Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5

Abstract

There is intense interest in induction and characterization of strain-transcending neutralizing Ab against antigenically variable human pathogens. We have recently identified the human malaria parasite Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) as a target of broadly neutralizing Abs, but there is little information regarding the functional mechanism(s) of Ab-mediated neutralization. In this study, we report that vaccine-induced polyclonal anti-PfRH5 Abs inhibit the tight attachment of merozoites to erythrocytes and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 mAbs, we provide evidence of the following: 1) the ability to block the PfRH5-basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; 2) neutralizing mAbs bind spatially related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; 3) a brief exposure window of PfRH5 is likely to necessitate rapid binding of Ab to neutralize parasites; and 4) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly neutralizing anti-malaria Abs and further encourage anti-PfRH5-based malaria prevention efforts.

Figure 1. Localization of PfRH5 by indirect IFA

Localization of PfRH5 was assessed by indirect IFA using anti-PfRH5 polyclonal rabbit serum (green). (A) Fixed and permeabilized schizonts with (+) or without (−) E64 treatment or free merozoites (inset), of 3D7 clone P. falciparum were co-stained with mouse mAbs (red) to mark various organelles: PfAMA1 (microneme), PfRAP1 (rhoptry body), or PfRON4 (rhoptry neck); or polyclonal mouse serum against further antigens: PfRH4 and PfRipr. Figures show the merge of the dual staining antibodies and nuclei stained with DAPI (blue), as well as the brightfield view. Scale bars = 1 μm. (B) Localization of PfRH5 and PfAMA1 was assessed by indirect IFA using antigen-specific polyclonal rabbit serum (green) on the surface of free, fixed merozoites isolated from E64-treated schizonts either permeabilized with Triton X100 (+ TX100) or non-permeabilized ( − TX100). Nuclei were stained with DAPI, and the merge of the images is shown. (C) Localization of PfRH5 and PfAMA1 using antigen-specific polyclonal rabbit serum (green) on the surface of non-fixed non-permeabilized merozoites adherent to RBC in the presence of 1μM cytochalasin D. Scale bars = 1 μm. (D) Localization of PfRH5 was assessed by indirect IFA using PfRH5-specific polyclonal rabbit serum (green) on fixed and permeabilized merozoites adherent to RBC in the presence of 1μM cytochalasin D. Co-staining with mouse antibodies (red) was used to identify PfRON4, PfRipr and PfRAP1. Nuclei were stained with DAPI, and the merge of the images together with the brightfield view is shown. RBCs, though present, are only detected as faint membrane structures due to the loss of hemoglobin during paraformaldehyde fixation and Triton X100 permeabilization.

Figure 2. Inhibition of merozoite attachment and effect of anti-PfRH5 antibodies on the PfRH5-BSG interaction measured by AVEXIS

(A) Three independent assays of merozoite attachment to erythrocytes were conducted. Lines link each observation of the percentage of RBCs with attached merozoites (left axis) from a single assay in the presence of 10mg/mL purified IgG pooled from rabbits immunized with a non-malaria antigen (‘Control’), and in the presence of 10mg/mL purified IgG that was pooled from three representative PfRH5-immunized rabbits (‘Anti-PfRH5’). Right-hand column indicates the percentage reduction in attachment induced by anti-PfRH5 IgG (calculated from the same data; right axis). (B,C) An AVEXIS assay was performed with plate-bound PfRH5 protein and soluble pentameric basigin. Prior to application of basigin, wells were incubated with serial dilutions of antisera from (B) mice or (C) rabbits immunized with viral vector vaccines expressing full-length PfRH5, a previously described PfRH5 fragment, PfAMA1, or a non-malaria antigen. Points and error bars indicate median and range of three replicate wells. Upper horizontal dashed line indicates the mean minus three standard deviations (SD) of results in control wells with PfRH5, BSG and substrate but no antibody. Lower dashed line indicates mean plus three SD of results in control wells with PfRH5 and substrate but no BSG. (D) Each sub-panel depicts the effect of an individual mAb tested at a range of concentrations by AVEXIS (x-axis). Two sub-panels are shown for each mAb, depicting the results obtained with different orientations of the bait and prey proteins used in the assay: the left hand sub-panels show the assay when PfRH5 protein was immobilized to the plate prior to application of soluble pentameric BSG, while the right hand sub-panels show data obtained with the reverse orientation. The solid lines (both sub-panels, left-hand y-axis) show the OD₄₈₅ readouts for the AVEXIS assay following pre-incubation of the PfRH5 protein either on the plate (left-hand panels) or in solution (right-hand panels) with a dilution gradient of each mAb. To permit comparison of the effect in the AVEXIS assay with the level of binding of mAb, total IgG ELISA was also carried out using replicate wells with an identical gradient of concentration of each mAb (broken line, left hand sub-panels, right-hand y-axis). Error bars for each line indicate the mean and range of the OD readings for n = 2-3 replicate wells within an experiment.

