Figure 3.
Epitope Binning Reveals that All Relevant Neutralizing Epitopes Lie within PfRH5ΔNL
(A) Epitope bins determined by BLI from a matrix of sequential PfRH5FL-binding assays for different mAbs, with data used to construct these bins in Figure S3A and Table S1.
(B) Potency of anti-PfRH5 mAb invasion inhibition grouped by epitope bin. EC30 values were interpolated from data in Figure 2B.
(C) The effect of mAbs on binding of PfRH5FL to BSG, PfCyRPA, and PfP113Nt, as determined by SPR. Black bars show binding in the absence of mAb as a control. The colors used in (B) and (C) match those of the epitope bins in (A).
(D) Binding of anti-PfRH5 mAbs to PfRH5ΔNL by ELISA. Bars show the mean of 4 replicate wells.
(E) GIA of purified total IgG from seven different PfRH5FL-vaccinated human volunteers alone or with 0.5 μM of PfRH5FL or PfRH5ΔNL protein (30 μg/mL and 20 μg/mL, respectively). Bars show the mean of duplicate wells.
(F) GIA of purified total IgG from rabbits immunized with PfRH5FL or PfRH5ΔNL (n = 6 rabbits per group). Concentrations of PfRH5FL-specific polyclonal IgG were measured by ELISA using a conversion factor determined by calibration-free concentration analysis. Individual data points show the mean of triplicate wells.
(G) Comparison of EC50 for rabbit sera. EC50 values were determined for each rabbit by interpolation after fitting the data from (F) to a four-parameter dose-response curve. Black horizontal bars represent the mean.
(H) Intravital luminescence signal of humanized mice infected with transgenic P. falciparum (NF54-luciferase) following passive transfer of 15 mg of pre-vaccination or PfRH5ΔNL-vaccinated rabbit IgG at day (d)6 post-infection. Starting groups were n = 2 for the PBS group and n = 4 for the IgG passive transfer groups. Mice that died before the experiment endpoint (d13) were: one at d7 and one at d13 in the PBS group, one at d9 and one at d12 in the pre-vaccination IgG group and one at d6, one at d10 and one at d12 in the vaccinated IgG group. Individual data points are connected by a line representing the mean.
(I) Concentration time course of PfRH5-specific rabbit IgG in the passive transfer experiment shown in (H), as determined by ELISA binding to PfRH5FL. Data points show the mean. All GIAs used 3D7 clone P. falciparum. All error bars show the SEM.