title: Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG.
abstract: 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.
authors.affiliation: Centro de Biotecnologia, Campus Universitário, Universidade Federal de Pelotas, Pelotas, RS 96010-900, Brazil.
authors.forename: Fabiana K
authors.initials: FK
authors.lastname: Seixas
authors.forename: Sibele
authors.initials: S
authors.lastname: Borsuk
authors.forename: Michel Q
authors.initials: MQ
authors.lastname: Fagundes
authors.forename: Daiane D
authors.initials: DD
authors.lastname: Hartwig
authors.forename: Everton F
authors.initials: EF
authors.lastname: Da Silva
authors.forename: Gustavo M
authors.initials: GM
authors.lastname: Cerqueira
authors.forename: Odir A
authors.initials: OA
authors.lastname: Dellagostin
mesh_terms: D000818:Animals; D001500:BCG Vaccine / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics* / Q000276:immunology; D007921:Leptospira interrogans / Q000235:genetics* / Q000276:immunology; D008074:Lipoproteins / Q000235:genetics / Q000276:immunology; D009163:Mycobacterium bovis / Q000235:genetics* / Q000276:immunology; D010957:Plasmids / Q000235:genetics / Q000276:immunology
chemical_list: D001500:BCG Vaccine; D001425:Bacterial Outer Membrane Proteins; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins