Chlamydia trachomatis (Ct) is the most frequent bacterial sexually transmitted pathogen, and can lead to ectopic pregnancy and infertility. Antibiotics cure infection but may not ameliorate disease and a vaccine is urgently needed. Success in preclinical animal studies is essential for vaccines to move to human clinical trials. Pigs are affordable, highly biologically relevant, and the natural host to the prevalent pathogen Chlamydia suis (Cs) – a chlamydia species closely related to Ct. Pigs are also susceptible to Ct; and protective CD4 T cells from Cs pre- exposed pigs cross-react to Ct. This cross-reaction makes Cs pre-exposed pigs a highly valuable animal model for Ct vaccine development: Vaccination of Cs-pre-exposed outbred pigs can simulate Ct vaccination of Ct pre- exposed humans – the target population of clinical phase III Ct vaccination trials. Dr. Kaeser has expertise in performing Cs and Ct vaccination and challenge trials in Cs pre-exposed pigs and in detailed analyses of Ct infection and immunity. Our long-term goal is to use this model to develop Ct vaccines and to offer the model to other researchers as a Ct vaccine testing platform. This study has two main goals: Use Cs pre-exposed pigs to develop a Ct vaccine candidate with novel vaccine formulations and Ct antigens, and provide an in-depth understanding of protective vaccine-induced immune mechanisms. The study is divided into three phases: Phase I will determine the most immunogenic parenteral/mucosal, prime/ boost vaccination strategy using whole-cell inactivated Ct either in a nanoemulsion or adjuvanted with the TriAdj adjuvant. Phase II will then use the most immunogenic vaccination strategy to determine the immunogenicity of proven and novel antigen combinations – likely MOMP, CFAMP, OmcB, and pgp3. Vaccine immunogenicity and the humoral and cell mediated immune responses will be analyzed both systemically and locally using state-of- the art technologies including multi-peptide antibody ELISAs, multi-color T cell flow cytometry, tissue clearance in combination with fluorescent immunohistochemistry and single-cell RNAseq analyses of adaptive T cells. These first two phases will develop our vaccine candidate - the most immunogenic vaccination strategy and Ct antigen combination. In Phase III, we will test the efficacy and immunogenicity of this vaccine candidate in two identical animal trials, demonstrating rigor and reproducibility to evaluate our Ct vaccine candidate. This study will contribute in three ways to further Ct vaccine research and human health: i) It will improve our understanding of the immune response relevant for protection against Ct; ii) it will develop a novel Ct vaccine candidate; iii) it will provide a proof-of-principle vaccination study to fully establish the Cs pre-exposed pig model. This highly relevant large animal model can be used to test new candidates and improve on current regimens with the goal to develop a vaccine that results in sterilizi...