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.

Keywords: Plasmodium falciparum; antibodies; bioinformatics; merozoite; protein microarray; vaccine candidates.

Figure 1

Configuration of KILchip v1.0. Individual slides contain 21 identical mini-arrays comprised of protein spots and a barcode for identification. Each mini-array contains 384 individual spots that include proteins and controls printed in triplicate. Four slides are assembled onto a hybridization cassette, four of which are simultaneously processed in a microarray hybridization work-station (image from Arrayit Corporation, used with permission).

Figure 2

P. falciparum merozoite protein panel included in KILchip v1.0. Proteins were either selected from the literature or from a combination of proteomics and bioinformatics analysis. Details of the parasite proteins are provided in Supplementary Table 1. ^*Protein fragments refers to specific amino acid regions selected from within a full-length protein ectodomain. ^#The protein fragments based on the 3D7 allele include MSP1-19 (Block 17), MSP1 Block 2 full, MSP1 Block 2 repeat, MSP3, MSPDBL1 N-terminus, MSPDBL1 C-terminus, MSPDBL2 N-terminus, MSPDBL2 C-terminus, 2 extracellular loops of PF3D7_0629500, Pf SEA1, SURFIN 4.2 3D7A, SURFIN 4.2 3D7B and the C-terminus of SURFIN 4.2. ^%Thirteen polymorphic variants of the P. falciparum merozoite proteins MSP1, MSP2, MSP3, and SURFIN 4.2 were included in KILchip v1.0. These included MSP1 Block 2 from the K1, MAD20, PaloAlto, Wellcome and RO33 alleles, the CH150/9 and DD2 alleles of MSP2, the K1 allele of MSP3 and the K1A and K1B alleles of SURFIN 4.2. ^&Protein tags refer to specific amino acids or polypeptides fused to target proteins to facilitate their subsequent affinity purification. These include the CD4 hexa-histidine, MBP and GST tags. ^@Technical controls for the assay included Alexafluor⁶⁴⁷ human IgG, purified human IgG and protein printing buffer.

Figure 3

Detection of heat-labile and conformational epitopes in a subset of recombinant proteins. Native and heat-denatured recombinant proteins were tested for reactivity against malaria immune sera (MIS), malaria naïve sera (MNS) and 5 monoclonal antibodies targeting conformational-dependent epitopes. (A) The monoclonal antibodies targeting conformational and disulfide-constrained epitopes on AMA1 (humAbAMA1), the F2 domain of EBA175 region II (mAb R217), the F1 domain of EBA175 region II (mAb R218), MSP1 (mAb 5.2), MSP4 (mAb 2.44), RH5 (mAb QA1 and mAb 2AC7) and rat CD4 domain (OX68) were used to measure reactivity against recombinant proteins by ELISA. All recombinant proteins demonstrated high reactivity with their respective monoclonal antibodies only. Low/negligible reactivity was observed after heat-denaturation of the recombinant proteins. (B) High antibody reactivity was detected with native recombinant protein. Decreased reactivity was observed with the heat-denatured proteins. Low reactivity was detected with malaria naïve sera (MNS) in both native and heat-denatured proteins.

Figure 4

Specificity of KILchip v1.0. Positive and negative controls were used to confirm specificity. (A) High antibody reactivity was detected using malaria immune sera (MIS) and low or negligible reactivity was observed with malaria naïve sera (MNS). No reactivity was observed with the sample buffer with the exception of the technical controls. Blue, landmarks; green, commercial human IgG and yellow, printing buffer. (B) Antibody reactivity to selected well-characterized merozoite antigens AMA1, MSP2 (3D7), MSP3 (3D7), Rh5, and RIPR varied as expected in MIS (blue) and serum samples from adult residents of Kilifi. Kenya (red). High reactivity to AMA1, MSP2 (3D7), and MSP3 (3D7) and low reactivity to Rh5 and RIPR was observed in the serum samples. (C) Antibody reactivity to well-characterized antigens AMA1, MSP2, MSP3, Rh5, and RIPR were all low/negligible when tested in serum samples from Swedish residents.

Figure 5

Intra-assay variability of antibody detection by KILchip v1.0. A comparison of antibody responses to triplicate readings of AMA1, MSP1, and MSP2 was measured in 66 serum samples from adults living in the malaria endemic region of Kilifi, Kenya. A strong positive correlation of >0.98 was observed in all three-way scatter plots tested for the three antigens. Correlation between triplicate readings for recombinant AMA1 (A–C), MSP1 (D–F) and MSP2 (G–I).

Figure 6

Inter-assay variability of antibody detection by KILchip v1.0. Positive and negative controls were used to measure antibody responses to 5 protein arrays printed on 5 consecutive days using identical printing conditions and the same batch of recombinant proteins. Data presented are the mean MFI values of triplicate readings obtained with MIS (top graph) and MNS (bottom graph) to a subset of the proteins (n = 26) printed on each mini-array. Individual proteins are represented as dots and lines have been included to aid visualization.

Figure 7

Stability of KILchip v1.0. A reference reagent, MIG, consisting of purified immunoglobulins (98% Total IgG) was used to generate a standard curve by testing a tripling dilution against all antigens printed on the protein array. A batch of slides printed at the same time was tested 24, 40, 54, and 86 days post printing. The standard curve showed good concordance without significant variation. Red, blue, black, and green lines were obtained from measurements obtained on day 24, day 40, day 54, and day 86 post printing of the slides.

Figure 8

Concordance between KILchip v1.0 and the gold standard ELISA. A comparison of antibody responses to 12 antigens measured simultaneously in KILchip v1.0 and individually by ELISA. Sixty-six serum samples from adults living in the malaria endemic region of Kilifi, Kenya were used to facilitate comparisons. A strong positive correlation of >0.8 was observed in 8 of the antigens (A–H) while four antigens (I–L) showed a correlation coefficient between 0.65 and 0.78. Red box - serum samples in which antibody responses reached the upper limit of detection when measured by ELISA, yet distinguishable by microarray with MFI values ranging from 40,000 to 60,000.