Abstract
Besides being the most widely used vaccine directed against tuberculosis (TB) worldwide, Mycobacterium bovis BCG is also the most controversial vaccine in current use. Its protective efficacy varies widely in different parts of the world. One approach to improving the current BCG vaccine might be to produce recombinant BCG strains that express major antigens encoded by genes that are present in the M. tuberculosis-specific region of difference 1 (RD1), such as pe35, cfp10, and esat6. In this study, pe35, cfp10, and esat6 genes were cloned into shuttle plasmid pDE22 to generate the recombinant plasmids PDE22-PE35, PDE22-CFP10, and PDE22-ESAT6, which were electroporated into BCG to generate recombinant BCGs (rBCGs). The cellular immune responses (antigen-induced proliferation and secretion of selected T helper 1 [Th1], Th2, and anti-inflammatory cytokines, i.e., gamma interferon [IFN-γ], interleukin 5 [IL-5], and IL-10, respectively) that are specific to the proteins of cloned genes were studied by using spleen cells from mice immunized with native BCGs and rBCGs and synthetic peptides covering the protein sequence of the cloned genes. The results showed that the spleen cells did not secrete IL-5, whereas IL-10 was secreted in response to peptides of all three proteins from mice immunized with rBCGs only, suggesting expression of the cloned genes and in vivo priming of spleen cells to the expressed proteins. However, in Th1 cell assays that correlate with protective cellular immune responses, i.e., antigen-induced proliferation and IFN-γ secretion, only mice immunized with rBCG-pDE22-PE35 yielded positive responses to the peptides of PE35. These results suggest that rBCG-PDE22-PE35 is the only one of the three vaccines used in this work that is worthy of consideration as a new vaccine candidate against TB.