ABSTRACT An efficacious vaccine response requires that the host generates a specific response to antigenic epitopes without any prior knowledge of their composition. While intrinsic components of immunity such as Tregs regulate inflammatory mechanisms, they may also impede protective immune responses to infection and vaccination. Regulatory T cells (Tregs) comprise one of the major mechanisms underlying immunological homeostasis and self-tolerance and they play a role in suppressing vaccine-induced immunity. We developed an immunoinformatic tool, JanusMatrix, that identifies bacterial and viral class II T cell epitopes that may induce regulatory T cell responses. We propose to evaluate and validate the selection of Treg epitopes by this tool by calibrating tolerance-associated determinants such as sequence homology with the human genome, epitope presentation in the thymus, tissue-specific expression in immune privileged sites, abundance, and subcellular location, to improve the precision of this tool for Treg epitope selection and exclusion in vaccine design. In Aim 1 we will test the hypothesis that vaccine antigens have sequences that may induce tolerance, and these sequences can be uncovered using our JanusMatrix tool. A JanusMatrix threshold has been established for “immunogenicity” and “tolerogenicity” based on the extent of cross-conservation between a given T cell epitope and similar-HLA-binding T cell epitopes in the human proteome. We will determine additional thresholds associated with protein prevalence, thymic expression, immune privilege, subcellular location, and presence among HLA ligands presented in benign tissues to assess properties of the protein in silico and link those characteristics to a JanusMatrix Score that would enable us to predict Tregitopes. In Aim 2, we will test the new JanusMatrix thresholds by predicting Treg epitopes in an antigen associated with placental malaria and assessing their immunosuppressive properties using a Tetanus Toxoid Bystander Suppression Assay (TTBSA). This project will help to elucidate new thresholds to predict T reg inducing epitopes, leading to optimized Treg epitope selection and exclusion in vaccine design.