Project Summary/Abstract Malaria kills over 400,000 people annually, underscoring the need for a highly effective malaria vaccine. A better understanding of immunity to Plasmodium falciparum, the most lethal of the human malaria parasites, in naturally exposed populations can inform the rational design of more effective vaccines. However, reliable immune correlates of protection against malaria remain elusive, and relatively little is known about immunity that prevents P. falciparum infection given that sustained protection from parasitemia through natural malaria exposure is rarely observed. In our K08-funded project, we have applied systems biology approaches to a unique subset of children who remained free of P. falciparum parasitemia during a single malaria season despite intensive surveillance consisting of PCR-based screening of blood collected every 2 weeks and during sick visits as well as antibody evidence for parasite exposure during a 7-month surveillance period. This rarely observed “aparasitemic” phenotype represents the closest approximation to sterile immunity in a naturally exposed population and a unique opportunity to study host control of parasitemia. The goal of this R21 proposal is to expand on findings from our prior bulk transcriptomic analysis of whole blood obtained from these aparasitemic children using higher resolution technologies that include single-cell (sc) sequencing and multiparameter flow cytometry. We propose using scRNA-seq on baseline peripheral blood mononuclear cells (PBMCs) obtained from these children just prior to the surveillance period. When integrated with single-cell assay for transposase- accessible chromatin sequencing (scATAC-seq), which assesses genome-wide chromatin accessibility to identify “open” chromatin regions, scRNA-seq can also provide insight into the regulation of gene expression. Combining integrated single-cell approaches with functional assays can help identify gene regulatory networks within specific cell types that could serve as immune correlates of protection against malaria infection. We hypothesize that, relative to parasitemic children, aparasitemic children will demonstrate increased antigen receptor signaling within memory B and T cell subsets and enhanced P. falciparum-specific T-cell memory responses despite having decreased proportions of T cells, which may reflect trafficking of T cells out of the peripheral blood to target organs such as the liver or spleen. We will address this hypothesis with the following specific aims: 1) to determine host immune genes that are differentially regulated and expressed using integrated single-cell gene expression and chromatin accessibility profiling of PBMCs from malaria-exposed children who differ in susceptibility to P. falciparum parasitemia and 2) to identify and characterize the immunophenotypic and functional differences in PBMCs from children who differ in susceptibility to P. falciparum parasitemia. Successful completion of t...