SUMMARY The overall project goal is to identify key characteristics of immune responses to vaccination with attenuated Plasmodium falciparum sporozoites (aPfSPZs) which results in sterilizing immunity. We hypothesize that this immunity is due to cellular and humoral responses to specific to Pf antigens (Ags) that are presented during the liver stage (LS) of infection. We propose a detailed longitudinal analysis of innate and adaptive immune responses using cutting-edge immunological analyses, associated informatics, and systems immunology. We will identify aSPZ vaccine-induced cellular and humoral responses that correlate with protection in malaria naïve and malaria experienced subjects. Complementary orthogonal analytical tools and approaches will identify relevant immune cell subsets, Ag receptor (AR) repertoires, lineages, and specificities to protective Ags. The functional characteristics of the immune cells and products, including antibodies, that correlate with protection will be identified and validated. This will be accomplished by: Aim 1: Identifying aSPZ vaccine induced cellular and humoral responses that are associated with protection. Unbiased and Ag specific cellular profiling will characterize the kinetics of protection associated responses to vaccination and CHMI. Follow-on analyses will profile identified Ag-specific adaptive and innate cellular responses by mass cytometry (CyTOF), multiplexed pMHC/Ag multimer staining, and multi-omic single cell sequencing to determine protection- associated gene and protein expression signatures and functional antibody responses, which will be extended in later aims. Aim 2: Identify protection associated T and B cell Ag receptor (AR) repertoires at key times after vaccinations and CHMI. Natively paired and single ARs chains from T and B cells will be sequenced to determine repertoire attributes and identify vaccine induced Pf Ag specific ARs. Cellular transcriptomic profiles and Ag specificities will be linked by joint single cell and TCR/BCR sequence analyses. Novel AR-Ag and antibody specificities will be identified by yeast display methods. This aim will identify T and B cell ARs and antibody Ag specificities that are correlated with protection. Aim 3: Identifying functional characteristics of cellular and antibody responses that confer protection. Sorted reactive T cells will be assayed by Ag-specific CyTOF-based intracellular cytokine staining (ICS), activation-induce marker (AIM), and single-cell sequence based multi-omics analyses. The biophysical and functional analyses of monoclonal antibodies will be used to assess B cell roles in protection. The comprehensive project data will undergo integrated multi-factorial bioinformatic analysis, curation and sharing using established platforms. Overall, this project will identify key characteristics of the intricate immune responses to the complex antigenic composition of aSPZ Pf vaccination and the effector processes that correlate with protection and provide a set of immune correlates of protective immunity against malaria to guide vaccine development.

PROJECT NARRATIVE Malaria kills hundreds of thousands and sickens hundreds of millions of people globally each year. This project will use cutting edge molecular and immunological analyses of samples from clinical trials with live attenuated parasite vaccines to identify new vaccine antigens and characterize vaccine induced antibody and cellular immune responses. The materials and knowledge from this project can aid malaria vaccine development.