Cloning of the gene encoding a 22-kilodalton cell surface antigen of Mycobacterium bovis BCG and analysis of its potential for DNA vaccination against tuberculosis

Using spleen cells from mice vaccinated with live Mycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-k Da 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 lambda gt11, The g ene encoding a 233-amino-acid (aa) protein, including a putative 26-aa sign al sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contai ned a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 rec ognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also hig hly expressed in the bacterial cell wall and membrane compartment but not i n the cytosol, C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid D NA encoding the 22-kDa protein and analyzed for immune response and protect ion 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 interf eron secretion, was only modest, and no protection against intravenous M. t uberculosis challenge was observed in any of the three mouse strains tested , Therefore, the 22-kDa antigen seems to have little potential for a DNA va ccine against tuberculosis, but it may be a good candidate for a mycobacter ial antigen detection test.