AbstractThere is an urgent need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis. Advax™ is a novel adjuvant based on delta inulin microparticles that enhances immunity with a minimal inflammatory profile and has entered human trials to protect against viral pathogens. In this report we determined if Advax displays broad applicability against important human pathogens by assessing protective immunity against infection with M. tuberculosis. The fusion protein CysVac2, comprising the M. tuberculosis antigens Ag85B (Rv1886c) and CysD (Rv1285) formulated with Advax provided significant protection in the lungs of M. tuberculosis-infected mice. Protection was associated with the generation of CysVac2-specific multifunctional CD4⁺ T cells (IFN-γ⁺TNF⁺IL-2⁺). Addition to Advax of the TLR9 agonist, CpG oligonucleotide (Advax^CpG), improved both the immunogenicity and protective efficacy of CysVac2. Immunisation with CysVac2/Advax^CpG resulted in heightened release of the chemoattractants, CXCL1, CCL3, and TNF, and rapid influx of monocytes and neutrophils to the site of vaccination, with pronounced early priming of CysVac2-specific CD4⁺ T cells. As delta inulin adjuvants have shown an excellent safety and tolerability profile in humans, CysVac2/Advax^CpG is a strong candidate for further preclinical evaluation for progression to human trials.