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. 2019 Jul;21(7):e13030.
doi: 10.1111/cmi.13030. Epub 2019 Apr 24.

Neutralising Antibodies Block the Function of Rh5/Ripr/CyRPA Complex During Invasion of Plasmodium Falciparum Into Human Erythrocytes

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Neutralising Antibodies Block the Function of Rh5/Ripr/CyRPA Complex During Invasion of Plasmodium Falciparum Into Human Erythrocytes

Julie Healer et al. Cell Microbiol. .
Free PMC article

Abstract

An effective vaccine is a priority for malaria control and elimination. The leading candidate in the Plasmodium falciparum blood stage is PfRh5. PfRh5 assembles into trimeric complex with PfRipr and PfCyRPA in the parasite, and this complex is essential for erythrocyte invasion. In this study, we show that antibodies specific for PfRh5 and PfCyRPA prevent trimeric complex formation. We identify the EGF-7 domain on PfRipr as a neutralising epitope and demonstrate that antibodies against this region act downstream of complex formation to prevent merozoite invasion. Antibodies against the C-terminal region of PfRipr were more inhibitory than those against either PfRh5 or PfCyRPA alone, and a combination of antibodies against PfCyRPA and PfRipr acted synergistically to reduce invasion. This study supports prioritisation of PfRipr for development as part of a next-generation antimalarial vaccine.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Domain structure of PfRipr and expression of recombinant proteins. (a) Domain structure of PfRipr showing the location of the EGF‐like domains (blue) and a protease processing site (scissors). Also shown are the boundaries of the recombinant proteins expressed to represent different domains of the PfRipr protein with residue range shown in brackets. (b) Expression and purification of Fl‐Ripr (amino acids 20–1086) from S2 Drosophila cells. Shown in the left panel is a SDS‐PAGE gel stained with Coomassie blue with molecular weight markers. Supern, culture supernatant; Input, concentrated supernatant loaded for affinity purification; Ft, flow through from affinity column; Elution, fractions eluted from affinity column. (c) Recombinant domains of PfRipr (as depicted in a) purified and stained with Coomassie blue. (d) SEC analysis of the PfRh5/CyRPA/PfRipr complex detected using Coomassie blue staining (lower panel)
Figure 2
Figure 2
Identification of invasion neutralising monoclonal antibodies and mapping of epitopes on PfRipr. (a) Immunoblots of FL‐Ripr under reduced (R) and nonreduced (NR) conditions after SDS‐PAGE using monoclonal antibodies raised to CT‐Ripr. Monoclonal antibodies used were 1C4, 3C3, 4A8, 5G6, and 1G12. (b) Growth inhibition assays (GIA) of the anti‐Ripr monoclonal antibodies using 3D7 Plasmodium falciparum parasites. (c) Immunoblots of PfRipr recombinant protein domains to map the monoclonal antibody epitopes (1C4, 3C3, 4A8, 5G6, and 1G12). The final panel was screened using rabbit anti‐Fl‐Ripr polyclonal antibodies. (d) Domain structure of PfRipr and mapping of the spatial location of monoclonal antibody epitopes
Figure 3
Figure 3
Neutralising monoclonal antibodies against PfRh5, PfRipr, and CyRPA and effect on PfRh5/CyRPA/PfRipr complex formation. (a) SEC analysis of inhibitory monoclonal antibody 2AC7 which binds to PfRh5 showing it blocks PfRh5/CyRPA/PfRipr complex formation. (b) SEC analysis of inhibitory monoclonal antibody 5G6 which binds to PfRipr but does not block PfRh5/CyRPA/PfRipr complex formation. Proteins are stained with Coomassie blue. SEC analysis for 1G12 and 1C4 are shown in Figure S5. (c) SEC analysis of inhibitory monoclonal antibody 8A7 which binds to CyRPA and blocksPfRh5/CyRPA/PfRipr complex formation. (d) SEC analysis of inhibitory monoclonal antibody 3D1 which binds to CyRPA but does not block PfRh5/CyRPA/PfRipr complex formation. Proteins in all SDS‐PAGE gels are stained with Coomassie blue. SEC analysis for 5B12 is shown in Figure S5. Traces shown above SEC gel profiles in all panels give elution volumes of molecular weight standards in kDa. Models of mAb binding to antigens and their effect on complex formation are shown underneath respective SEC panels
Figure 4
Figure 4
Antibodies against PfRh5, CyRPA, and PfRipr inhibit Plasmodium falciparum growth. (a) Growth inhibition assays using IgG rabbit antibodies raised to equal concentrations of Ct‐Ripr, Fl‐Ripr, CyRPA, PfRh5/CyRPA/PfRipr complex or PfRh5 with P. falciparum strains 3D7, W2mef, FCR3, and FVO. Plots are data from two biological replicates with mean and SEM shown. (b) Combined summary of GIA assays at 5 mg/ml IgG for 3D7, W2mef, FCR3, and FVO. (c) GIA assays using 3D7 parasites where anti‐Fl‐Ripr, CyRPA, and PfRh5 antibodies have been added in combinations. Total IgG concentration shown on x‐axis. (d) GIA assays using 3D7 parasites where polyclonal antibodies against Rh5, CyRPA and PfRipr have been combined to a total IgG concentration of 6 mg/ml. * Denotes significantly different activity p < .05 (t test). (e) Expected and actual level for inhibition of parasite growth when CyRPA and PfRipr monoclonal antibodies have been combined. Monoclonal antibodies against Ripr (1G12 and 5G6) were combined with those against CyRPA (5G6 and 5B12). Shown in black bars is the expected level of inhibition derived by adding the GIA activity of the individual mAbs each at a concentration of 0.25 mg/ml. In grey bars is the actual GIA activity of the combined mAbs at a total concentration of 0.5 mg/ml (0.25 mg/ml of each)
Figure 5
Figure 5
Antibodies to PfRh5 and PfRipr are associated with protection from clinical malaria. (a) Time to first light microscopy positive parasitemia with ≥5,000 parasites/μl in individuals with low (L), medium (M), or high (H) titers of antibody to PfRh5, PfRh5/CyRPA, PfRh5/CyRPA/PfRipr, CyRPA, PfRipr, CyRPA/PfRipr at study enrolment, as determined with Kaplan–Meier survival analysis. (b) Hazard ratios were determined to assess associations of antibody responses with time to first clinical episode and (c) incidence rate ratios were determined to assess associations between antibody responses and incidence of clinical episodes. Age‐adjusted ratios with 95% confidence interval are shown. Clinical episode was defined by the presence of fever plus light microscopy positive parasitemia with ≥5,000 parasites/μl. * p ≤ .05, ** p ≤ .01

