MODULARIZING MANUFACTURE OF PFSPZ VACCINES: OOKINETE PRODUCTION FOR PFSPZ MANUFACTURE IN MOSQUITOES AND IN VITRO - IN 2022 WHO CALLED FOR HIGHLY EFFICACIOUS VACCINES AGAINST MALARIA THAT PREVENT PLASMODIUM FALCIPARUM (PF) INFECTION IN > 90% OF RECIPIENTS. ALONE AMONG MALARIA VACCINES, SANARIA PFSPZ (PF SPOROZOITE (SPZ)) VACCINES HAVE SHOWN > 90% VACCINE EFFICACY (VE) AGAINST CONTROLLED HUMAN MALARIA INFECTION AND VE AGAINST INTENSE FIELD TRANSMISSION OF MALARIA TO PREGNANT WOMEN FOR TWO TRANSMISSION YEARS WITHOUT BOOSTING. OUR GOALS ARE TO FURTHER INCREASE POTENCY, INCREASE EFFICIENCY AND CONSISTENCY OF MANUFACTURE AND REDUCE COST OF GOODS (COGS) TO BE ABLE TO USE A PFSPZ VACCINE FOR ELIMINATION OF MALARIA. PFSPZ VACCINES ARE MANUFACTURED USING STAGE V PF GAMETOCYTES FED TO MOSQUITOES, A PROCESS THAT INCLUDES TIGHT COORDINATION BETWEEN CULTURE OF GAMETOCYTES AND MOSQUITO REARING. MOST IMPORTANTLY, SANARIA HAS DEVELOPED METHODS FOR PRODUCING PFSPZ WITHOUT MOSQUITOES IN BIOREACTORS (NATURE, 2022). TRANSITIONING TO BIOREACTOR PRODUCTION WOULD INCREASE EFFICIENCY OF MANUFACTURE BY AT LEAST 10-FOLD, REDUCE COGS BY >90%, AND ALLOW FOR PFSPZ VACCINE PRODUCTION WORLDWIDE. THIS PROPOSAL IS INTENDED TO PROVIDE MASS-PRODUCED, ASEPTIC, PURIFIED, CRYOPRESERVED OOKINETES THAT CAN BE USED TO INCREASE EFFICIENCY OF PRODUCTION OF MOSQUITO- AND BIOREACTOR-PRODUCED PFSPZ VACCINES. WE WILL OPTIMIZE PRODUCTION, PURIFICATION, CRYOPRESERVATION, QC RELEASE, AND USE IN MANUFACTURING OF OOKINETES. THE EARLY STEPS IN MANUFACTURING THAT END WITH OOKINETES CAN THEN BE DE- LINKED FROM THE LATER STEPS THAT COMMENCE WITH OOKINETES, ALLOWING BOTH TO PROCEED SEPARATELY, UNCONSTRAINED BY TIMING OR PHYSICAL LOCATION. THE WORK WILL BE ADDRESSED IN 4 SPECIFIC AIMS. 1: OPTIMIZE OOKINETE PRODUCTION. MEDIA AND CULTURE CONDITIONS FOR STAGE V GAMETOCYTES WILL BE OPTIMIZED FOR CONVERSION TO OOKINETES. READOUTS WILL INCLUDE FEMALE:MALE RATIO, MACROGAMETOCYTE DENSITY, AND STAGE SPECIFIC ANTIGEN EXPRESSION. OUR TARGET IS A METHOD THAT CONSISTENTLY RESULTS IN 25% CONVERSION OF STAGE V GAMETOCYTES TO OOKINETES. 2: DEVELOP AND OPTIMIZE METHODS FOR OOKINETE PURIFICATION. WE WILL TEST MULTIPLE METHODS, SELECTING THOSE THAT CAN BE SCALED. OUR TARGET IS PF OOKINETES THAT ARE 100% FREE OF RBCS WITH A YIELD OF 80% USING A METHOD THAT CAN BE ADAPTED TO LARGE SCALE MANUFACTURING. 3: DEVELOP A PROTOCOL FOR OOKINETE CRYOPRESERVATION. SANARIA ROUTINELY CRYOPRESERVES PFSPZ WITH CONSISTENT, EXCELLENT RESULTS. OOKINETES ARE SIMILAR TO PFSPZ. BOTH ARE MOTILE, HAVE A MULTIMEMBRANE PELLICULAR COMPLEX, EXPRESS A DOMINANT MEMBRANE PROTEIN, AND ARE SIMILAR IN SIZE. A RANGE OF CRYOPROTECTANT ADDITIVES (CPA), COOLING AND WARMING RATES, AND CPA DILUTION METHODS WILL BE TESTED. OUR TARGET IS A SURVIVAL RATE OF AT LEAST 50%. 4: DEVELOP ASSAYS FOR OOKINETE VIABILITY AND POTENCY. 4 APPROACHES WILL BE TESTED: A) MEMBRANE INTEGRITY, B) MOTILITY, C) INFECTIVITY TO MOSQUITOES TO PRODUCE PFSPZ, AND D) IN VITRO CONVERSION OF OOKINETES TO 3- AND 8-DAY OOCYSTS AND IPFSPZ.