Sadamu Nagai

Identification of immunodominant antigens during infection with Mycobacterium tuberculosis.

T lymphocytes isolated from mice infected with Mycobacterium tuberculosis respond vigorously to proteins secreted by the bacilli and these antigens may be of importance in the generation of protective immunity against the disease. In this study, short‐term culture filtrate (ST‐CF), which constitutes a complex mixture of secreted proteins, was fractionated by a modified preparative SDS‐PAGE technique. The ability of each fraction to be recognized by T cells isolated from infected mice was evaluated by quantifying proliferation and IFN‐γ production in cell cultures. Two molecular mass regions 4–11 and 26–35 kDa were found to possess marked stimulatory properties. Four potent single antigens were mapped within the stimulatory regions. These purified antigens stimulated T cells isolated from mice at the height of a tuberculous infection to produce large amounts of IFN‐γ, Two of these stimulatory antigens belonged to lhe antigen 85 complex.

A localization index for distinction between extracellular and intracellular antigens of Mycobacterium tuberculosis

A series of mycobacterial antigens were quantified by immunoelectrophoresis, enzyme-linked immunosorbent assays, or SDS-PAGE with immunoblotting using antisera against purified mycobacterial antigens. The antigens showed a characteristic distribution profile. Some had a marked quantitative dominance in the culture fluid while others had a marked dominance in sonicates of whole washed bacilli. The majority of the antigens tested could thus be located and grouped as either secreted or cytoplasmic in terms of a localization index (LI) which is described. A 5-week-old Mycobacterium tuberculosis culture fluid preparation with a low degree of lysis was valuable in the delineation of localization indexes. The various secreted antigens showed a great span in LI values, from 5 to 1000. This variation may express different degrees of secretion efficiency or differences in tendency to adhere to the bacterial surface. The identification of proteins as extracellular or cytosolic according to their LI values was in agreement for cultures of M. tuberculosis with a low degree of lysis and cultures of M. bovis BCG and M. bovis AN-5 with significant lysis of the bacterial cells.

Heterogenous Expression of the Related MBP70 and MPB83 Proteins Distinguish Various Substrains of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv

MPB70 and MPB83 are homologous cross‐reactive secreted mycobacterial proteins with very limited species distribution. The expression of these two proteins was compared between several substrains of Mycobacterium bovis BCG, virulent M. bovis and Mycobacterium tuberculosis H37Rv. A polyclonal antibody specific for MPB70 in Western blotting, and a monoclonal antibody, MBS43, found to be specific for MPB83 in ELISA and Western blotting, were used for the comparison. The previously established pattern of high‐ and low‐producing substrains of BCG for MPB70 is only partially applicable for MPB83. MPB70 low‐producing strains are also MPB83 low‐producing, but the expression of MPB83 is much more variable than the expression of MPB70 in the MPB70 high‐producing strains. Purified MPB83 (23 kDa) was found to be glycosylated. A band in SDS‐PAGE at 1–2 kDa lower than that of purified MPB83 may represent unglycosylated MPB83. Furthermore, it was confirmed that purified MPB70 (22 kDa) is unglycosylated. There is cross‐reactive antigen at 26 kDa. The MPB83 related antigen at 26 kDa was found to be the most abundant. These findings indicate greater heterogeneity between different substrains of BCG than previously realized. Virulent M. bovis produce and secrete large amounts of MPB70 and MPB83 while both these proteins occur in a far lower concentration in M. tuberculosis

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.

A family of cross-reacting proteins secreted by Mycobacterium tuberculosis.

Cross‐reactions between live proteins actively secreted by Mycohacierium tuberculosis were studied by crossed immunoelectrophoresis, SDS ‐PAGE with immuiioblotting. and ELISA using polyelonal rabbit aniisera and mouse monoclonal aniibodies to the purified proteins. The monoclonal antibody HBT4 was demonstrated to react with the MPT5I protein. The 85A, 85B and 85C constituents of the M. tuberculosis and Mycobacterium bovis BCG antigen 85 complex cross‐react extensively, each of the components containing component‐specific as well as cross‐reacting epilopes. These components also cross‐reacted with MPT5I and MPT64.

Homology Between the MPB70 and MPB83 Proteins of Mycobacterium bovis BCG

Isolation of MPB83 from Mycobacterium bovis BCG Tokyo culture fluid is described. MPB70 and MPB83 have similar molecular mass as judged by SDS‐PAGE but differ in isoelectric points. Peptides isolated after CNBr cleavage of MPB83 revealed extensive homology as well as distinct differences from corresponding parts of the amino acid sequence deduced from the mpb70 gene cloned by Terasaka et al. Antibodies produced by immunization with MPB70 and MPB83 had distinctly different fine specificity revealing cross‐reactivity between the proteins. These findings indicate that two distinct, homologous genes code for these proteins. Sensitization with live BCG Tokyo also induced T cell responses to MPB83 with development of delayed type hypersensitivity in guinea pigs.

Delayed-type hypersensitivity to a recombinant mycobacterial antigen, MPB64, in guinea pigs sensitized to Mycobacterium tuberculosis or Mycobacterium bovis BCG.

Recombinant MPB64 (rMPB64), a mycobacterial antigen, was obtained from an Escherichia coli clone transformed with a recombinant expression vector, pMAL64c. The rMPB64 was examined for the activity to elicit delayed‐type hypersensitivity (DTH) in guinea pigs injected with live Mycobacterium tuberculosis H37Rv or live M. bovis BCG Tokyo. It was found that rMPB64 has the same reactivity as native MPB64 (nMPB64) or MPT64 (nMPT64) and the potency to elicit DTH was 13.4 times higher than that of PPD. Because MPB64 is secreted only by living M. tuberculosis and some strains of BCG, it is possible to use this antigen for the diagnosis of tuberculosis. J. Leukoc. Biol. 57: 221–225; 1995.

Immunogenic protein MPB57 from Mycobacterium bovis BCG: Molecular cloning, nucleotide sequence and expression

Using a single‐probe method, we have cloned the gene for an immunogenic MPB57 protein of Mycobacterium bovis BCG. The nucleotide sequence includes an ORF of 300 base pairs encoding a protein of 99 amino acids with an M r of 10 818. This cloned gene was expressed in an Escherichia coli expression vector to give a mature protein which reacted with a polyclonal antibody raised against MPB57.

Secretion of MPB64 antigen by a recombinant clone of Mycobacterium smegmatis:characterization and application for the diagnosis of tuberculosis.

MPB64, a specific antigen to Mycobaeterium tuberculosis complex (TB complex), was produced and secreted by a clone of M. smegmatis‐MPB64 where the structural gene of MPB64 was inserted using a new mycobacteria, E. coli shuttle plasmid pNIS vector. Antibodies against the recombinant MPB64 (rMPB64) were used for the reverse particle latex agglutination (RPLA) test to detect the MPB64 antigen rapidly. RPLA tests were applied to the stock cultures and the clinical isolates of mycobacteria to identify TB complex. RPLA with anti‐MPB64 antibody‐coated latex beads completely distinguished TB complex from other mycobacteria. Thus, it is suggested that RPLA with anti‐MPB64 antibody would be a new, easy and inexpensive method for the rapid diagnosis of tuberculosis.