Cloning of the Gene Encoding a 22-Kilodalton Cell Surface Antigen ofMycobacterium bovisBCG and Analysis of Its Potential for DNA Vaccination against Tuberculosis

Abstract

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 gt11. 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.Tuberculosis remains a widespread and lethal infectious disease affecting millions of people worldwide. The World Health Organization estimates that there are about 8 million new cases each year and that the disease is responsible for at least 3 million deaths annually (5,12,23,34). The eradication of tuberculosis by early diagnosis, efficient therapy, and effective prophylactic vaccination remains an important goal. For the latter purpose, characterization of purified antigens implicated in protective immunity against mycobacterial infections is essential. The protective antigens of Mycobacterium tuberculosis are still not precisely defined, but several observations in experimental animal models have indicated that they predominantly reside within a pool of secreted or surface-exposed proteins that can be found in mycobacterial culture filtrate (CF) (2,15,16). Using the powerful technique of DNA vaccination, we and others have shown so far that at least four antigens present in M. tuberculosis CF are able to induce a protective immunity in mice against M. tuberculosis H37Rv infection: the 38-kDa PstS-1 and 40-kDa PstS-3 phosphate binding proteins (36, 41) and the 30-and 32-kDa components of the antigen 85 complex (17,22). Protective immune responses can also be induced with plasmid DNA encoding the 65-kDa heat shock protein, which is generally considered to be a cytosolic protein but is very abundant in CF from stressed mycobacterial cultures (37)...

LppX is a lipoprotein required for the translocation of phthiocerol dimycocerosates to the surface of Mycobacterium tuberculosis

“…This gene encodes a protein with a signal sequence and lipoprotein lipid attachment motif (PS00013) typically found in prokaryotic lipoproteins and thus presumably encodes a lipoprotein precursor. LppX, which was studied earlier for its immunogenic properties (Lefèvre et al , 2000; Al‐Attiyah and Mustafa, 2004), was found in the membrane, cell wall, cell surface and culture medium of Mycobacterium bovis BCG and M. tuberculosis H37Rv (Lefèvre et al , 2000). Thus, as a potential lipoprotein, LppX seems not to remain anchored into the cytoplasmic membrane but, instead, to be exported to the outermost layers of the mycobacterial cell envelope.…”

Virulence factors of theMycobacterium tuberculosiscomplex

“…The protein is secreted and is also highly expressed in the bacterial cell wall and membrane compartment but has not been found in the cytosol 126 . STM strategy has established the link of this Lpp to virulence.…”

Lipoproteins ofMycobacterium tuberculosis: an abundant and functionally diverse class of cell envelope components

“…Rv1411c induced strong delayed-type hypersensitivity and Th1-type immune responses in immunised mice [96]. Likewise, DNA vaccination of mice with the M. bovis homologue of Rv2945c was unsuccessful in conferring protective immunity [95]. Likewise, DNA vaccination of mice with the M. bovis homologue of Rv2945c was unsuccessful in conferring protective immunity [95].…”

“…It remains to be determined if significant numbers of Lpp are associated with the outer mycolate-based lipid layers of the mycobacterial cell envelope. has been surface-localised by flow cytometry and this antigen is also released into culture supernatants [95]. Thus, the Rv0432 SOD has been immunolocalised to the peripheral layers of M. tuberculosis H37Rv [59] and the MPB83 homologue of MPT83 has been localised to the surface of M. bovis BCG (see Section 4.3.1; [73,74]).…”