Development of a recombinant protein vaccine for Chlamydia trachomatis

There is a clear need for a vaccine to protect against Chlamydia trachomatis (Ct), a sexually transmitted bacterium causing an ongoing epidemic associated with reproductive complications. IFNγ-producing CD4 T cells (Th1 & Th1/17 cells) are required for protection from Ct, which grows and replicates intracellularly, whereas antibodies provide complementary protection in the presence of IFNγ. Our studies of Chlamydia-infected women and mice have identified Chlamydial Protease Activity Factor (CPAF) as an immunoprevalent and immunodominant antigen for CD4 T cells and B cells, making 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 requires potent adjuvants and mucosal vaccination to imprint FGT homing or generate resident memory T cells. Our preliminary data indicate that intranasal immunization of female mice with CPAF conjugated to the TLR9 agonist, CpG, combined with the STING agonist, cyclic-di-AMP (CDA) in the squalene oil-in-water nanoemulsion AddaS03 (AS03), induces high levels of CPAF-specific CD4 T cells and protection from infection and pathology. Our novel covalent CPAF-adjuvant conjugation approach enhances antigen- presenting cell activation through coincident stimulation leading to the potential for dose sparing, reduced toxicity, enhanced immunogenicity, superior efficacy, and reduced cost of goods. This proposal incorporates a tiered approach to determine if refinements using innovative new generation TLR7/9 and STING agonists delivered via mucosal routes will enhance protection via the following specific aims: Aim 1. Complete product development activities of 2nd generation dual agonist CPAF vaccines. (i) Refine the multi-adjuvant CPAF-CpG+CDA+AS03 vaccine. (ii) Covalently conjugate TLR7, -9 and STING agonists to CPAF. (iii) Perform in-vivo formulation, toxicology, and preliminary immunogenicity analysis of candidate vaccines. Aim 2. Evaluate efficacy of the 2nd generation candidates in the female murine challenge model and immunogenicity in male mice. (i) Determine most efficacious vaccine regimen (candidate and route). (ii) Determine protection against female infertility and vaccine immunogenicity in males. (iii) Determine duration of humoral and cell- mediated responses by top candidate, and the contribution of additional boosting. Aim 3. Vaccine process development. (i) Optimize upstream production, downstream purification, and conjugation of CPAF. (ii) Develop analytical assays and qualify as suitable for intended use. (iii). Develop formulation and evaluate vaccine stability. (iv) Conduct a non-GLP toxicology assessment of the final vaccine candidate. Accomplishing these three Specific Aims will result in a Chlamydia vaccine candidate ready for tech transfer to a qualified CMO, GMP production, and early-phase human clinical trials.