Figure 3. Effects of anti-PfRH5 mAbs in the assay of GIA

Anti-PfRH5 mouse mAbs were tested in the in vitro assay of GIA at a range of concentrations against (A) 3D7 clone P. falciparum parasites and (B) FVO parasites. Results show the mean of two experiments with triplicate wells. Error bars indicate inter-well SEM.

Figure 4. Anti-BSG mAbs which do not block the PfRH5-BSG interaction by AVEXIS are capable of in vitro parasite invasion inhibition

(A) Anti-BSG mAbs were tested for their ability to inhibit the PfRH5-BSG interaction in the AVEXIS assay. mAbs MEM-M6/6 and TRA-1-85 potently blocked the PfRH5-BSG interaction. mAbs P2C2-1-D11, MEMM6/1, and 8J251 did not. Assays for mAbs MEM-M6/6, TRA-1-85, MEM-M6/1, and 8J251 were performed simultaneously in triplicate; points and error bars indicate median and range of replicate wells. The assay using P2C2-1-D11 was performed separately in singlet. (B) Anti-BSG mAbs were tested at a range of concentrations in a flow cytometric invasion inhibition assay against 3D7 clone parasites. All tested mAbs potently inhibited invasion. Points and error bars indicate median and range of three replicate wells. Data for the BSG-PfRH5 interaction blocking mAbs MEM-M6/6 and TRA-1-85 are in agreement with previously published data (16).

Figure 5. Mapping of linear and minimal epitopes for mAb binding

(A) Assessment by ELISA of mAb binding to overlapping PfRH5-derived 20mer peptides. Binding to native PfRH5 protein is also shown as a control. Peptides are indicated in this graph by the single-letter code and numerical position of their first αα, e.g. E26 indicates the peptide ENAIKKTKNQENQLTLLPIK, beginning with glutamic acid 26 (the first amino acid residue after PfRH5’s 25 amino acid residue signal peptide). * indicates significant binding (OD₄₀₅ values greater than 3 SD above the mean for an individual mAb). (B) Binding of mAb QA5 to peptides progressively truncated from the N-terminus (white) and the C-terminus (red) of its 20mer-epitope (beginning with Y194: YHKSSTYGKCIAVDAFIKKI). Results are the mean of two replicate assays. Truncation of the N-terminus beyond Y200 abrogated binding; truncation of the C-terminus beyond I213 progressively reduced binding, which was completely abrogated by truncations beyond K211. Binding to a 20mer with the FVO parasite strain sequence (YHKYSTYGKYIAVDAFIKKI; green) was similar to that to the 3D7-clone 20mer Y194 (blue). Other controls shown in blue include a non-recognized 20mer peptide (beginning with H178) and the 9AD4 20mer binding peptide (beginning Y346). (C) Binding of 9AD4 to progressively truncated peptides derived from its 20mer epitope beginning with Y346 (YNNNFCNTNGIRYHYDEYIH), as for (B). (D) Binding of QA5 to peptides with each of the internal amino acids in the sequence YGKCIAVDAFIKK progressively mutated to alanine (or glycine in the case of the two alanines in the native sequence). Results are the mean of two replicate assays. Results obtained with these ‘alanine walk’ peptides are shown in grey; results with peptides from the N- and C-terminal truncation set are shown in white and red respectively; results obtained with the originally identified 20mer peptide are shown in blue. The lettering "YGK(C/Y)IAVDAFIKKI" indicates the inferred linear epitope for QA5, with sizes of lettering proportional to the reduction in binding resulting from mutation of each amino acid and (C/Y) indicating the polymorphism between 3D7 (red) and FVO (green) parasites. (E) Binding of 9AD4 to the series of ‘alanine walk’ peptides within the inferred minimal epitope. Colours as in (D); one of the original 20mer peptides (beginning NGIRY...; bar second from left; blue) was used to infer the role of the initial threonine in the epitope.

