Deletion Of Nuog From The Vaccine Candidate Mycobacterium Bovis Bcg Delta Urec::Hly Improves Protection Against Tuberculosis

The current tuberculosis (TB) vaccine, Mycobacterium bovis Bacillus Calmette-Guerin (BCG), provides insufficient protection against pulmonary TB. Previously, we generated a listeriolysin-expressing recombinant BCG strain, which to date has successfully completed phase I and phase IIa clinical trials. In an attempt to further improve efficacy, we deleted the antiapoptotic virulence gene nuoG, encoding NADH dehydrogenase 1 subunit G, from BCG Delta ureC::hly. In vitro, deletion of nuoG unexpectedly led to strongly increased recruitment of the autophagosome marker LC3 to the engulfed vaccine, suggesting that nuoG also affects xenophagic pathways. In mice, BCG Delta ureC::hly Delta nuoG vaccination was safer than BCG and improved protection over that of parental BCG Delta ureC::hly, significantly reducing TB load in murine lungs, ameliorating pulmonary pathology, and enhancing immune responses. Transcriptome analysis of draining lymph nodes after vaccination with either BCG Delta ureC::hly or BCG Delta ureC::hly Delta nuoG demonstrated earlier and stronger induction of immune responses than that with BCG SSI and suggested upregulation of inflammasome activation and interferon-induced GTPases. In summary, BCG Delta ureC::hly Delta nuoG is a promising next-generation TB vaccine candidate with excellent efficacy and safety.IMPORTANCE Autophagy and apoptosis are fundamental processes allowing cells to degrade their components or kill themselves, respectively. The immune system has adopted these mechanisms to eliminate intracellular pathogens. Residing in host cells, the causative agent of TB, Mycobacterium tuberculosis, has evolved strategies to set cellular programs of autophagy and apoptosis "on hold." The mycobacterial gene nuoG was found to prevent host cell apoptosis. We have deleted nuoG in the live vaccine candidate BCG Delta ureC::hly, which is in phase II clinical development, to leave cellular apoptosis "on go" upon immunization. In preclinical models, this strategy boosted immunity and improved protection from M. tuberculosis infection. Unexpectedly, we obtained compelling evidence that mycobacterial nuoG facilitates inhibition of autophagic pathways, suggesting a new role for this gene in the host-pathogen interplay in TB.