Construction and characterization of novel Mycobacterium tuberculosis-derived triple and quadruple knockout vaccines against tuberculosis

Tuberculosis (TB) is a deadly disease that claims the lives of over a million people each year worldwide. The Bacille Calmette-Guérin vaccine has long been used to protect against TB, but it produces variable effects across different populations and fails to protect against adult pulmonary TB. Therefore, there is an urgent need for alternative vaccines that can offer better protection. We have developed a strategy for the rational deletion of virulence-related genes in Mycobacterium tuberculosis (Mtb) to create hyperattenuation that also enhances immunogenicity. Previously, we generated both single (∆fbpA) and double knockout (DKO) (∆fbpA-∆sapM) mutants of Mtb and assessed their immunogenicity and efficacy using mice. Herein, we have created triple knockout (TKO) and quadruple knockout (QKO) strains to enhance the immunogenicity and safety of the DKO strain by deleting the zmp1 and dosR genes. The resulting TKO strains, TKO-Z (∆fbpA-∆sapM-∆zmp1) and TKO-D (∆fbpA-∆sapM-∆dosR), and the QKO strain (∆fbpA-∆sapM-∆zmp1-∆dosR), were evaluated for their immunogenicity and safety in mice. Whereas TKO-Z and QKO strains exhibited superior immunogenicity compared to the DKO strain, their protective efficacy in mice was comparable. However, survival studies involving SCID mice indicated that the QKO strain was highly attenuated. Therefore, rational deletion of genes in Mtb seems to be an innovative approach for developing safer and more efficacious vaccines against TB.