PROJECT SUMMARY/ABSTRACT Coccidioidomycosis or Valley Fever (VF), is caused by the fungus Coccidioides immitis and C. posadasii with highest reported prevalence in Arizona and California. Due to similar clinical presentations, VF is frequently misdiagnosed and mistreated as bacterial or viral pneumonia, resulting in unnecessary antibiotics and longer times to resolve illness. Patients lose productivity and experience symptoms for many months. Severe CM manifests in <5% of symptomatic cases but can be life-threatening. T cell responses plays an important role in vaccine induced protection in animal models. In fact, this activity appears to be critical for defense against Coccidioides in humans, as evidenced by the fact that humans with low CD4+ T cell counts due to HIV infection are at increased risk of severe VF. The objective of this proposal is to identify T cell clones and their associated epitopes that are generated in human patients and animal models. For Coccidioides spp. epitopes, we will focus on previously confirmed and novel in vivo early expressed antigens and we will link these epitopes to their TCRs using a T cell peptide stimulation strategy. Our hypothesis is that epitopes from these Coccidioides antigens drive patient TCR profiles and, hence, will contain “public” TCR sequences that can be used for VF diagnosis and prioritize vaccine candidates. We predict that the TCR profiles are indicative of immune and disease states. We will address this hypothesis by deep TCR sequencing of activated T cells stimulated with Coccidioides peptide epitopes. We will identify TCRs specific to these and other Coccidioides proteins in patient samples from both Arizona (C. posadasii) and California (C. immitis). We will also confirm that mouse and macaque T cells detect epitopes from these antigens to aid in Coccidioides challenge model development. In addition, the immunogenic Coccidioides antigens will be evaluated for protection in these animal models after vaccination. Finally, we will generate a TCR sequencing diagnostic test to detect VF patients. Specifically, we aim to: 1) Characterize C. posadasii and C. immitis epitopes and associated T cell clones (TCR) in human specimens using T cell receptor sequencing and standard cell sorting techniques, 2) Determine TCRs and epitopes recognized during animal model (mouse and pig-tailed macaque) challenge, natural exposure, and RNA/DNA vaccinations, and 3) Compare the sensitivity between traditional serology and TCR sequencing using endemic and non-endemic populations. If successful, this project will make available to the community a novel set of T cell epitopes for VF in humans, mice, and macaques. The TCR sequences can be used to develop next generation diagnostics and aid in antigens identification for vaccination. Finally, T cell clonotypes specific to these epitopes will be correlated to protection through the models and disease outcomes in VF patients.