Abstract PfSPZ Vaccine, a radiation-attenuated, whole-organism vaccine against P. falciparum (Pf) malaria, based on the Pf NF54 strain, has consistently shown durable, high (>85%) protective efficacy against a homologous parasite strain after controlled human malaria infection (CHMI). This result represents a major milestone in the fight against a disease that kills >400,000 people annually. At current dosages, PfSPZ Vaccine has been shown to have significant but still incomplete protective efficacy against naturally acquired malaria infection, as well as against CHMI with a heterologous strain. The most expedited, informed path to broaden the efficacy of this vaccine relies on the knowledge of the parasite proteins against which the immune system mounts a protective response following immunization with PfSPZ Vaccine. Here, we propose to use parasites, specimen samples and clinical outcomes from the three largest efficacy field trials of PfSPZ Vaccine, to identify and validate parasite protein targets of the vaccine-induced protective immune response. We will generate whole genome sequence (WGS) data from ~400 Pf infections from controls and vaccinees in three field efficacy trials conducted, under separate funding, in Kenya, East Africa, and in Gabon, Central Africa. In AIM 1 we will develop new Pf genome reference assemblies from recently collected strains for East and West Africa, to increase the quality and quantity of sequence variants (SNPs and indels) identified in these clinical samples. In AIM 2, we will use the genome-wide genotype calls in these 400 samples to compare infections in vaccinees vs. controls, in order to identify parasite antigens targeted by PfSPZ Vaccine-induced protective immunity (“target loci”), under the assumption that, in these target loci, allele frequency distributions will differ in malaria infections in the vaccine vs. control arms of field efficacy trials, including a lower frequency of NF54-like alleles in vaccinees than in controls. We will use similar samples from control and vaccine arms of a new PfSPZ Vaccine field trial, to start in Equatorial Guinea in 2018, to validate these results. Finally, in AIM 3, we will conduct a high-throughput immunologic validation of Pf targets of PfSPZ Vaccine-induced protection that will determine whether or not allele-specific efficacy is indeed a phenomenon that impacts whole organism-based vaccines. These results will be used to inform the choice of additional P. falciparum strains with which to design multivalent vaccines with broader efficacy, and hopefully lead to a decrease in disease burden and improve the prospects of malaria eradication. By defining the targets of the immune response to a malaria parasite, this research may also expand our understanding of host immune responses to whole- organism vaccines against parasitic diseases, and accelerate future developments in this field.