Project Summary/Abstract Malaria is a globally devastating, mosquito-borne disease which infects hundreds of millions of people each year, resulting in ~435,000 deaths. The continual emergence of resistance to frontline antimalarials, due to the considerable genetic plasticity of the malaria parasite P. falciparum, has made development of a highly effective malaria vaccine an important part of the global eradication campaign. However, efforts to date have been largely unsuccessful, likely owing to the extraordinarily high threshold for protection. Thus in order to develop a vaccine which confers both robust and durable immunity, it is necessary to understand both the immune mechanisms required to induce a protective response, and the mechanisms by which this response confers protection. The humoral immune response to malaria parasites is dominated by antibodies (Abs) against the central repeat region of an intrinsically disordered P. falciparum surface protein PfCSP, and a small percentage of these repeat- targeting Abs confer potent, sterile protection in animal models. The overall goal of this proposal is to elucidate the mechanism by which these Abs protect from malaria infection, and in doing so define new, more precise immune correlates to enable rational vaccine design. To achieve this, this proposal outlines an interdisciplinary approach utilizing in vivo protection assays and a synthesis of low and high resolution light and electron microscopy. These experiments will test the overall hypothesis that protective PfCSP repeat-targeting Abs induce a structural phenotype on PfCSP which is both correlated with and underlies the mechanism of protection. I have solved a number of high-resolution cryo-EM structures of protective Abs bound to PfCSP, which form multivalent helical structures stabilized by affinity-matured Ab-Ab contacts. In Aim 1, I will test the role of these interactions in the mechanism of protection by directly comparing the protective efficacy of mutant and WT Ab forms with in vivo protection assays in our malaria mouse model. In Aim 2, I will use a suite of imaging methods to determine how protective and non-protective Abs engage PfCSP on the surface of live P. falciparum sporozoites. Light microscopy experiments will determine the overall effect of these Abs on sporozoite biological function and PfCSP distribution. Then I will use cryo-electron tomography (cryo-ET) to discern fine differences in the structural phenotypes on sporozoites induced by Ab binding, and correlate these with protective efficacy. This will be complimented by room temperature immuno-EM experiments, which will allow high contrast imaging and the ability to confirm the molecular identity of specific ultrastructural features of these structural phenotypes. Overall, these experiments will greatly enhance our understanding the mechanism by which Abs protect from malaria infection, and may revolutionize our understanding of basic P. falciparum biology. This w...