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References

    1. Baum J., Chen L., Healer J., Lopaticki S., Boyle M., Triglia T., … Cowman A. F. (2009). Reticulocyte‐binding protein homologue 5—An essential adhesin involved in invasion of human erythrocytes by Plasmodium falciparum . Int. J. Parasitol., 39, 371–380. 10.1016/j.ijpara.2008.10.006 - DOI - PubMed
    1. Bustamante L. Y., Bartholdson S. J., Crosnier C., Campos M. G., Wanaguru M., Nguon C., … Rayner J. C. (2013). A full‐length recombinant Plasmodium falciparum PfRH5 protein induces inhibitory antibodies that are effective across common PfRH5 genetic variants. Vaccine, 31, 373–379. 10.1016/j.vaccine.2012.10.106 - DOI - PMC - PubMed
    1. Bustamante L. Y., Powell G. T., Lin Y. C., Macklin M. D., Cross N., Kemp A., … Rayner J. C. (2017). Synergistic malaria vaccine combinations identified by systematic antigen screening. Proc. Natl. Acad. Sci. U S A, 114, 12045–12050. 10.1073/pnas.1702944114 - DOI - PMC - PubMed
    1. Chen L., Lopaticki S., Riglar D. T., Dekiwadia C., Uboldi A. D., Tham W. H., … Cowman A. F. (2011). An EGF‐like protein forms a complex with PfRh5 and is required for invasion of human erythrocytes by Plasmodium falciparum . PLoS Pathog, 7, e1002199 10.1371/journal.ppat.1002199 - DOI - PMC - PubMed
    1. Chen L., Xu Y., Healer J., Thompson J. K., Smith B. J., Lawrence M. C., & Cowman A. F. (2014). Crystal structure of PfRh5, an essential P. falciparum ligand for invasion of human erythrocytes. Elife, 3 10.7554/eLife.04187 - DOI - PMC - PubMed

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