CpG-mediated TLR9 signaling pathway enhances the efficacy of the OprF/PcrV DNA vaccine with cGAS-STING-activating properties.

Aims: This study aimed to overcome the limited protective efficacy of the bivalent DNA vaccine (DNA-OprF/PcrV) against Pseudomonas aeruginosa (PA) infection by developing a complexed adjuvant strategy to enhance immunogenicity and protection, providing a novel clinical candidate vaccine.

Materials And Methods: We formulated a bivalent DNA vaccine encoding PA antigens OprF and PcrV (DNA-OprF/PcrV) complexed with the TLR9 agonist CpG adjuvant (DNA-OprF/PcrV + CpG). In vitro mechanistic studies assessed synergistic pathway activation and dendritic cell maturation, while murine models evaluated humoral immunity (antibody titers), cellular immunity (Th1/CTL responses), and protective efficacy via pulmonary infection models with bacterial burden quantification and inflammation analysis.

Key Findings: The CpG-adjuvanted vaccine synergistically activated both cGAS-STING (vaccine-mediated) and TLR9 (CpG-mediated) pathways, significantly enhancing dendritic cell maturation and innate immunity. Consequently, it amplified adaptive immunity, including potentiated Th1 polarization and cytotoxic T lymphocyte (CTL) activity alongside elevated PA-specific antibody titers. In pulmonary infection models, this formulation conferred superior protection marked by significantly reduced bacterial burden and attenuated inflammation compared to the non-adjuvanted vaccine.

Significance: This work demonstrates that the CpG-adjuvanted DNA vaccine complex overcomes DNA vaccine limitations through dual-pathway synergy, providing a clinically translatable strategy against multidrug-resistant PA infection and pioneering a cGAS-STING/TLR9 co-activation paradigm for antibacterial vaccine design.