We propose to further enhance Plasmodium falciparum (Pf) Sporozoite (SPZ)-based vaccines against malaria that are the only immunogens proven to induce >90% short term (3 weeks) and long term (at least 14 months) protection against controlled human malaria infection with Plasmodium falciparum (Pf) in humans. Using a unique glycolipid adjuvant 7DW8-5, the goal is to prolong the duration of vaccine efficacy (VE) and to increase efficacy in endemic settings. In the mouse model using P. yoelii (Py) sporozoites (SPZ) we achieved > 80% protection at 16 weeks with 2 dose and 4 dose accelerated regimens of irr PySPZ plus 7DW8-5 adjuvant administered by direct venous inoculation (DVI) representing a 2-fold enhancement over irr PySPZ without adjuvant. The adjuvant could be mixed with irr PySPZ. High level (>80%) protection of mice persisted at 16 weeks with irr PySPZ by DVI, in the presence of 7DW8-5, but not by non-DVI routes. Manufacturing of 7DW8-5 under cGMPs was completed and in a pilot study with P. knowlesi (Pk) SPZ, irr PkSPZ we achieved 50% VE and no improvement with the adjuvant, likely attributable to sub-optimal comparative dose or dosing regimens, or the short-term infectious challenge design. Due to the excellent demonstrable safety record of the combined SPZ-adjuvant vaccine in NHPs, and comparable bioactivity on co- culture human iNKT cells in vitro, we propose further optimization of dosing regimens for durable immunity in pig-tailed macaques, the natural host for Pk, along with protection studies to assess adjuvant effects on chemically attenuated (PySPZ-chemoprophylaxis vaccine CVac) and genetically attenuated (PySPZ-LARC) in mice. Using humanized HISA2/ hCD1d mice possessing functional human CD8+ T cells and human iNKT cells (cellular targets of 7DW8-5) we will investigate whether a PfSPZ-7DW8-5 combination immunogen can enhance the human CD8+ T-cell response to PfSPZ. Adjuvant-associated biomarker discovery studies are also planned in mice. Towards clinical use of the PfSPZ-7DW8-5 combination vaccine, we will establish stability criteria and formulation methodologies for 7DW8-5, and further comparability testing of GMP-grade 7DW8-5 for bioactivity in vitro as a lot release attribute and humanized HISA2/ hCD1d mice in vivo, compile a pre-IND package in preparation for pre-clinical and clinical evaluation of the safety and efficacy of 7DW8-5-PfSPZ combinations, and manufacture GMP 7DW8-5 for clinical use. Because studies outlined in this project will be conducted with clinical grade, well characterized 7DW8-5, a positive outcome in our studies will place us in a position to rapidly design and conduct formal pre-clinical toxicology and/or biodistribution studies in compliance with FDA mandates for a speedier path to the clinic. Completion of this project will mark the first development of an adjuvant for a live eukaryotic parasite vaccine.