Carlo Cocito

Composition and immunological properties of the protein fraction of A36, a major antigen complex of Mycobacterium paratuberculosis.

TMA (thermostable macromolecular antigens) are major mycobactcrial complexes present in all mycobticteria. We have purified A36, the TMA complex of M. paratuberculosis, the etiological agent of paratuberculosis (Johnes disease), and shown by the immune electron microscopy approach its presentation at the cell surface. The immunodominance of the A36 complex in Johnes disease was suggested by comparative ELISA analysis of infected bovine sera, using either A36 or M. paratuberculosis total soluble sonicate as antigens. The cross‐reactivity of TMA complexes from diifcrcnt tnycobacteria was evaluated by immunocn/ymometri c measurements. Percentage of shared epifopes was high tor the couple M. paratuberculosis‐ M. avium. and somewhat lower for the couple M. paratuberculosis and M. bovis Immunological kinship between M. paratuberculosis and M. leprae was suggested by the finding that oul of eleven anti‐M. lepraemonoclonals, four cross‐reacted with A36 proteins. The specificiiy missing al the level of the whole A36 complex was sought at the level of its protein components. Comparative immunoblot analysis of electrophoresed A36 proteins indicated three of them to contain epitopes not shared by M. bovis proteins, and one of them to contain epitopes specific wilh respect lo M. avium, M. bovis and M. phlei. The latter component, a 34‐kDa protein, could be an ideal reagent for a serological tesi for, Johnes disease, being immunodominant in infected cattle and endowed with species‐specific epitopes.

Serological Analysis of Human Tuberculosis by an ELISA with Mycobacterial Antigen 60

An ELISA method for detecting serum antibodies against A60, an antigen prepared from the cytoplasm of Mycobacterium bovis BCG, has been applied to 385 subjects, namely 197 controls (neonates, healthy adults, and tuberculin negative, nontuberculous patients), and 188 subjects at various stages of tuberculous infection and disease. Most IgM determinations gave negative results. While the neonates and normal adults had titers of IgG anti-A60 antibodies below the cut off value, wide variations in antibody titers were observed among the various types of subjects infected by M. tuberculosis. The results obtained with nontuberculous subjects were: 100% negative IgG in neonates and healthy adult individuals and 6.4% false positive cases among 124 non-tuberculous patients. The percentage of serologically positive cases of tuberculosis was: 5.9% in latent active primary forms, 42.8% in patent active primary forms, and 82.8% in active postprimary forms. Tuberculous infections had a positively rate of 14.7%, while inactive postprimary tuberculosis had a positivity rate of 50%. The results obtained with A60 can favourably be compared with other serum ELISA tests for tuberculous antibodies against purified or semipurified mycobacterial antigens. Anti-A60 ELISA IgG antibody test can be useful to monitor the kinetics of humoral immunological response during tuberculous infection, disease and chemotherapy. A positive IgG ELISA test may support the diagnosis of active tuberculous disease.

Chemical Composition of Antigen 60 from Mycobacterium bovis BCG

Antigen 60 (A60). the main thermostable immunogen of tuberculin and PPD. has been purified from Mycobacterium bovis BCG cytoplasm, and identified by crossed Immunoelectrophoresis with anti‐BCG polyclonal antiserum. Two A60 fractions, free lipids and lipid‐conjugated compounds, have been recognized. The free lipids represented about 30% (dry weight), and consisted essentially of C16‐C18, fatly acids, and of phosphatidyl‐inositol‐mannosides. Lipocon‐jugates, upon DEAE‐cellulose chromatography and gel filtration, yielded two main fractions of neutral and polar components. Chromatography of delipidated and deproieinizd A60 on Sephadex G‐100yielded: a high molecular weight fraction (Al, 18%, a lipoglucan of ± 106) and a low molecular weight fraction (B, 10%, a lipopeptidoglycan of ± 104) containing mannose, glucose, and small amount of arabinose. The polysaccharide moieties of fractions Al and B were submitted to aeetylalion, methylation, and acid hydrolysis, and ihe structure of the hydrolysed polymer was deduced by combined gas chromalography/mass spectrometry analysis. The results indicated a branched structure involving 1. 4‐, 1, 6‐, and 1, 4, 6‐linked D‐gluco‐ or n‐manno‐pyranosyl residues. Glucan‐ and peptidoglycan‐bound fatty acids were identified as saturated (C16‐C18) and monounsaturated linear acids (C12, C18). Immunodiffusion on agarose gel indicated that delipidalion and proteolysis did not suppress the ability of A60 to yield immunoprrecipitates with anti ‐A60 antiserum. The high polymer fractions obtained by chromatography on DEAF cellulose and Sephadex G‐100 were also reactive. It is concluded that A60 is made of tree lipids and of hpopcptidoglycans of high molecular weights (106‐107) endowed with immunogenic properties.

