Characterizing TLR4 agonist EmT4™ as an anti-Mycobacterium tuberculosis vaccine adjuvant

Tuberculosis (TB) has once again become the leading cause of death from infectious disease worldwide, highlighting the urgent need for more effective vaccines. An ideal subunit TB vaccine requires immunogenic, affordable, and widely accessible antigens and adjuvants. In this study, we evaluated a synthetically produced TLR4 agonist, Monophosphoryl lipid A (SyMLP), formulated in an oil-in-water emulsion (EmT4™), combined with selected fusion proteins, for its ability to elicit protective immune responses in C57BL/6 mice challenged with Mycobacterium tuberculosis (M.tb) strains HN878 and H37Rv.

We found that EmT4™ enhanced the activation of bone marrow-derived macrophages and dendritic cells from C57BL/6 mice, as shown by increased expression of CD40, CD86, and MHCII markers via flow cytometry. A scaled SAG agonist tolerability study confirmed that EmT4™ did not trigger any adverse safety signals.

In immunogenicity studies, mice immunized three times at three-week intervals with the ID93 antigen formulated with EmT4™ showed significantly elevated levels of proinflammatory cytokines and ID93-specific IgG antibodies, both before and after M.tb challenge, compared to saline controls. Upon ex vivo restimulation with ID93, splenocytes and lung cells demonstrated robust polyfunctional CD4+ T-helper 1 responses.

Importantly, vaccination with ID93 + EmT4™ significantly reduced bacterial loads in C57BL/6 mice four weeks after infection. Furthermore, EmT4™ combined with the next-generation fusion protein ID91 provided protective immunity against M.tb HN878 in both young (6–8 weeks) and aged (20-month-old), immunocompromised Beige mice.

These findings support EmT4™ as a promising synthetic adjuvant candidate that can help replenish the preclinical TB vaccine pipeline and meet the broader needs of global TB prevention efforts.