KILchip v1.0: A Novel Plasmodium falciparum Merozoite Protein Microarray to Facilitate Malaria Vaccine Candidate Prioritization

Abstract

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 < 0.0001). The inter-assay and intra-assay variability was minimal, with reproducible results obtained in re-assays using the same chip over a 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.

Systematic Serological Diversity of a Putative Malaria Vaccine Candidate and Broad Protection

“…6) were removed from the final data. To reduce batch effect and systematic variations, we normalized the data using ComBat function (SVA package in R) and VSN methods 56,57 , respectively, as previously reported 16…”

“…We prepared 27 constructs to express antigen variants as full-length proteins in the Expi293 expression system (Invitrogen) as previously described 16,23 . Briefly, protein sequences were designed following the removal of the predicted signal peptide and GPI modification site.…”

Reassessing therapeutic antibodies for neglected and tropical diseases

“…In studies of the merozoite protein RH5 (reticulocytebinding protein homologue 5) as a potential vaccine, some antibodies were described that could block the cycle of erythrocyte infection [135]. The specific merozoite epitopes are now being characterized, and this could form the basis of a second-generation antibody [136,137].…”

Controlled Human Malaria Infection in Semi-Immune Kenyan Adults (CHMI-SIKA): a study protocol to investigate in vivo Plasmodium falciparum malaria parasite growth in the context of pre-existing immunity

“…Immune responses, antibody and cell-mediated, will be assayed to determine signatures of immunity using plasma and peripheral blood mononuclear cells (PBMCs). For assessment of antibody immunological responses, techniques will include protein microarrays 44 , antibody-dependent assays of functional immunity such as (GIAs) 45– 47 , opsonic phagocytosis assays (OPA) 48 , and antibody dependent respiratory burst (ADRB) 46, 47, 49 . In further exploratory analysis we will also undertake principal component analyses to determine if there are typical “signatures” of protective responses, and analyses for combinations of protective antigens as previously described by Osier et al .…”