Induction of cellular immune reactions by A36, an antigen complex of Mycobacterium paratuberculosis : comparison of A36 and Johnin components

Paratuberculosis (Johnes disease) is a chronic enteritis syndrome of ruminants, which is due to infection by Mycobacterium paratuberculosis. Cutaneous testing with proteins extracted from a mycobacterial culture fluid (johnin‐PPD) is currently used to evaluate the cellular immune status. We have compared the components of johnin‐PPD with those of the A36 complex, a thermostable macromolecular antigen (TMA) present in the cytoplasm and associated with the cell wall of M. paratuberculosis. The presence in the johnin‐PPD of fifteen A36 components has been shown by Western blotting. Moreover, monoclonal antibodies, which bind respectively to the 65‐kDa M. leprae heat shock protein, the 28‐kDa M. leprae superoxide dismutase, and M. tuberculosis lipoarabinomannan, recognized components of the johnin‐PPD. The ability of A36 to trigger delayed hypersensitivity reactions in sensitized rabbits, and to induce the proliferation of T lymphocytes from the lymph nodes of A36‐sensitized mice, matched that of johnin‐PPD. The homology levels of T epitopes between A36 and the TMA complexes of M. phlei, M. bovis, M. tuberculosis and M. avium were estimated, in a lymphoproliferation assay, to be 51, 52, 59 and 94% respectively. A strong cross‐reactivity of A36 with an M. leprae sonicate was also observed by cutaneous testing. The A36 components within the 45.2–26.8‐kDa and the 21.6–19.8‐kDa ranges were proved to induce the proliferation of T lymphocytes from sensitized mice. This work supports the possible use of the A36 complex, and of some of its components, for cutaneous tests and lymphocyte proliferation assays, in order to monitor cellular immunity in Johnes disease.

Immunization of Mice With the Antigen-a60 of Mycobacterium-bovis Bcg

Antigen 60 (A60) is a thermostable component of the cytoplasm of Mycobacterium tuberculosis and BCG which can be fractionated into at least 15 protein bands when analysed by Western blot. Normal B6D2 mice were immunized subcutaneously with 20 μg of the A60 protein suspended in Freunds incomplete adjuvant (FIA) or in saline. Three weeks later the mice received a second dose of vaccine followed 2 weeks later by an aerogenic challenge with approximately 103 CFU of M. tuberculosis Erdman. The mice receiving the adjuvanled A60 showed a significant reduction (P < 0·05) in the number of viable organisms recovered from the lungs and the spleen 3 weeks after challenge. However, this response was less than that seen in BCG vaccinated controls.

Delayed hypersensitivity reactions by the mycobacterial antigen A60 and cutaneous testing in tuberculosis.

Antigen A60 has been purified from the cytoplasm of Mycobacterium bovis BCG, and its composition has been determined: it has proved to be able to elicit immune reactions of both humoral and cellular type. Inoculation of A60 into the footpad of mice previously sensitized with the same antigen, or with whole mycobacterial cells produced a footpad swelling showing a peak at 24 h. Similar delayed hypersensitivity reactions were induced in sensitized guinea-pigs by subcutaneous injection of an A60 dose of 0.01 μg (minimal revealing dose). A quantity thousandfold higher (15 μg A60) was unable to induce in unsensitized guinea pigs the mounting of a cellular immunisation against A60, as shown by negative cutaneous testings 1 month later. Our results show that A60 preparations satisfied the requirements of the European Pharmacopoeia Commission and met the WHO recommandations for new tuberculins. Handicaps of old tuberculin and PPD (heterogeneous mixtures titrated biologically and unstable in solution) can be overcome by A60 preparations (a single antigen spectrophoretically measurable and stable).