Figure 6. Analysis of anti-PfRH5 mAb epitope overlap by sandwich SPR binding assay

A sandwich SPR-based assay was used to assess overlap of epitopes bound by mAbs 2AC7, 4BA7, 6BF10, 8BB10, 9AD4, QA1 and QA5. (A) Levels of binding (response units) of each mAb when injected as a secondary mAb (‘mAb B’) over PfRH5 captured onto chip by binding to a primary mAb (‘mAb A’). See Figure S3A for illustration of assay configuration, and Figure S3B-H for the data from which these binding levels were calculated. To assess binding competition, levels of binding were compared both to the maximum level of binding of that specific ‘mAb B’ to PfRH5 captured by any ‘mAb A’, and to the maximum level of binding of any ‘mAb B’ to PfRH5 captured by the same ‘mAb A’. Complete competition (red), partial competition (orange) and non-competitive binding (green) were defined as in Methods. (B) Schematic model of the arrangement of epitopes on the PfRH5 surface, with summary of inhibitory binding interactions: solid lines indicate complete competition; dashed lines indicate partial competition; absence of a line indicates non-competitive binding. The blue-outlined area indicates the putative BSG-binding region, as defined by ability of these mAbs to block the PfRH5-BSG interaction in AVEXIS (Figure 2D); the red-outlined area indicates the region susceptible to neutralizing antibodies (Figure 3).

Figure 7. Kinetics of mAb – RH5 interactions

(A-D) Binding data from single cycle kinetic measurements of the interactions between mAbs 2AC7, 9AD4, QA1 and QA5 are depicted respectively. Curves represent captured mAb-specific binding of PfRH5 in solution (after subtraction of the PfRH5 binding to the reference cell and refractive index changes due to dummy injections of buffer). In each case, replicate results are shown, obtained on two different days with independently captured mAb chip surfaces (and independently prepared PfRH5 dilution series). Solid lines indicate observed binding; dashed lines indicate model-fitted binding. The maximal level of achievable binding (R_max) varied between replicates; these differences in R_max were proportionate to differences in the quantity of captured mAb, which varied between 130RU and 230RU for different mAbs and on different days. (E) Results of single cycle kinetic measurement of interactions of mAbs with PfRH5. To aid interpretation, equilibrium dissociation constants, K_D, are shown both in molar units, and expressed as the μg/mL concentration of mAb which would be expected to achieve 50% saturation of antigen at equilibrium (assuming monovalent binding; the molar K_D multiplied by 1.5×10⁸). To aid interpretation of the dissociation rate constant, k_d, the interaction half-life in minutes is also shown (−ln[0.5]/[60k_d]). Standard errors (SE) of model-fitted kinetic parameters and chi-squared measures of model goodness-of-fit (<10% of R_max in all cases) are also presented.

Figure 8. In vitro GIA with anti-PfRH5 Fabs as compared to GIA with intact IgG

GIA was assessed against 3D7 clone P. falciparum parasites with (A) mAb 2AC7, (B) mAb 9AD4 and (C) mAb QA5, and (D) purified polyclonal rabbit anti-PfRH5 IgG. In each case, GIA with intact IgG is plotted as well as GIA with the respective Fabs. GIA using control purified rabbit IgG and control Fab samples is also shown. A control IgG1 mouse mAb was used for 2AC7 and QA5, whilst RB3 (an IgG2a mAb) was used for 9AD4. The RB3 Fab showed negative GIA within the range of concentration (up to 250μg/mL) in which intact RB3 mAb is GIA-negative (Figure 3). Results show the mean of two assays, each with three replicate wells; error bars plot inter-well SEM. Concentrations are shown as ‘IgG equivalent’. For Fabs, this is the weight/volume concentration of IgG which would have the same molar concentration of antigen-binding sites (i.e. 1.5x the w/v Fab concentration).