AbstractTuberculosis (TB) remains a major cause of mortality and morbidity worldwide, yet current control strategies, including the existing BCG vaccine, have had little impact on disease control. The tubercle bacillus modifies protein expression to adapt to chronic infection of the host, and this can potentially be exploited to develop novel therapeutics. We identified the gene encoding the first step of the Mycobacterium tuberculosis sulphur assimilation pathway, cysD, as highly induced during chronic infection in the mouse lung, suggesting therapies based on CysD could be used to target infection. Vaccination with the composite vaccine CysVac2, a fusion of CysD and the immunogenic Ag85B of M. tuberculosis, resulted in the generation of multifunctional CD4⁺ T cells (interferon (IFN)-γ⁺TNF⁺IL-2⁺IL-17⁺) in the lung both pre- and post-aerosol challenge with M. tuberculosis. CysVac2 conferred significant protection against pulmonary M. tuberculosis challenge and was particularly effective at controlling late-stage infection, a property not shared by BCG. CysVac2 delivered as a booster following BCG vaccination afforded greater protection against M. tuberculosis challenge than BCG alone. The antigenic components of CysVac2 were conserved amongst M. tuberculosis strains, and protective efficacy afforded by CysVac2 was observed across varying murine MHC haplotypes. Strikingly, administration of CysVac2 to mice previously infected with M. tuberculosis reduced bacterial load and immunopathology in the lung compared with BCG-vaccinated mice. These results indicate that CysVac2 warrants further investigation to assess its potential to control pulmonary TB in humans.