Auxotrophic complementation as a selectable marker for stable expression of foreign antigens in Mycobacterium bovis BCG

Borsuk, Sibele; Mendum, Tom A.; Fagundes, Michel Quevedo; Michelon, Marcelo; Cunha, Cristina W.; McFadden, Johnjoe; Dellagostin, Odir A.

doi:10.1016/j.tube.2007.07.006

Cited by 34 publications

(43 citation statements)

References 25 publications

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“…A difference would be expected as BCG ΔleuD cells that lose the plasmid fail to survive due to the unavailability of exogenous leucine inside macrophage cells. In a similar experiment performed in mice, a high level of functional stability was also observed as all the recovered BCG ΔleuD transformed with vectors carrying lacZ gave rise to blue colonies when plated onto medium containing X-gal (Borsuk et al, 2007).…”

Section: Resultsmentioning

confidence: 60%

“…A BCG auxotrophic for the amino acid leucine was constructed by knocking out the leuD gene by unmarked homologous recombination as previously described (Borsuk et al, 2007). Expression of leuD on a plasmid (pUP410) not only allowed complementation, but also acted as a selectable marker (Borsuk et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

“…Expression of leuD on a plasmid (pUP410) not only allowed complementation, but also acted as a selectable marker (Borsuk et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

“…To assess functional stability of the leuD complementation system in vivo, the coding sequences for the L. interrogans antigens LipL32 and the non identical region of the LigA antigen (LigAni), together with the 18 kDa gene promoter from M. Leprae, were amplified by PCR as expression cassettes from pUS2000/lipL32 and pUS2000/ligAni vectors (Seixas et al, 2007b), and cloned into pUP410, an E. coli-mycobacteria shuttle vector that carries the leuD gene, which allows auxotrophic complementation of the BCG ΔleuD strain (Borsuk et al, 2007). PCR products were ligated into the KpnI site of the pUP410 vector, replacing the hyg R gene.…”

Section: Methodsmentioning

confidence: 99%

“…This instability is observed mainly when replicative vectors are used, which are lost during BCG replication in vivo (Edelman et al, 1999). Integrative vectors are more stable (Dellagostin et al, 1993 (Borsuk et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

et al. 2010

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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.

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Section: Resultsmentioning

confidence: 60%

Section: Methodsmentioning

confidence: 99%

“…Expression of leuD on a plasmid (pUP410) not only allowed complementation, but also acted as a selectable marker (Borsuk et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

et al. 2010

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Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro

Begnini

Rizzi

Campos

et al. 2012

Appl Microbiol Biotechnol

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BCG therapy remains at the forefront of immunotherapy for treating patients with superficial bladder cancer. The high incidence of local side effects and the presence of non-responder diseases have led to efforts to improve the therapy. Hence, we proposed that an auxotrophic recombinant BCG strain overexpressing Ag85B (BCG ∆leuD/Ag85B), could enhance the cytotoxicity to the human bladder carcinoma cell line 5637. The rBCG was generated using an expression plasmid encoding the mycobacterial antigen Ag85B to transform a BCG ∆leuD strain. The inhibitory effect of BCG ∆leuD/Ag85B on 5637 cells was determined by the MTT method, morphology observation and a LIVE/DEAD assay. Gene expression profiles for apoptotic, cell cycle-related and oxidative stress-related genes were investigated by qRT-PCR. Bax, bcl-2 and p53 induction by BCG ∆leuD/Ag85B treatment was evaluated by Western blotting. BCG ∆leuD/Ag85B revealed a superior cytotoxicity effect compared to the control strains used in this study. The results showed that the expression level of pro-apoptotic and cell cycle-related genes increased after BCG ∆leuD/Ag85B treatment, whereas the mRNA levels of anti-apoptotic genes decreased. Interestingly, BCG ∆leuD/Ag85B also increased the mRNA level of antioxidant enzymes in the bladder cancer cell line. Bax and p53 proteins levels increased following treatment. In conclusion, these results suggest that treatment with BCG ∆leuD/Ag85B enhances cytotoxicity for superficial bladder cancer cells in vitro. Therefore, rBCG therapy may have potential benefits in the treatment of bladder cancer.

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Recombinant Mycobacterium bovis BCG for immunotherapy in nonmuscle invasive bladder cancer

Begnini

Buss

Collares

et al. 2015

Appl Microbiol Biotechnol

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In the past three decades, intravesical instillation of Mycobacterium bovis bacille Calmette-Guérin (BCG) has been used for treating bladder cancer and it still remains at the forefront of immunotherapy for cancer patients. Although BCG-based therapy is the most effective intravesical therapy for this kind of tumor and represents the only agent known to reduce progression into muscle invasive bladder cancer, BCG is ineffective in approximately 30-40 % of cases and disease recurs in up to 50 % of patients. Since that BCG is considered an effective vehicle for delivery of antigens due to its unique characteristics, the genetic manipulation of these mycobacteria has been appealing in the search for less toxic and more potent therapeutic agents for bladder cancer immunotherapy. Herein, we discuss current advances in recombinant BCG construction, research, concerns, and future directions to promote the development of this promising immunotherapeutic approach for bladder cancer.

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Auxotrophic complementation as a selectable marker for stable expression of foreign antigens in Mycobacterium bovis BCG

Cited by 34 publications

References 25 publications

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro

Recombinant Mycobacterium bovis BCG for immunotherapy in nonmuscle invasive bladder cancer

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