for the development of an alternative subunit vaccine against tuberculosis.

Immune Responses Stimulated by Percutaneous and Intradermal Bacille Calmette-Guerin

Healthy volunteers were randomized to receive percutaneous or intradermal bacille Calmette-Guérin (BCG) vaccination. Delayed-type hypersensitivity (DTH) to tuberculin, as well as proliferative and interferon-gamma responses in peripheral blood mononuclear cells stimulated by whole cell lysates and culture filtrates of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare, were compared before and after vaccination. Positive DTH reactions were detected in 83% of intradermal and 40% of percutaneous BCG recipients 3 months after vaccination (P < .004). M. tuberculosis-specific proliferation was increased after intradermal BCG (P < .01) but not after percutaneous BCG compared with prevaccination responses. In addition, M. tuberculosis-specific interferon-gamma production was increased after intradermal BCG compared with both prevaccination responses (P < .04) and those measured after percutaneous BCG (P < .05). Predominant immune responses stimulated by BCG were directed against antigens present in mycobacterial whole cell lysate. These results indicate that the BCG vaccination route can affect both in vivo and in vitro immune responses.

Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.

A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.

Identification by two-dimensional gel electrophoresis of a 58-kilodalton tumor necrosis factor-inducing protein of Mycobacterium tuberculosis.

We have previously identified proteins in fractions of culture filtrate of Mycobacterium tuberculosis with the capacity to induce cytokine production in monocytes, by using a technique we defined as "monocyte Western blotting" (immunoblotting). In this series of experiments, we have extended this technique to two-dimensional gel electrophoresis and have identified a novel 58-kDa protein of M. tuberculosis which induces production of tumor necrosis factor by human monocytes. Nitrocellulose particles bearing this protein were used to develop murine monoclonal antibodies by the technique of intrasplenic immunization. The protein was purified by preparative isoelectric focusing and gel electrophoresis and subjected to N-terminal amino acid sequence analysis. As tumor necrosis factor is a mediator of both pulmonary necrosis and macrophage activation for intracellular killing, this 58-kDa protein may play an important role both in the immunopathogenesis of tuberculosis and in mycobacterial immunity.

Isolation of two antigens from the culture filtrates of Mycobacterium tuberculosis and their applications in the laboratory diagnosis of the tuberculous meningitis

Two antigens were isolated from the culture filtrates of H37Ra Mycobacterium tuberculosis by immunoabsorbent affinity chromatography, M. tuberculosis antigen 5 and immunoabsorbent affinity column-purified antigen (IAP). The potential application of these two mycobacterial antigens in the laboratory diagnosis of tuberculous meningitis was evaluated by indirect enzyme-linked immunoabsorbent assay in cerebrospinal fluid specimens. IAP antigen was more sensitive than antigen 5, although antigen 5 was more specific than IAP antigen in detecting tuberculous aetiology. Technical aspects of immunoabsorbent affinity chromatography have been highlighted in this study.

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.

The Detection by Immunoassay of Antibody to Mycobacterial Antigens and Mycobacterial Antigens in Bronchoalveolar Lavage Fluid from Patients with Tuberculosis and Control Subjects

Bronchoalveolar lavage (BAL) samples from patients with pulmonary tuberculosis and from control subjects were studied using direct and inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgA antibody to Mycobacterium tuberculosis culture filtrate and purified antigen 5 and for the detection of mycobacterial antigens. The IgG and IgA antibodies were found in the majority of samples from tuberculous patients. The IgG titers were higher than were IgA titers. Substantial nonspecificity was observed for antibodies to crude M tuberculosis culture filtrate. Antibodies to purified M tuberculosis antigen 5 were more specific. The detection of antibody in BAL did not offer advantages over previously described serodiagnostic assays. Antigen was found in some BAL samples from tuberculous patients. Greater specificity was observed with an assay based on purified antigen 5 than on crude mycobacterial culture filtrate.

Biological activity of protein antigens isolated from Mycobacterium tuberculosis culture filtrate.

Mycobacterium tuberculosis culture filtrate (MTCF) protein antigens were isolated from mid-logarithmic-phase cultures grown in liquid medium and examined by high-pressure liquid chromatography and Western blot (immunoblot) analysis. A major protein band with a molecular mass of about 68 kilodaltons (kDa) and several fainter bands in the 38- and 24-kDa range were observed. The MTCF protein produced a significant delayed footpad hypersensitivity response in Mycobacterium bovis BCG-vaccinated C57BL/6 mice, comparable to that observed with standard purified protein derivative (PPD). The same proteins induced a blastogenic response in tuberculin-sensitive human peripheral blood monocytes and in T-cell clones developed from these cells. The proliferative responses to the MTCF antigens were equivalent to those observed following stimulation with PPD or M. tuberculosis sonic extracts. However, the MTCF sensitins were not recognized by five monoclonal antibodies directed against killed M. tuberculosis antigens in an enzyme immunoassay, although some response was seen with a monoclonal antibody (ML34) directed against M. leprae antigens. The ability of the MTCF to stimulate T-cell responses both in vivo and in vitro while not being recognized by antibodies directed against dead mycobacterial antigens suggests that they may be of interest as potential protective immunogens.

