Search

Using spleen cells from mice vaccinated with live Mycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687-2693, 1993). These monoclonal antibodies were used to screen an M. bovis BCG genomic library made in phage lambdagt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contained a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosis challenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosis challenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test.

We describe the identification of the gene encoding an immunodominant 32-kilodalton (kDa) protein of Mycobacterium tuberculosis. The 32-kDa antigen is abundantly secreted into the culture supernatant of a variety of mycobacteria and appears to be a major stimulant of cellular and humoral immunity against mycobacteria. Recombinant clones expressing a 140- or 125-kDa beta-galactosidase fusion protein reactive with rabbit polyclonal anti-32 kDa protein serum were detected. The corresponding DNA sequence contains a 1,008-base-pair coding region. The deduced amino acid sequence corresponds to a 336-residue protein including the previously determined NH2-terminal sequence of the 32-kDa protein (J. De Bruyn, K. Huygen, R. Bosmans, M. Fauville, R. Lippens, J. P. Van Vooren, P. Falmagne, M. Weckx, H. G. Wiker, M. Harboe, and M. Turneer, Microb. Pathog. 2:351-366, 1987). Upstream of this NH2-terminal region, the gene codes for a signal peptide required for the secretion of a 294-amino-acid-long mature protein. A putative promoter sequence could be located upstream of the open reading frame. Comparison of the M. tuberculosis 32-kDa antigen with the Mycobacterium bovis BCG alpha-antigen (K. Matsuo, R. Yamaguchi, A. Yamazaki, H. Tasaka, and T. Yamada, J. Bacteriol. 170:3847-3854, 1988) revealed 73.8% homology between DNA sequences and 72.8% homology between amino acid sequences (signal and mature protein). Finally, the 140-kDa fusion protein could selectively be recognized by human tuberculous sera. This result confirms our previous finding that the 32-kDa antigen could be a valuable tool for the serological diagnosis of tuberculosis. Moreover, the availability of recombinant proteins opens perspectives for the localization of relevant B- and T-cell epitope regions on the 32-kDa antigen.