Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis

Attenuated mutants of Mycobacterium tuberculosis represent potential vaccin e candidates for the prevention of tuberculosis, It is known that auxotroph s of a variety of bacteria are attenuated in vivo and yet pro,ide protectio n against challenge with wild-type organisms. A leucine auxotroph of M. tub erculosis was created by allelic exchange, replacing wild-type leuD (Rv2987 c), encoding isopropyl malate isomerase, with a mutant copy of the gene in which 359 bp had been deleted, creating a strain requiring exogenous leucin e supplementation for growth in vitro. The frequency of reversion to protot rophy was <10(-11). In contrast to wild-type M. tuberculosis, the Delta leu D mutant was unable to replicate in macrophages in vitro. Its attenuation i n vivo and safety as a vaccine were established by the fact that it caused no deaths in immunodeficient SCID mice. Complementation of the mutant with wild-type leuD abolished the requirement for leucine supplementation and re stored the ability of the strain to grow both in macrophages and in SCID mi ce, thus confirming that the attenuated phenotype was due to the Delta leuD mutation. As a test of the vaccine potential of the leucine auxotroph, imm unocompetent BALB/c mice, susceptible to fatal infection with wild-type M. tuberculosis, were immunized with the Delta leuD mutant and subsequently ch allenged with virulent M. tuberculosis by both the intravenous and aerosol routes. A comparison group of mice was immunized with conventional Mycobact erium bovis BCG vaccine. Whereas all unvaccinated mice succumbed to intrave nous infection within 15 weeks, mice immunized with either BCG or the Delta leuD mutant of M. tuberculosis exhibited enhanced and statistically equiva lent survival curves. However, the leuD auxotroph was less effective than l ive BCG in reducing organ burdens and tissue pathology of mice challenged b y either route. We conclude that attenuation and protection against M. tube rculosis challenge can be achieved with a leucine auxotroph and suggest tha t to induce optimal protection, attenuated strains of M. tuberculosis shoul d persist long enough and be sufficiently metabolically active to synthesiz e relevant antigens for an extended period of time.