Characterization of Mycobacterium tuberculosis antigen 5 epitopes by using a panel of 19 monoclonal antibodies

Antigen 5 is a 35,000-dalton protein which has been purified from culture filtrates of Mycobacterium tuberculosis and shown by immunoprecipitation to be restricted in distribution to M. tuberculosis and M. bovis among 14 mycobacterial species studied. We raised 19 antigen 5-reactive monoclonal antibodies and used them to characterize epitopes of this antigen by enzyme-linked immunosorbent assay and immunoabsorbent affinity chromatography. Fifteen monoclonal antibodies, all immunoglobulin M (IgM), cross-reacted in two major patterns with culture filtrates from five species of mycobacteria and with purified mycobacterial arabinomannan and arabinogalactan. All 15 monoclonal antibodies also reacted with M. tuberculosis antigen 6. Immunoabsorbent affinity columns prepared with these antibodies yielded principally arabinomannan and arabinogalactan. Four monoclonal antibodies, three IgG2a and one IgM, reacted by enzyme-linked immunosorbent assay with antigens 5 and 6 exclusively and not with mycobacterial culture filtrates or polysaccharides. All four monoclonal antibodies yielded antigen 5 and small amounts of antigen 6 when used for immunoabsorbent affinity chromatography. We conclude from these studies that antigen 5 has two nonspecific epitopes, possibly carbohydrate in nature, and one apparent species-specific epitope which is shared with antigen 6.

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.

Carbohydrate analysis of concanavalin A-reactive and concanavalin A-nonreactive mycobacterial polysaccharides.

Concanavalin A-nonreactive polysaccharide and 2 concanavalin A-reactive polysaccharides of differing concanavalin A affinities that had been purified from culture filtrates of Mycobacterium tuberculosis were subjected to carbohydrate analysis by gas and gas-liquid chromatography. Concanavalin A-nonreactive polysaccharide was found to be D-arabinogalactan, and the concanavalin A-reactive polysaccharide of highest affinity was found to be D-arabinomannan. The lower affinity concanavalin A-reactive polysaccharide was rich in arabinose and probably represented a mixture, with more than one polysaccharide present. Glucan was found to be a significant contaminant of all 3 polysaccharides.

The purification of mycobacterial polysaccharides with concanavalin A.

Concanavalin A was demonstrated to react with 3 antigenic polysaccharide constituents of culture filtrates of Mycobacterium tuberculosis. Using direct precipitation and affinity chromatography methods, concanavalin A-reactive polysaccharides could be separated from concanavalin A-non-reactive polysaccharide in a partially purified, polysaccharide-rich fraction of mycobacterial culture filtrate. By the use of appropriate dissociation conditions, 2 concanavalin A-reactive polysaccharides of differing concanavalin affinities could be separated and purified. Four polysaccharides thus studied were identified with the United States-Japan mycobacterial antigen nomenclature. Antigen 1 was found to be a concanavalin A-reactive polysaccharide of high affinity. Antigen 2 was found to identify both with a concanavalin A-reactive polysaccharide of low affinity and with a concanavalin A-nonreactive polysaccharide. Antigen 3 was found to represent the large molecular weight polysaccharide II of Seibert and to react with...

Purification and Characterization of Two Proteins from Culture Filtrates of Mycobacterium tuberculosis H 37 Ra Strain

Two proteins have been purified from culture filtrates of Mycobacterium tuberculosis, H(37)Ra strain by a procedure combining gel filtration, diethylaminoethyl (DEAE)-cellulose chromatography, and zone electrophoresis. The two proteins are similar in molecular weight but differ slightly in charge. The faster migrating protein, designated a(1), is not antigenic. The slower migrating protein, designated a(2), is antigenic both with respect to antisera and as a skin-testing antigen.

