Broadly inhibitory antibodies against severe malaria virulence proteins

Abstract

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

Figure 1:. Isolation of monoclonal antibodies against the PfEMP1 CIDRα1 domain.

A) Schematic representation of CIDRα1-containing multi-domain PfEMP1 proteins with the N-terminal domain complex comprised of the N-terminal segment (NTS), DBLα, and CIDRα1 domain indicated. The EPCR binding site is also shown. B) Overview of the experimental strategy to isolate monoclonal antibodies against CIDRα1 domains. C) Heatmap showing monoclonal antibody reactivity and inhibition of EPCR binding to a panel of CIDRα1 variants (Luminex assay). Controls include bovine serum albumin (BSA) and CD36-binding CIDRα2, CIDRα5, and CIDRα6 variants. D) Titration of monoclonal antibody reactivity and inhibition of EPCR binding to CIDRα1 variants representative of each of the six subclasses for C7 and C74. MFI, median fluorescence intensity.

Figure 2:. C7 and C74 reactivity to and inhibition of P. falciparum-infected erythrocytes.

A) Flow cytometry analysis showing C7 and C74 (FITC) staining of live P. falciparum-infected erythrocytes expressing PfEMP1 variants containing CIDRα1.1 (IT4VARl9), CIDRα1.4 (HB3VAR03), and CIDRα1.6 (IT4VAR18). IgG from rats immunized with the respective PfEMP1 variants were used as positive controls. IgG samples from rats immunized with a heterologous PfEMP1 variant was included as negative controls. Dashed lines indicate the cutoff for positive cells, as determined using non-infected erythrocytes in the same sample. B) Binding of P. falciparum-infected erythrocytes expressing CIDRα1.4 PfEMP1 (HB3VAR03) to recombinant EPCR under static conditions in culture medium, or in presence of 50 μ/ml C7 or C74. As a negative control, mAb PAM1.4 targeting the non-CIDRα1-containing VAR2CSA PfEMP1 was used. Recombinant soluble EPCR was included as positive control. Binding levels from seven independent experiments are shown. Within each experiment, binding was normalized to the medium only condition that was indexed to 100. A repeated-measures one-way ANOVA followed by Dunnet’s test was used to evaluate differences compared to the negative control. P values shown are from Dunnet’s post-hoc test and are corrected for multiple comparisons. + denotes the mean. C) Binding of CIDRα1.4 PfEMP1-expressing P. falciparum-infected erythrocytes (HB3VAR03) to human brain endothelial cells in 3D microvessels under flow conditions. Top: schematic of device components used to generate a 13-by-13 3D microfluidic network. Bottom: volumetric reconstruction of a microvessel cross section (120 μm diameter) after immunofluorescence labelling with an anti-human VE-cadherin antibody (magenta) and nuclear staining by DAPI (blue). Parasite nuclei can be identified as smaller, brighter blue foci attached to the bottom endothelial surface (DAPI). D) Percentage of endothelial area occupied by sequestered P. falciparum-infected erythrocytes in the 3D microvessels at regions exposed to different wall shear stress rates. Dots indicate the median values, and the shaded regions show the interquartile range, from a total of 9, 7, and 9 independent biological replicates for C7 (0.47 mg/ml), C74 (0.4 mg/ml), and the isotype control IgG₁ (neg., 0.47 mg/ml), respectively. Statistical analyses were performed for binned regions (dotted vertical lines) using a Kruskal-Wallis test, followed by comparisons between IgG₁ and C7 or C74 using Dunn’s post-hoc test, corrected for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 3:. Structural analysis of C7 Fab in complex with recombinant PfEMP1.

A) Schematic of the single CIDRα1.4 domain (HB3VAR03). EPCR-binding (EB) and supporting (EBS) helices of the CIDRα1.4 domain are shown in yellow. B) X-ray crystallography structure of the single CIDRα1.4 domain (HB3VAR03) in complex with C7 Fab shown in cartoon representation. C) Overlay of X-ray structure from panel B with the CIDRα1.4 (HB3VAR03):EPCR structure (PDB: 4V3D). EPCR is shown in gray surface representation. D) Schematic of the IT4VAR22 three-domain protein. The domain architecture is shown in the top, with EB and EBS helices of CIDRα1.7 highlighted in red. E) Cryo-EM map of the IT4VAR22 three domain protein in complex with C7 Fab. F) Superimposition of CIDRα1.4 (HB3VAR03) and CIDRα1.7 (IT4VAR22) domains in complex with C7 Fab. The root mean square deviation (RMSD) for alpha-carbon atoms (Cα) in both structures is shown. G) Molecular interaction of C7 Fab with EB and EBS helices of CIDRα1.7 (IT4VAR22). π-stacking interactions can be seen between CIDRα1.7 residues F661 and F662 and heavy chain residues Y100E, Y100I, and F100J.

Figure 4:. Cryo-EM structure of C74 Fab complexed with the CIDRα1.7 PfEMP1 variant.

A) Cryo-EM map of C74 Fab complexed with the three-domain protein derived from IT4VAR22 PfEMP1. EPCR binding (EB) and supporting (EBS) helices of CIDRα1.7 are colored in red. B) Molecular interaction of C74 Fab with the CIDRα1.7 EB and EBS helices. C) Superimposition of aromatic π-stacking interaction with the CIDRα1.7 FF motif (left panel) and superimposition of the Y-S residues of C7 and C74 targeting E67l of IT4VAR22 CIDRα1.7 (right panel). D) Key antigen-contacting aromatic residues of C7 and C74 shown in their respective H-CDR conformation.

Figure 5:. Conservation and surface exposure of C7 and C74 epitope residues.

A) CIDRα1 residues contacting C7 and C74 mapped onto to the IT4VAR22 CIDRα1.7 domain (circled amino acid residues are mAb specific). B) Surface exposure of C7 and C74 contact residues mapped onto the unbound three-domain protein of the CIDRα1.4 PfEMP1, HB3VAR03 (PDB: 8C3Y). Numbering is based on the CIDRα1.7 IT4VAR22 sequence. C) Sequence logo plot showing amino acid conservation (colored by chemistry) of the EB and EBS helices in CIDRα1.4 – 1.8 and CIDRα1.1 (numbered relative to the IT4VAR22 sequence). D) ELISA reactivity of C7, C74 and recombinant EPCR to the mutated CIDRα1.4 (HB3VAR03) and CIDRα1.1 (IT4VAR20) domains. Wild type OD values >1; data indexed to WT=1.

Figure 6:. Analysis of C7 germline mAbs.

A) Depiction of inferred germline antibody designs, showing mAb C7 (top), C7 containing an inferred germline heavy chain variable region (iGL_H, middle), and C7 containing inferred heavy and light chain variable regions (iGL_HL, bottom). B) Reactivity of C7, iGL_H C7 and iGL_HL C7 across a panel of recombinant CIDRα1 protein domains and negative control (neg.) CD36-binding CIDRα2/5 domains. Blue squares indicate positive reactivity. C) All-atom molecular dynamics simulation of the antigen-bound structure of C7, iGL_H C7 and iGL_HL C7 with the number of distinct structural clusters indicated. D) Reactivity of C7 with germline-reverted H-CDR1 or H-CDR2 as well as iGL_H C7 with matured H-CDR1 or H-CDR2 to select representative CIDRα1 protein domains and a negative control (neg.).