BioBricks: desenho racional de vacinas de Mycobacterium bovis BCG recombinante

Abstract

Since the 90s, Mycobacterium bovis BCG has been genetically engineered for use as a vaccine vehicle. However, the lack of standardization in cloning sequences from mycobacteria to construct rBCG results in poorly reproducible protocols and unpredictable efficacy. BioBricks strategy allows to standardize the construction of basic parts of DNA by the use of prefix and suffix tags flanking the target sequence. In this thesis, the BioBrick technology was applied to develop a kit, named a toolbox, containing mycobacterial DNA sequences, important for construction of rBCG strains. Additionally, BioBrick-compatible episomal vectors able to replicate in BCG were developed and characterized by fluorescence microscopy and flow cytometry using the reporter protein eGFP. These vectors were applied for the expression of chimeric proteins from Leptospira interrogans in rBCG, which were evaluated as vectorized vaccines against leptospirosis in hamsters. The kit constructed for mycobacteria according to the BioBricks standard contains ten promoters, two coding sequences, a base plasmid, a reporter gene, a kanamycin resistance gene and the origin of replication in mycobacteria. All these DNA sequences are functional and compatible with each other. In addition, the functionality of seven episomal vectors constructed using different promoters was demonstrated by detecting eGFP expression in BCG grown in vitro and inside macrophages. These vectors are available for expression of any heterologous antigens in BCG, cloned with the BioBricks standard. Vaccines vectorized by BCG using the plasmids constructed in this study were able to protect hamsters against lethal challenge with L. interrogans and from the renal carrier status of this spirochete. Taken together, these findings represent substantial progress for construction of rBCG strains, bringing facility for this process, as well as highlight the potential of BCG as a vaccine vehicle against leptospirosis.