An effective malaria vaccine is urgently needed, but progress toward this goal has been hindered by our limited understanding of the mechanisms underlying immunity to malaria and a lack of reliable in vitro correlates of protection. It has recently been shown that protective immunity to malaria can be achieved by experimental infection with viable sporozoites under "cover" of antimalarial drugs that eradicate blood-stage parasites but allow the pre-erythrocytic parasite stages to develop. This regimen induces CD4 T cells that produce IL-2, TNFα, and IFNγ and results in sterile protection from homologous challenge that persists for at least 2 years. In the prior funding period, we tested the hypothesis that chemoprevention coupled with natural exposure to malaria could yield similar protection when given to children in a high endemnicity setting, leveraging a randomized trial of monthly dihydroartemisinin-piperaquine (DP) chemoprevention given to young children in Uganda. We found that children highly adherent to monthly DP exhibited sustained protection against naturally occurring malaria strains during one year of follow-up after the intervention ended. Children randomized to DP developed higher frequencies of malaria-specific CD4 T cells co-producing IL-2/TNFα, which were associated with prospective protection, and lower frequencies of CD4 cells co-producing IL10/IFNγ, which were associated with increased risk. These data suggest that the functional quality of the CD4 T cell response is influenced by chemoprevention and is a critical determinant of protective immunity. In the renewal period, we wish to more fully characterize the functional, phenotypic, and transcriptional differences in T cell responses associated with protection by leveraging samples and data from a new trial, currently underway, in which pregnant women and their infants are randomized to receive monthly DP chemoprevention during pregnancy and the first 2 years of life. We will assess how chronic malaria antigen exposure impacts both CD4 T cells and Vδ2+ γδ T cells, a semi-innate lymphocyte population with intrinsic reactivity to malaria antigens. In the first aim, we will characterize CD4 T cell responses associated with protection from malaria in children receiving monthly DP chemoprevention, and determine how the phenotype, function, and transcriptional program of malaria-specific CD4 cells is impacted by chronic malaria antigen exposure. In the second aim, we will determine how malaria exposure impacts the Vγ9Vδ2 T cell population, including their frequency, differentiation status, clonotype composition, and expression of activating and inhibitory NK receptors (NKRs). We will also examine the impact of NKR expression on functional responsiveness to malaria antigens. In the third aim, we will measure associations between malaria-specific T cell responses (CD4 and Vγ9Vδ2) and protection from P. falciparum parasitemia and symptomatic malaria. These studies will address fundamental gaps in our understanding of the cellular and molecular mechanisms underlying protective immunity to malaria, and could provide a strong public health rationale for implementing malaria chemoprevention during pregnancy or early infancy.