kilchip v1 0 a novel plasmodium falciparum merozoite protein microarray to facilitate CORD-Papers-2022-06-02 (Version 1)

Title: 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-nave individuals. The intensity of antibody reactivity varied as expected from strong to weak across well-studied antigens such as AMA1 and RH5 (KruskalWallis 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 (0.650.95)]. 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.
Published: 2018-12-11
Journal: Front Immunol
DOI: 10.3389/fimmu.2018.02866
DOI_URL: http://doi.org/10.3389/fimmu.2018.02866
Author Name: Kamuyu Gathoni
Author link: https://covid19-data.nist.gov/pid/rest/local/author/kamuyu_gathoni
Author Name: Tuju James
Author link: https://covid19-data.nist.gov/pid/rest/local/author/tuju_james
Author Name: Kimathi Rinter
Author link: https://covid19-data.nist.gov/pid/rest/local/author/kimathi_rinter
Author Name: Mwai Kennedy
Author link: https://covid19-data.nist.gov/pid/rest/local/author/mwai_kennedy
Author Name: Mburu James
Author link: https://covid19-data.nist.gov/pid/rest/local/author/mburu_james
Author Name: Kibinge Nelson
Author link: https://covid19-data.nist.gov/pid/rest/local/author/kibinge_nelson
Author Name: Chong Kwan Marisa
Author link: https://covid19-data.nist.gov/pid/rest/local/author/chong_kwan_marisa
Author Name: Hawkings Sam
Author link: https://covid19-data.nist.gov/pid/rest/local/author/hawkings_sam
Author Name: Yaa Reuben
Author link: https://covid19-data.nist.gov/pid/rest/local/author/yaa_reuben
Author Name: Chepsat Emily
Author link: https://covid19-data.nist.gov/pid/rest/local/author/chepsat_emily
Author Name: Njunge James M
Author link: https://covid19-data.nist.gov/pid/rest/local/author/njunge_james_m
Author Name: Chege Timothy
Author link: https://covid19-data.nist.gov/pid/rest/local/author/chege_timothy
Author Name: Guleid Fatuma
Author link: https://covid19-data.nist.gov/pid/rest/local/author/guleid_fatuma
Author Name: Rosenkranz Micha
Author link: https://covid19-data.nist.gov/pid/rest/local/author/rosenkranz_micha
Author Name: Kariuki Christopher K
Author link: https://covid19-data.nist.gov/pid/rest/local/author/kariuki_christopher_k
Author Name: Frank Roland
Author link: https://covid19-data.nist.gov/pid/rest/local/author/frank_roland
Author Name: Kinyanjui Samson M
Author link: https://covid19-data.nist.gov/pid/rest/local/author/kinyanjui_samson_m
Author Name: Murungi Linda M
Author link: https://covid19-data.nist.gov/pid/rest/local/author/murungi_linda_m
Author Name: Bejon Philip
Author link: https://covid19-data.nist.gov/pid/rest/local/author/bejon_philip
Author Name: Frnert Anna
Author link: https://covid19-data.nist.gov/pid/rest/local/author/frnert_anna
Author Name: Tetteh Kevin K A
Author link: https://covid19-data.nist.gov/pid/rest/local/author/tetteh_kevin_k_a
Author Name: Beeson James G
Author link: https://covid19-data.nist.gov/pid/rest/local/author/beeson_james_g
Author Name: Conway David J
Author link: https://covid19-data.nist.gov/pid/rest/local/author/conway_david_j
Author Name: Marsh Kevin
Author link: https://covid19-data.nist.gov/pid/rest/local/author/marsh_kevin
Author Name: Rayner Julian C
Author link: https://covid19-data.nist.gov/pid/rest/local/author/rayner_julian_c
Author Name: Osier Faith H A
Author link: https://covid19-data.nist.gov/pid/rest/local/author/osier_faith_h_a
sha: db2773bfab2b81fdcf13212b9c7b4fef0af57fcf
license: cc-by
license_url: https://creativecommons.org/licenses/by/4.0/
source_x: Medline; PMC
source_x_url: https://www.medline.com/https://www.ncbi.nlm.nih.gov/pubmed/
pubmed_id: 30619257
pubmed_id_url: https://www.ncbi.nlm.nih.gov/pubmed/30619257
pmcid: PMC6298441
pmcid_url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298441
url: https://www.ncbi.nlm.nih.gov/pubmed/30619257/ https://doi.org/10.3389/fimmu.2018.02866
has_full_text: TRUE
Keywords Extracted from Text Content: merozoite antigens AMA1 P. falciparum merozoite MSP3 (3D7 leakproof vaccine antigen Figure 3B Papua New Guinea (73 MSPDBL1 MSP2-CH150/9 surface P. vivax alanine serum mAb R217 Alexafluor mAb SERA1 ONCYTE SuperNOVA RH5 Vrije Universiteit Brussels Figure 3A −1 Yellow-MSP2 HBS RhopH3 Coomassie https://www.frontiersin.org/articles/10.3389/fimmu Mass-spectrometry CD4-hexa-histidine Cell Supplementary Figure 3 | Antibody Supplementary Figure 1 | KEMRI/SERU/CGMR-C/001/3139 humAbAMA1 combiSlide KCl Spearman's R MNS RAP1-2 PF3D7_1343700 SigmaFAST GenePix glycerol malariaendemic λ SEA1 mouse kappa light chain E. coli micro-arrayer Kilifi MBP MSP4 Plasmodium falciparum MSP1 glutamine Figure 4A mDeg μl/min × SURFIN4.2 Figure 8B upper Orange-RH5 