Fig 1: Genotype–phenotype associations identified that dominant SNPs in PfRh5 do not influence invasion inhibition. Next Generation Sequencing (NGS) analysis of samples from successful ex vivo invasion assays. (A) Description of the single nucleotide polymorphisms (SNPs) identified in patient samples from Kédougou. Specific site of collection is abbreviated as follows: Bandafassi (BF), Bantaco (BAT), Camp Militaire (CM), Dalaba (DAL), Mako (MAK). Both nucleotide and corresponding nonsynonymous amino acid changes are indicated. SNP frequency and read coverage at the SNP site (SNP Read Coverage) are also indicated. Sequences are accessible in Genbank (MW042085-MW042101). Of the 11 samples with SNPs at the dominant threshold (25%), 9 (52.9%) contained the C203Y SNP, 2 (11.8%) contained I407V, 1 (5.9%) contained K429N, 1 (5.9%) contained V371I. Sample 320558 is the only one to contain more than one SNP. Six samples contained no SNPs. These SNPs have been previously described in both published9,11,13,19,20 and unpublished data generated by the Pf3k project (www.malariagen.net/pf3k), representing 3,248 samples from 40 separate locations in 20 countries9. (B) Genotype–phenotype associations were performed for samples with the C203Y SNP and those without (C203Y vs C203 respectively), at each concentration of anti-BSG (MEM-M6/6) antibody. Medians and 95% confidence intervals are shown. No statistically significant difference between either genotype was observed at any of the antibody concentrations tested.
Fig 2: Identification of BSG and heparan sulfate as independent receptors for P. falciparum RH5 on HEK293 cells. (A) Biotinylated RH5 was clustered around a streptavidin–PE conjugate and binding to HEK293 cells was analyzed by flow cytometry. RH5 binding is only partially reduced by a blocking anti-BSG mAb relative to controls. (B) Rank-ordered genes identified from gRNA enrichment analysis required for cell surface display of an anti-BSG mAb (left) and RH5 binding (right). Significantly enriched genes with a FDR < 0.05 are colored (full screening results available in Supplemental Data S2); genes encoding the receptor (BSG) and chaperone (SLC16A1) were common to both screens, and a gene involved in GAG-biosynthesis (SLC35B2) was additionally required for RH5 binding. (C) Binding of RH5 to cells is reduced when transduced with lentiviruses encoding gRNAs targeting either the receptor (BSG) or enzymes required for HS synthesis (SLC35B2, EXTL3) relative to controls. Transduced polyclonal lines were used for this experiment. (D) RH5 binding to SLC35B2-targeted HEK293 cells could be completely prevented if preincubated with a blocking anti-BSG mAb but not an isotype-matched control. (E) RH5 binding to BSG-targeted HEK293 cells could be completely blocked if preincubated with 200 µg/mL heparin but not 200 µg/mL CS. A representative of three independent (A and C) or technical (D and E) replicate experiments is shown.
Fig 3: Monoclonal antibodies targeting BSG potently inhibit ex vivo Plasmodium falciparum isolates in a dose-dependent manner. The data from the 17 successful ex vivo invasion assays are shown here. PMR is calculated as: Final parasitemia (RPMI alone)/Initial parasitemia (RPMI alone). Invasion inhibition was calculated as 100- [(Average Percent invasion in anti-BSG MEM-M6/6 antibody wells)/(Average parasitemia in IgG1 isotype control wells) * 100]. Parasitemia was counted using a Miller reticle and 500 erythrocytes were counted for each assay. (A) Distribution of inhibition at each concentration: 0.1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu$$\end{document}µg/ml, 1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu$$\end{document}µg/ml, and 10 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu$$\end{document}µg/ml, demonstrating overall conservation in inhibition at each concentration. Box and whisker plots show the median, interquartile range, and whiskers indicate the minimum and maximum. (B) Dose-response curves for inhibition with anti-BSG MEM-M6/6 antibody for each sample, including 3D7 for reference. (C) Dose-response curves for inhibition with anti-BSG MEM-M6/6 antibody for samples from the bottom 25% of inhibition (calculated from the 10 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu$$\end{document}µg/mL concentration), including 3D7 for reference. (D) Dose-response curves for inhibition with anti-BSG MEM-M6/6 antibody for samples from the top 25% of inhibition (calculated from the 10 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu$$\end{document}µg/mL concentration), including 3D7 for reference.
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