Vaccination with the combination of Ag85B-TB10.4 and IC31 generated a high amount of polyfunctional CD4 + T cells expressing high levels of IFN-³, TNF-±, and IL-2. This in turn led to significant protection against infection with M. tuberculosis in the mouse aerosol challenge model of tuberculosis. Importantly, our results also showed that both the immunogenicity of the vaccine and its ability to protect against TB infection was highly dependent on the antigen dose. Thus, whereas the standard antigen dose of 5.0µg, as well as 15.0µg, did not induce significant protection against M. tuberculosis, reducing the dose to 0.5µg increased both the immunogenicity of the vaccine as well as its protective efficacy to a level comparable to that observed in BCG vaccinated mice. Thus, the adjuvant IC31®, with the optimal antigen dose, can induce a strong protective Th1 response against M. tuberculosis.