Burghaus Alexafluor 647 Expi293 antigen RH4 MNS sera donkey immunoglobulins Yann MSV000083144 horseradish ArrayJet 1.4 M NaCl PF3D7_1229300 HumAbAMA1 Green-RIPR transmembrane glutathione-S-transferase ftp://massive.ucsd.edu/ MSV000083144 Nterminal signal peptide threedimensional Codon-optimized serum samples serine landmarks Expifectamine 293 mAb R218 Cells phosphate buffer Tris-HCL pH Sweden mAb QA1 PF3D7_1252300 Blue-MSP3 BL21 silicone P. falciparum merozoite proteins 50mM NaH2PO4 P10 c18 pipette CD4 Supplementary Figure 3 miniarrays Red-AMA1 apical organelles Tween solid-surface formic acid Figure 7 Expi293F cells Supplementary Table 1 sub-cellular erythrocyte HEPES P. falciparum proteins SVA package in R Plasmodium falciparum recombinant FTP samples sera patient Addgene mammalian cells naïve sera EBA175 H 2 R218 tissues merozoite mAb QA1 monoclonal antibodies offtarget recombinant proteins MSP3 (B) proteins Supplementary Figure 4 phosphate buffered saline left methionine disulphide expressors ARYC cells Figures 8A-L B PF3D7_1025300 transmembrane domain(s pEXP5-NT/TOPO PXD011746 P. falciparum surface proteins H 2 O 2 P. falciparum P. falciparum antibody PF3D7_0629500_SEG2 heat-labile epitopes PF3D7_0830500 GST MSP2 (3D7 acetonitrile × 10 6 human IgG human PF3D7_0424400 children pTT3 rat Cd4 domains 3 5e4 o-phenylenediamine dihydrochloride MassIVE Figure 4B MSP3 were 23629 mAb 2AC7 Tween 20/HBS NaH 2 PO 4 SERU ∼30 Rh5 green Tris-HCL extracellular loop RIPR pGEX-2T MSP2 merozoite antigens P. falciparum 3D7 coomassie GraceBio KILchip merozoites ∼450,000 C. PF3D7_1237900 EntreMed/NIAID MSP3 LC conformation-dependent antibody Jackson ImmunoResearch immune sera −1 · SERA6 KEMRI-Wellcome ProteomeXchange Consortium51 distilled water AMA1 mini-arrays AGC GLURP multimembrane dithiothreitol masses SARS-coronavirus Immunolon
Extracted Text Content in Record: First 5000 Characters:Protein microarrays are increasingly used in the "omic" era of research in multiple formats that share the basic requirement to investigate interactions of tens to thousands of proteins simultaneously (1) . They have had important translational applications in biomarker discovery to guide patient diagnosis, treatment and prognosis, as well as in drug discovery and vaccine antigen identification (2) . Protein microarrays have facilitated a rapid, systematic and high-throughput approach to probing an entire pathogens' proteome or fraction thereof for immunoreactivity, in an approach that forms part of a reverse vaccinology workflow. These have aided in the discovery of potential diagnostic markers for Mycobacterium tuberculosis and SARS-coronavirus as well as potential vaccine candidates in over 30 human pathogens including Plasmodium falciparum (2, 3) . P. falciparum malaria causes ∼450,000 deaths per year (4), and is of major public health importance to sub-Saharan Africa (5) . Recent gains in reducing the burden appear to have stalled despite ongoing control efforts (4, 6) . Efforts to design a highly effective vaccine that would protect against this disease have been hampered by the complexity of the organism and its' multi-stage life cycle: its genome encodes >5,300 proteins that are expressed variably in different tissues as the infection develops in the host (7) . Coupled to this is an impressive array of strategies for generating protein polymorphisms or protein variants and redundant erythrocyte invasion pathways, which facilitate immune evasion (8) (9) (10) . Consequently, although efforts to develop a highly effective malaria vaccine have been on-going for over a century, this goal has yet to be achieved. The current leading vaccine candidate against P. falciparum malaria has limited efficacy and induces only short-lived protective immunity (11, 12) . Multiple P. falciparum and/or P. vivax protein arrays have been designed over the past decade to help identify and prioritize potential malaria vaccine antigen candidates. The majority of these arrays have been manufactured using either the E. coli-based or the wheat germ cell free in-vitro transcription/translation expression system, with the largest to date including ∼30% of the entire P. falciparum proteome (13) (14) (15) (16) (17) . Protein selection was based on stage-specific transcription or protein expression, sub-cellular localization, secondary protein structures or documented immunogenicity in human and animal models. However, the in-vitro transcription/translation systems are relatively poor at generating functional surface proteins, which frequently require disulphide bonding and/or post-translational modification to attain their correct