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 wellstudied 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.