Immunogenicity and Protective Effects of an Ag85B Tuberculosis Subunit Vaccine Formulated with Synthetic TLR4 Agonists in BCG-Boosted Mice

Background/objectives Tuberculosis (TB) remains a major global health challenge, and the Bacillus Calmette-Guérin (BCG) vaccine has limited efficacy against adult pulmonary disease. Protein subunit vaccines are a promising alternative but require strong adjuvants to induce cell-mediated immunity. Synthetic agonists targeting toll-like receptor 4 (TLR4) and stimulators of interferon genes (STINGs) have emerged as effective immunostimulants. Therefore, we aimed to evaluate the immunogenicity and protective efficacy of Ag85B-based subunit vaccines formulated with synthetic TLR4 and STING agonists in a BCG-boosted mouse model. Methods Three synthetic adjuvants-QTP709-1, QTP709-3, and QTP701-were formulated as oil-in-water emulsions containing distinct surfactant and immunostimulant components. The potential of vaccine formulations to activate dendritic cells (DCs) and elicit Ag85B-specific immune responses, including IgG subclass levels, interferon-γ (IFN-γ) enzyme-linked immunosorbent spots, and polyfunctional T-cell responses, was assessed by flow cytometry. Protective efficacy was evaluated based on pulmonary bacterial burden and histopathology following Mycobacterium tuberculosis (M. tb) Erdman challenge. Results All formulations promoted DC maturation and enhanced antigen-specific immune responses. Each adjuvant elicited strong Ag85B-specific humoral immunity, increased IFN-γ secretion, and polyfunctional CD4 + and CD8 + T cells co-producing IFN-γ, TNF-α, and interleukin-2. Among them, QTP709-1 was associated with increased levels of chemokine receptor 5-associated chemokines and showed a trend toward reduced lung bacterial burden and histopathological inflammation following M. tb challenge. Conclusions Synthetic TLR4 and STING agonists were associated with enhanced immunogenicity of TB subunit vaccines and showed evidence of protective potential, with TLR4-based formulations exhibiting more pronounced immunological responses. QTP709-1 exhibited strong immunostimulatory and protective effects, supporting its potential as a candidate adjuvant for next-generation TB vaccines.