threedimensional structure. Nevertheless, subsequent studies have down-selected proteins from this initial panel (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) , indicating that essentially >75% of the parasite genome has yet to be evaluated in the context of immunity. A few additional proteins have been tested independently in smaller scale studies accounting for only a marginal increase in the proportion of the parasite proteome evaluated to date (32) (33) (34) . These studies have rationally selected merozoite proteins that were established or plausible targets of antibodies, and evaluated antibody associations with protection in longitudinal studies using standard ELISA-based approaches (32, 33) . They highlighted the importance of evaluating a broad repertoire of antigens and combinations of antibody responses in studies of acquired immunity. However, there still remains a need for a common platform with standardized protein expression and high-throughput antibody detection methods that can be applied widely across different clinical studies (35) . This would accelerate identification of protective antibody targets and facilitate the comparisons between studies and populations. To contribute to vaccine candidate discovery, as well as the validation and prioritization of existing candidates for clinical trials, we designed a novel protein microarray. We focused on the merozoite stage that is a target of immunity that can prevent or reduce the clinical symptoms of malaria. As per the case with other infectious diseases (36, 37) , we hypothesized that proteins on or associated with the surface of the invasive P. falciparum merozoite would be accessible targets for protective antibodies (33) . We mined the literature to identify multiple potential surface-associated merozoite proteins (32) (33) (34) (38) (39) (40) (41) (42) (43) and added new proteins that were identified as immunogenic in adults from malariaendemic countries and had proteomic and/or bioinformatic features suggestive of merozoite surface-localization, secretion and/or involvement in erythrocyte invasion (44) . We expressed and purified these proteins and printed them on a custom microarray, which we refer to as KIL
Keywords Extracted from PMC Text: mAb ArrayJet human mouse kappa light chain Spearman's R extracellular pTT3 Tris-HCL P. falciparum humAbAMA1 horseradish HEPES MSP4 SERA1 KEMRI/SERU/CGMR-C/001/3139 HBS vaccine antigen sera mini-arrays Kilifi 52–54 AGC methionine phosphate buffer EBA175 P. falciparum antibody alanine SigmaFAST merozoite mAb QA1 sub-cellular MNS mDeg·cm2·dmol−1·res−1 MSP2-CH150/9 Figure 3B micro-arrayer Cell serine P. falciparum proteins MSP2 rat Cd4 domains 3 silicone gaskets serum H2O2 PF3D7_0424400 Papua New Guinea (73 P. falciparum surface proteins https://www.addgene.org BL21 Coomassie Tris-HCL pH donkey LC SERA6 disulphide ONCYTE SuperNOVA Mass-spectrometry Figure 4A immune sera GraceBio Figure 3A glutamine RH5 P10 c18 pipette formic acid × landmarks heat-labile epitopes RH4 children Jackson ImmunoResearch CO2 Figures 8A–L Crosnier mammalian cells P. falciparum 3D7 conformation-dependent antibody Burghaus RAP1-2 × 2 55–58 GenePix Plasmodium falciparum CD4 solid-surface serum samples Figures 8A–H GST human IgG mAb R218 MSPDBL1 Expi293F cells pEXP5-NT/TOPO multi-membrane mDeg RIPR immunoglobulins PXD011746 SERU acetonitrile B Immunolon combiSlide Expi293 " green MSP3 were 23629 FTP SARS-coronavirus apical organelles MassIVE phosphate buffered saline patient KEMRI-Wellcome SEA1 Addgene Figure 4B merozoites P. vivax μ ms2 ProteomeXchange Consortium51 ftp://massive.ucsd.edu/MSV000083144 Tween 20/HBS Expifectamine 293 transmembrane domain(s ARYC Figure 8B KCl E. coli MSP3 glutathione-S-transferase o-phenylenediamine dihydrochloride GLURP P. falciparum merozoite proteins M NaCl MSP1 P. falciparum merozoite Supplementary Figure 4 RhopH3 glycerol upper erythrocyte Sweden naïve sera tissues transmembrane Tween ms1 MSV000083144 surface 1.4 M NaCl left merozoite antigens mAb R217 samples Cells mAb 2AC7 MBP AMA1 pGEX-2T 5e4 ~450,000 dithiothreitol Codon-optimized Supplementary Table 1 antigen SURFIN4.2 Figure 7 Supplementary Figure 3
