Engineering a pro-apoptotic BCG strain to improve efficacy of the current tuberculosis vaccine

Bacillus Calmette-Guérin (BCG), introduced almost 100 years ago, is the only vaccine designed to prevent tuberculosis (TB). BCG effectively protects newborns from meningeal TB yet fails to prevent adult pulmonary TB. In fact, TB kills 1.3 million people annually in areas where BCG vaccination is widely practiced. Thus, more efficient TB vaccines are urgently needed. We and others have shown that BCG possesses the same virulence traits of Mycobacterium tuberculosis, in particular attenuation of essential macrophage functions such as phagosome maturation and antigen presentation. One of these studies revealed that defect in antigen presentation is largely due to down-regulation of the macrophage’s cysteine protease cathepsin S (CatS), which leads to prevention of MHC II molecule maturation and proper antigen presentation. Recent studies also suggested a potential role for cysteine proteases in the regulation of apoptosis, a key cellular process used by the macrophage to i) contain and process ingested bacteria and ii) facilitate cross-talk antigen presentation between the macrophage and dendritic cells. To reverse the phenotype of vaccine-mediated macrophage attenuation, we engineered a novel BCG strain that expresses and secretes active CatS (rBCG-CatS). Since caspase-3 plays a central role in the execution of apoptosis, we also constructed a BCG strain that secrets an active form of caspase-3 (rBCG-C3). Macrophages infected with either recombinant strain elicited a pro-apoptotic phenotype as indicated by increased levels of annexin V surface staining, PARP degradation, and caspase-3 cleavage compared to parental BCG. Furthermore, macrophage transcriptomic profiling revealed that rBCG-CatS up-regulates key pro-apoptotic genes and down-regulates anti-apoptotic genes, which were further confirmed by RT-qPCR analyses. Consistent with these findings, mice vaccinated with rBCG-CatS or rBCG-C3 showed increased antigen-specific CD4+ and CD8+ T-cell responses, as well as enhanced cytokine production and proliferation upon ex vivo re-stimulation. Of particular note, immunogenicity responses from mice vaccinated with rBCG-C3 exceeded the effects observed with rBCG-CatS, demonstrating that induction of apoptosis is key to achieving high immunogenicity of TB vaccines. Collectively, we have shown that by modifying BCG we can promote key host traits that confer high potential in improving efficacy of the TB vaccine.Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat