AbstractWe designed and produced a self-assembling protein nanoparticle. This self-assembling protein nanoparticle contains five CD8⁺ HLA-A03-11 supertypes-restricted epitopes from antigens expressed during Toxoplasma gondii’s lifecycle, the universal CD4⁺ T cell epitope PADRE, and flagellin as a scaffold and TLR5 agonist. These CD8⁺ T cell epitopes were separated by N/KAAA spacers and optimized for proteasomal cleavage. Self-assembling protein nanoparticle adjuvanted with TLR4 ligand-emulsion GLA-SE were evaluated for their efficacy in inducing IFN-γ responses and protection of HLA-A*1101 transgenic mice against T. gondii. Immunization, using self-assembling protein nanoparticle-GLA-SE, activated CD8⁺ T cells to produce IFN-γ. Self-assembling protein nanoparticle-GLA-SE also protected HLA-A*1101 transgenic mice against subsequent challenge with Type II parasites. Hence, combining CD8⁺ T cell-eliciting peptides and PADRE into a multi-epitope protein that forms a nanoparticle, administered with GLA-SE, leads to efficient presentation by major histocompatibility complex Class I and II molecules. Furthermore, these results suggest that activation of TLR4 and TLR5 could be useful for development of vaccines that elicit T cells to prevent toxoplasmosis in humans.