Project description:Passive transfer studies in humans clearly demonstrated the protective role of IgG antibodies against malaria. Identifying the precise parasite antigens that mediate immunity is essential for vaccine design, but has proved difficult. Completion of the Plasmodium falciparum genome revealed thousands of potential vaccine candidates, but a significant bottleneck remains in their validation and prioritization for further evaluation in clinical trials. Focusing initially on the Plasmodium falciparum merozoite proteome, we used peer-reviewed publications, multiple proteomic and bioinformatic approaches, to select and prioritize potential immune targets. We expressed 109 P. falciparum recombinant proteins, the majority of which were obtained using a mammalian expression system that has been shown to produce biologically functional extracellular proteins, and used them to create KILchip v1.0: a novel protein microarray to facilitate high throughput multiplexed antibody detection from individual samples. The microarray assay was highly specific; antibodies against P. falciparum proteins were detected exclusively in sera from malaria-exposed but not malaria-naïve individuals. The intensity of antibody reactivity varied as expected from strong to weak across well-studied antigens such as AMA1 and RH5 (Kruskal-Wallis H test for trend: p-value <0.0001). The inter-assay and intra-assay variability was minimal, with reproducible results obtained in re-assays using the same chip over duration of 3 months. Antibodies quantified using the multiplexed format in KILchip v1.0 were highly correlated with those measured in the gold-standard monoplex ELISA (median (range) Spearman’s R of 0.84 (0.65-0.95)). KILchip v1.0 is a robust, scalable and adaptable protein microarray that has broad applicability to studies of naturally acquired immunity against malaria by providing a standardized tool for the detection of antibody correlates of protection. It will facilitate rapid high-throughput validation and prioritization of potential Plasmodium falciparum merozoite-stage antigens paving the way for urgently needed clinical trials for the next-generation of malaria vaccines.

Project description:Nilsson Bark SK, Ahmad R, Dantzler K, Lukens AK, De Niz M, Szucs MJ, Jin X, Cotton J, Hoffmann D, Bric-Furlong E, Oomen R, Parrington M, Milner D, Neafsey DE, Carr SA, Wirth DF, Marti M. Mol Cell Proteomics 2017. Despite recent efforts towards control and elimination, malaria remains a major public health problem worldwide. Plasmodium falciparum resistance against artemisinin, used in front line combination drugs, is on the rise, and the only approved vaccine shows limited efficacy. Combinations of novel and tailored drug and vaccine interventions are required to maintain the momentum of the current malaria elimination program. Current evidence suggests that strain-transcendent protection against malaria infection can be achieved using whole organism vaccination or with a polyvalent vaccine covering multiple antigens or epitopes. These approaches have been successfully applied to the human-infective sporozoite stage. Both systemic and tissue-specific pathology during infection with the human malaria parasite P. falciparum is caused by asexual blood stages. Tissue tropism and vascular sequestration are the result of specific binding interactions between antigens on the parasite-infected red blood cell (pRBC) surface and endothelial receptors. The major surface antigen and parasite ligand binding to endothelial receptors, PfEMP1 is encoded by about 60 variants per genome and shows high sequence diversity across strains. Apart from PfEMP1 and three additional variant surface antigen families RIFIN, STEVOR and SURFIN, systematic analysis of the infected red blood cell surface is lacking. Here we present the most comprehensive proteomic investigation of the parasitized red blood cell surface so far. Apart from the known variant surface antigens, we identified a set of putative single copy surface antigens with low sequence diversity, several of which are validated in a series of complementary experiments. Further functional and immunological investigation is underway to test these novel P. falciparum blood stage proteins as possible vaccine candidates.

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Plasmodium falciparum

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