Purified Protoplasmic Peptides of Mycobacteria: In Vivo and In Vitro Comparison of the Species Specificity of Purified Protoplasmic Peptides and Purified Protein Derivatives of Mycobacterial Culture Filtrates

The specificity of purified protein derivatives (PPD) prepared from the culture filtrates of Mycobacterium tuberculosis (PPD), M. kansasii (PPD-Y), M. intracellulare (PPD-B), and M. scrofulaceum (PPD-G) were compared to comparable protoplasmic extracts (PPP) of the same organisms by gel diffusion and delayed hypersensitivity reactions in sensitized guinea pigs. PPD and, to a lesser degree, PPD-Y demonstrated specificities sufficient to enable identification of homologously sensitized guinea pigs in the above group of four mycobacteria. PPD-B and PPD-G did not always elicit the largest reaction in homologously sensitized animals. The PPP sensitins from M. tuberculosis and M. kansasii produced as good skin reactions at 24 and at 48 hr as did their PPD counterparts. The PPP from M. scrofulaceum and M. intracellulare were more specific and more reactive than corresponding PPD, regardless of the time of comparison. Although based on different immunological mechanisms, the specificity of these two groups of sensitins, as demonstrated by delayed hypersensitivity, correlated well with serological comparisons in the gel diffusion test. The low degree of specificity of PPD-B and PPD-G in contrast to that of corresponding PPP was reflected in the precipitin bands in agar gel.

RESISTANCE-ENHANCING ACTIVITY OF CULTURE FILTRATES OF MYCOBACTERIUM TUBERCULOSIS.

Hedgecock, Loyd W. (Veterans Administration Hsopital, St. Louis, Mo.). Resistance-enhancing activity of culture filtrates of Mycobacterium tuberculosis. J. Bacteriol. 88:1349-1355. 1964.-It was demonstrated that filtrates of cultures of Mycobacterium tuberculosis exhibit resistance-enhancing activity against infection with Klebsiella pneumoniae and Diplococcus pneumoniae. Resistance was demonstrable when the culture filtrate was injected 3 hr prior to challenge with K. pneumoniae, and reached the highest level when injected 24 hr before challenge. Protection was maximal when the filtrate and challenging organisms were injected by the same route. After infection with K. pneumoniae, the bacterial titers in the tissues of filtrate-treated animals were significantly lower than those of untreated susceptible controls. Mice injected with the filtrate 24 hr previously demonstrated significantly increased rates of clearance of injected carbon from the blood. In examination of a variety of mycobacteria, resistance-enhancing activity was found only in culture-filtrates of pathogenic mycobacteria, the highest concentration occurring in filtrates of virulent organisms. The injection of a relatively large amount of phenol-killed cells of all of the mycobacteria examined enhanced resistance to infection with K. pneumoniae.

Comparison of In Vitro Leukocytolysis Produced with PPD and with Fractions from Culture Filtrate of Mycobacterium Tuberculosis

Comparison of In Vitro Leukocytolysis Produced with PPD and with Fractions from Culture Filtrate of Mycobacterium Tuberculosis

Comparison of in vitro leukocytolysis produced with PPD and with fractions from culture filtrate of Mycobacterium tuberculosis.

Comparison of in vitro leukocytolysis produced with PPD and with fractions from culture filtrate of Mycobacterium tuberculosis.

Multiple Precipitins to Cow's Milk in Chronic Respiratory Disease

In July, 1959, an 8-month-old white boy was admitted to the University of Arkansas Medical Center because of severe chronic cough, persistent and changing pulmonary infiltrations, recurrent diarrhea, poor weight gain, and anemia. He had been studied in a local hospital on several occasions, and sweat chloride determinations had twice been normal; but persistence of severe symptoms necessitated a reevaluation. After sweat electrolyte and duodenal intubation studies had excluded the diagnosis of cystic fibrosis of the pancreas, skin and serologic tests for tuberculosis and deep mycoses were performed. All skin tests were negative, as were precipitin tests for histoplasmosis,1blastomycosis,2and coccidioidomycosis. The patient's serum, however, did react strongly with a culture filtrate of virulent Mycobacterium tuberculosis. The serum was then tested for precipitins to 5 additional crude antigens prepared from mycobacterial culture filtrates, but in no instance was a reaction observed. These findings were unique, since none

A Study of Antigens Active in the Tannic Acid Hemagglutination Test Present in Filtrates of Culture of Mycobacterium Tuberculosis

Summary A number of different fractions from culture filtrates of Mycobacterium tuberculosis have been tested for ability to sensitize tanned red cells to agglutination by rabbit antiserum prepared against phenol-killed tubercle bacilli. The different fractions varied considerably in their potency in this respect, and some were inactive. The antigenic relationship of the fractions active in the test was studied in cross inhibition tests. Four active fractions from unheated culture filtrates were found to be completely distinct from each other serologically and showed very little cross reaction. Three preparations of PPD and other fractions from heated culture filtrates cross reacted with each other strongly and little difference could be seen among them. These heated fractions also cross reacted with one of the unheated preparations (that analogous to Seibert's C protein).