| Resum: |
The development of a safe, effective, and scalable bivalent vaccine targeting both HIV and tuberculosis (TB) remains a global health priority, particularly in low-resource settings where co-infection is prevalent. Recombinant Mycobacterium bovis BCG (rBCG) has emerged as a promising vaccine vector due to its proven neonatal safety, heat stability, and immunostimulatory properties. In this preclinical study, we propose the construction and evaluation of rBCG vaccine candidates expressing conserved regions of simian immunodeficiency virus (SIVmac251) as a surrogate for HIV, in the cynomolgus macaques (Macaca fascicularis) model. To address the genetic diversity and immune escape of HIV, we use two synthetic immunogens, SIVconsv. gag and SIVconsv. pol, previously designed by our group, comprising the most functionally conserved regions of the SIVmac251 proteome. These sequences, which contain the kanamycin resistance gene and the complementing lysine gene, were codon-optimized, HA-His-tagged, and individually cloned into the integrative shuttle vector pGV223. Mutant lysine auxotrophic M. bovis BCGΔLys strains were transformed via electroporation and selected using antibiotic resistance and lysine depletion. Recombinant colonies were phenotypically characterized and genetically confirmed by PCR analyses. Heterologous protein expression was evaluated by Dot-Blot and Western Blot assays. The resulting rBCG strains, rBCG:SIVconsv. gag and rBCG:SIVconsv. pol, will undergo in vitro genetic stability testing. A working vaccine stock will be generated and colony-forming units (CFU) will be titered for downstream in vivo immunogenicity studies in M. fascicularis, evaluating both SIV-specific and Mycobacterium tuberculosis-specific immune responses. This project aims to establish a versatile rBCG-based platform for bivalent vaccine development, integrating improved genetic stability. By targeting conserved viral regions and using a translational non-human primate model, this approach holds potential for future application in dual HIV/TB vaccination strategies. |
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