Intranasal immunization with CPAF combined with cyclic-di-AMP induces a memory CD4 T cell response and reduces bacterial burden following intravaginal infection with Chlamydia muridarum

(Ct) is the most common bacterial sexually transmitted infection globally, and a vaccine is urgently needed to stop transmission and disease. Chlamydial Protease Activity Factor (CPAF) is an immunoprevalent and immunodominant antigen for CD4 T cells and B cells, which makes it a strong vaccine candidate. Due to the tolerogenic nature of the female genital tract (FGT) and its lack of secondary lymphoid tissue, effective induction of protective cell-mediated immunity will likely require potent and safe mucosal adjuvants. To address this need, we produced CPAF in a cell-free protein synthesis platform and adjuvanted it with the TLR9-agonist CpG1826, STING (stimulator of interferon genes) agonist cyclic-di-AMP (CDA), and/or the squalene oil-in-water nanoemulsion, AddaS03. We determined that intranasal immunization with CPAF plus CDA was well tolerated in female mice, induced CD4 T cells that produced IL-17A or IFNγ, significantly reduced bacterial shedding, and shortened the duration of infection in mice intravaginally challenged with