Extracted PMC Text Content in Record: First 5000 Characters:Protein microarrays are increasingly used in the "omic" era of research in multiple formats that share the basic requirement to investigate interactions of tens to thousands of proteins simultaneously (1). They have had important translational applications in biomarker discovery to guide patient diagnosis, treatment and prognosis, as well as in drug discovery and vaccine antigen identification (2). Protein microarrays have facilitated a rapid, systematic and high-throughput approach to probing an entire pathogens' proteome or fraction thereof for immunoreactivity, in an approach that forms part of a reverse vaccinology workflow. These have aided in the discovery of potential diagnostic markers for Mycobacterium tuberculosis and SARS-coronavirus as well as potential vaccine candidates in over 30 human pathogens including Plasmodium falciparum (2, 3). P. falciparum malaria causes ~450,000 deaths per year (4), and is of major public health importance to sub-Saharan Africa (5). Recent gains in reducing the burden appear to have stalled despite ongoing control efforts (4, 6). Efforts to design a highly effective vaccine that would protect against this disease have been hampered by the complexity of the organism and its' multi-stage life cycle: its genome encodes >5,300 proteins that are expressed variably in different tissues as the infection develops in the host (7). Coupled to this is an impressive array of strategies for generating protein polymorphisms or protein variants and redundant erythrocyte invasion pathways, which facilitate immune evasion (8–10). Consequently, although efforts to develop a highly effective malaria vaccine have been on-going for over a century, this goal has yet to be achieved. The current leading vaccine candidate against P. falciparum malaria has limited efficacy and induces only short-lived protective immunity (11, 12). Multiple P. falciparum and/or P. vivax protein arrays have been designed over the past decade to help identify and prioritize potential malaria vaccine antigen candidates. The majority of these arrays have been manufactured using either the E. coli-based or the wheat germ cell free in-vitro transcription/translation expression system, with the largest to date including ~30% of the entire P. falciparum proteome (13–17). Protein selection was based on stage-specific transcription or protein expression, sub-cellular localization, secondary protein structures or documented immunogenicity in human and animal models. However, the in-vitro transcription/translation systems are relatively poor at generating functional surface proteins, which frequently require disulphide bonding and/or post-translational modification to attain their correct three-dimensional structure. Nevertheless, subsequent studies have down-selected proteins from this initial panel (18–31), indicating that essentially >75% of the parasite genome has yet to be evaluated in the context of immunity. A few additional proteins have been tested independently in smaller scale studies accounting for only a marginal increase in the proportion of the parasite proteome evaluated to date (32–34). These studies have rationally selected merozoite proteins that were established or plausible targets of antibodies, and evaluated antibody associations with protection in longitudinal studies using standard ELISA-based approaches (32, 33). They highlighted the importance of evaluating a broad repertoire of antigens and combinations of antibody responses in studies of acquired immunity. However, there still remains a need for a common platform with standardized protein expression and high-throughput antibody detection methods that can be applied widely across different clinical studies (35). This would accelerate identification of protective antibody targets and facilitate the comparisons between studies and populations. To contribute to vaccine candidate discovery, as well as the validation and prioritization of existing candidates for clinical trials, we designed a novel protein microarray. We focused on the merozoite stage that is a target of immunity that can prevent or reduce the clinical symptoms of malaria. As per the case with other infectious diseases (36, 37), we hypothesized that proteins on or associated with the surface of the invasive P. falciparum merozoite would be accessible targets for protective antibodies (33). We mined the literature to identify multiple potential surface-associated merozoite proteins (32–34, 38–43) and added new proteins that were identified as immunogenic in adults from malaria-endemic countries and had proteomic and/or bioinformatic features suggestive of merozoite surface-localization, secretion and/or involvement in erythrocyte invasion (44). We expressed and purified these proteins and printed them on a custom microarray, which we refer to as KILchip v1.0 for its origin at the KEMRI-Wellcome Trust Research Programme in Kilifi, Kenya where the majority of the work was carried out.
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