RESUMO
Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18 kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.
Assuntos
Vacina BCG/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Vetores Genéticos/genética , Leptospira interrogans/genética , Mycobacterium bovis/genética , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Cricetinae , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/imunologia , Leptospira interrogans/imunologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Mycobacterium bovis/imunologia , Plasmídeos/genética , Plasmídeos/imunologiaRESUMO
Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.
Assuntos
Animais , Cricetinae , Vacina BCG/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Vetores Genéticos/genética , Leptospira interrogans/genética , Mycobacterium bovis/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/imunologia , Leptospira interrogans/imunologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Mycobacterium bovis/imunologia , Plasmídeos/genética , Plasmídeos/imunologiaRESUMO
The search for leptospiral antigens (L. interrogans serogroup icterohaemorrhagiae) was carried out in 24 guinea pigs experimentally inoculated with 1 ml of culture containing 10(7)-10(8) leprospires and sequentially sacrificed from the first until the 6th day of infection. Semiquantitative analysis of histopathological variables comprising kidney interstitium, tubules and glomeruli was done in 1 micron sections of tissue embedded in glycolmetacrylate. Leptospiral antigen (LAg) and its glycolipoprotein (GLP) expression were detected through PAP in paraffin embedded tissue. The mild interstitial involvement of the kidney, manifested chiefly by oedema and focal interstitial nephritis seen at the 4th day, progressed to tubular damage at the 6th day, characterized by either swelling or cytoplasmic acidophilia of epithelial cells with loss of cell cohesion and sloughing of cells into the tubular lumina. Brush border alterations and mitochondrial changes were observed. Endothelial cell injury was noted in the interstitial vessels. LAg expression was parallel to the kidney changes: small deposits of elongated forms of LAg were detected at the 4th day either within the vascular lumen or free in the interstitium. A rise in the antigen expression was observed at the 5th day when it was seen either around tubules or in their walls. LAg was detected inside the tubular lumina at the 6th day of infection when granular LAg and GLP were abundant. This sequence reproduces the pathway of leptospires in the kidney and the crescent amounts of antigens detected toward the end of the experiment, with antigen concentration in cases of major tissue damage suggesting a direct action of the microorganisms and/or their products in the pathogesis of the lesions.