Characterization of CD4 T cells targeting liver and blood stage malaria epitopes

About This Grant

Abstract Malaria is a leading killer of children worldwide, and existing WHO-recommended vaccines offer only modest protection that is of short duration. Malaria vaccine innovation has been impeded by our limited knowledge of the antigens and epitopes targeted by T cells and the characteristics of malaria-specific CD4 T cells that are most critical for immune protection. We will address these critical gaps by identifying hundreds of novel P. falciparum CD4 T cell epitopes and using innovative T cell assays to characterize epitope-specific T cells at the individual cell level. P. falciparum has a complex life cycle, and the human immune response differs according to the stage of parasite development. The most successful malaria vaccine strategies identified to date require vaccination with live attenuated sporozoites, which infect hepatocytes but undergo developmental arrest at the liver stage and do not establish blood-stage parasitemia. Evidence from both human and animal studies of sporozoite vaccination indicates that CD4 T cells targeting liver-stage parasites are critical for this protection, but the antigens targeted by protective T cells are almost entirely unknown. In preliminary studies, we have developed a bioinformatic strategy to identify P. falciparum antigens transcriptome-wide that are highly and selectively expressed during the liver stage of infection. In the first aim, we will use a novel assay to generate large DNA-barcoded P. falciparum peptide libraries, including geographically relevant sequence variants, and perform highly multiplexed screens to identify peptides that bind HLA-DRB1 allotypes common in East Africa. We will then test the recognition of candidate epitopes using samples from a large cohort of highly malaria- exposed Ugandan children. In the second aim, we will use the peptide:HLA binding data to build a 1000-plex DNA-barcoded multimer probe-set to label cells for single-cell sequencing, which will enable us to simultaneously discern the epitope specificity, HLA restriction, full transcriptomes, and TCRα:β sequences of P. falciparum-specific CD4 T cells. We will use this novel genomic assay platform to compare the transcriptional and functional phenotype of CD4 T cells targeting liver- vs. blood-stage epitopes in children residing in a highly malaria-endemic region of Uganda. Lastly, we will use a unique set of samples from a randomized trial of childhood chemoprevention to test the hypothesis that selective suppression of blood-stage infection by antimalarial drugs fosters the development of a broader and more functionally robust CD4 T cell response to liver-stage antigens. Together, these novel assay platforms will enable us to identify hundreds of novel P. falciparum epitopes recognized by malaria-exposed Ugandan individuals and characterize the responding CD4 T cells in unprecedented detail to uncover the functional and phenotypic features critical for T cell-mediated protection.