Analysis of Deoxyribonucleic Acids from Armadillo-Derived Mycobacteria

Armadillos are used for propagation of Mycobacterium leprae and are hosts of mycobacteria of the ADM (armadillo-derived mycobacteria) group. The deoxyribonucleic acids (DNAs) of isolates of the five phenetic subgroups of the ADM were analyzed and compared with the genomes of related bacteria. The guanine-pluscytosine (G+C) contents of the DNAs were 62.2 to 67.1 mol% for different ADM strains, 56 mol% for M. leprae and LDC (leprosy-derived corynebacteria), and 62 to 71 mol% for reference mycobacteria. Restriction analysis showed neither adenine methylation in the GATC site (a specific trait of LDC strains) nor cytosine methylation in the CCGG and GGCC sites. Hybridization higher than 80% was obtained with DNA from isolates within the same ADM subgroups, whereas 17 to 50% hybridizations were observed with organisms from different ADM subgroups. Genomes of ADM strains and reference mycobacteria were 15 to 40% homologous, except for subgroups IV and V, whose DNAs were 54 to 62% homologous with Mycobacterium lepraemurium DNA. Little or no homology between M. leprae and ADM genomes was found. We concluded that single ADM subgroups can be considered as distinct species within the genus Mycobacterium; they are genetically unrelated to the other leprosy-associated bacteria.

Comparative analysis of the genomes of mycobacteriophages infecting saprophytic and pathogenic mycobacteria.

SummaryThe genomes of a series of mycobacteriophages have been analyzed to disclose possible relationships between genetic characteristics and host range. The percent guanine-plus-cytosine in the DNA of 14 phages was found to be 34.4 to 47.5, as determined by a double-labelling procedure, which is unaffected by the presence of modified bases. The DNA of few mycobacteriophages yielded discordant values when the G+C content was estimated by buoyant density determination and by the double labelling procedure. This observation suggests the possible presence of modified bases in these genomes. The reduced susceptibility of viral DNAs to several restriction endonucleases is suggestive of the occurrence of both methyladenine and methylcytosine in the genome of all the mycobacteriophages studied. Heterologous annealing among the 14 DNAs analyzed yielded 6 hybridization groups. Within one group, the homology level among viral genomes was estimated by comparing the electrophoretic mobilities of restriction fragments: values of 0.8 to 1.3% base substitution have thus been found. A comparison of the genomic characteristics and host range of the mycobacteriophages analyzed suggests a possible relationship between restriction pattern, G+C content, crosshybridization level and host range.

Comparative cutaneous testing with purified protein derivative and the antigen complex A60 in vaccinated subjects and tuberculosis patients.

Some 840 bacille Calmette-Guérin (BCG)-vaccinated healthy controls and tuberculosis patients from two Chinese hospitals were submitted to comparative skin tests with purified protein derivative of tuberculin (PPD; as reference) and with the antigen complex A60 from Mycobacterium bovis BCG. In a first trial, including 581 persons (185 healthy juveniles, 180 healthy adults and 216 tuberculosis patients), a limited dose of A60 (1μg) was used. Performance of the A60 test was similar to that of 5 I.U. PPD for controls (cut-off values=5 mm induration diameter), but lower than that seen for tuberculosis patients (10 mm cut-off values). A second survey was conducted on 259 persons (109 recently revaccinated healthy persons, considered as tuberculin-negative in the first trial, and 150 tuberculosis patients), using a higher dose of A60 (2 μg) and the same dose of PPD (5 I.U.). Similar results were obtained with the two tests in all cases, thus supporting the possibility of PPD replacement by A60 in cutaneous testing. The pattern of induration diameter distribution in healthy subjects who took part in the first testing round (64% positively rate) was displaced to the inactivity side (with a peak at 5 to 9-mm diameter), in comparison with the second round (90% positivity rate and peak at 10–14 mm). This indicates a progressive fading of cellular immunity reactions after BCG vaccination. In tuberculosis patients, no correlation was found among the following three parameters: positivity at cutaneous testing (with PPD or A60), titer of anti-A60 mycobacterial immunoglobulins in blood (IgG titer higher than cut-off line) and presence of mycobacteria in sputum.