Enhancing blockade of Plasmodium falciparum erythrocyte invasion: assessing combinations of antibodies against PfRH5 and other merozoite antigens

Abstract

No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50) values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.

Figure 1. Measurement of GIA EC₅₀ of antigen-specific anti-PfRH5 and anti-PfAMA1 polyclonal rabbit antibodies.

Panel A - CFCA-measured antigen-specific antibody as a proportion of total IgG (measured by spectrometry) for each of ten rabbits. Individual points indicate mean of three measurements. Panel B - GIA vs. total IgG concentration, with lines connecting data for each of five PfRH5-vaccinated rabbits (red) and five PfAMA1-vaccinated rabbits (black). Each point is the mean of three replicate wells in two independent experiments, i.e. n = 6. Error bars indicate SEM. Panel C - GIA (from the experiments depicted in panel B) vs. antigen-specific antibody concentration (calculated for each sample using the data in panel A), for each of five PfRH5-vaccinated rabbits (red) and five PfAMA1-vaccinated rabbits (black). Each point is the mean of triplicate wells in two independent experiments. Panel D - antigen-specific antibody EC₅₀ values for PfRH5 and PfAMA1, calculated by interpolation from the data in panel C. Individual data-points and the median are shown. Panel E - Dose-response curve fitted to all GIA vs. antigen-specific antibody concentration data for the 3D7 parasite clone (multiple IgG dilutions for each of five rabbits for PfRH5 and PfAMA1). Dashed vertical lines indicate the fitted EC₅₀ value for anti-PfRH5 (red) or anti-PfAMA1 (black) IgG. Each GIA value is the mean of triplicate wells in each of two experiments (n = 6). Red indicates anti-PfRH5 samples; black indicates anti-PfAMA1 samples.

Figure 2. Antigen-specific EC₅₀ estimation for anti-PfRH5 and anti-PfAMA1 IgG against short-term-adapted Cambodian parasite isolates.

Dose-response curves were fitted to all GIA versus antigen-specific antibody concentration data for A) CP803, B) CP806, C) CP830, D) CP845 and E) CP887 (multiple IgG dilutions for each of five rabbits for PfRH5 and each of four rabbits for PfAMA1). Dashed vertical lines indicate the fitted EC₅₀ value for anti-PfRH5 IgG (red) or anti-PfAMA1 (black) IgG for that isolate. Each value is the mean of three wells in a single experiment. Red indicates anti-PfRH5 samples; black indicates anti-PfAMA1 samples.

Figure 3. GIA effects of anti-PfRH5 IgG in combination with polyclonal antibody specific for other merozoite antigens.

Percentage GIA against the 3D7 parasite clone over increasing concentrations of total purified IgG from rabbits immunized with A) PfRAP3, B) PfMSP1, C) PfAMA1, D) Pf38, E) PfEBA175, F) PfRH2 and G) PfRH4, with (blue line) or without (solid black line) the addition of a fixed low concentration of PfRH5-immunized rabbit IgG (0.156 mg/mL) which, when used alone, gives approximately 25% GIA (dashed black line). Predicted additive effects were calculated according to Bliss independence (see Materials and Methods) and illustrated as the red line on each graph. Data points represent the mean of triplicates from two independent experiments. Bars indicate SEM for all six replicates over two experiments. Asterisks indicate that the predicted and observed values differed significantly (*P<0.05; **P<0.01; ***P<0.001, 2-way ANOVA with Bonferroni post-hoc testing).

Figure 4. Contour plots and isobolograms of GIA achieved by anti-PfRH5 IgG in combination with either anti-PfRH4 or anti-PfAMA1 IgG.

Panel A,B–Contour plots of GIA versus concentration of total IgG combined from rabbits immunized with either PfRH5 or PfRH4 (A), or with either PfRH5 or PfAMA1 (B). Each experiment was conducted independently. Black lines are contours linking anti-PfRH5 and anti-PfRH4 IgG combinations inducing 25%, 50% and 75% GIA (as labelled), obtained by interpolation between observed GIA values. Shaded area indicates 0–25% GIA (blue), 25–50% GIA (green), 50–75% GIA (orange), and 75–100% GIA (pink). Thin diagonal dashed line from origin indicates line of equal concentration of IgG from each component. Panel C,D – 50% GIA isobologram for anti-PfRH5 and anti-PfRH4 IgG (C) and anti-PfRH5 and anti-PfAMA1 IgG (D) combinations. Red line links the observed combination of anti-PfRH5 IgG and either anti-PfRH4 or anti-PfAMA1 IgG that induced 50% GIA, plotted on axes of anti-PfRH5 and anti-PfRH4/anti-PfAMA1 IgG concentration expressed as percentage of the EC₅₀. Dashed line illustrates 50% contour predicted if anti-PfRH5 IgG and the other antibody are Loewe additive. Diagonal x = y line from origin links points at which anti-PfRH5 and anti-PfRH4/PfAMA1 IgG concentrations (as proportion of EC₅₀) are equal; the letters M and N indicate the line intersections used to calculate Hewlett's synergy index. Panel E – GIA attained by mixing equal concentrations of anti-PfRH5 with anti-PfRH4 IgG (blue line), plotted against the total IgG concentration in the well shown on the lower x-axis (i.e. twice the concentration of each individual component). The solid red line indicates the GIA effect when anti-PfRH5 IgG is used alone at the concentrations on the lower x-axis (i.e. twice the concentration of anti-PfRH5 IgG in the antibody mixture), and the dashed red line indicates the GIA effect of anti-PfRH5 IgG alone at the concentration shown on the upper dashed x-axis (i.e. the concentration of anti-PfRH5 IgG present in the antibody mixture). The solid and dashed black lines indicate the same relationship for anti-PfRH4 IgG.

Figure 5. GIA synergies against the vaccine-heterologous FVO parasite clone.

Percentage GIA against the FVO parasite clone over increasing gradients of concentrations of IgG from rabbits immunized with A) PfEBA175, B) PfRH2 or C) PfRH4 with (blue line) or without (solid black line) the addition of a fixed low concentration of PfRH5-immunized rabbit IgG (0.156 mg/mL) which, when used alone, gives approximately 20% GIA (dashed black line). Predicted additive effects were calculated according to Bliss independence (see Materials and Methods) and illustrated as the red line on each graph. Data points represent the mean of triplicates from two independent experiments. Bars indicate SEM for all six replicates over two experiments. Asterisks indicate that the predicted and observed values differed significantly (*P<0.05; ** P<0.01; 2-way ANOVA with Bonferroni post-hoc testing).

Figure 6. GIA synergies with mixtures of rabbit antisera and mouse monoclonal antibodies.

Percentage GIA against 3D7 and FVO parasites using increasing gradients of concentrations of IgG from rabbits immunized with PfRH4 (Panels A–C) or PfEBA175 (Panel D) with (blue line) or without (solid black line) the addition of a fixed low concentration of either an anti-basigin mAb (TRA-1-85, 1 µg/mL) or anti-PfRH5 mAb (QA5, 10 µg/mL) which, when used alone, give approximately 15–25% GIA (dashed black line). Predicted additive effects were calculated according to Bliss independence (see Materials and Methods) and illustrated as the red line on each graph. Results are mean of two independent experiments for the anti-PfRH4/TRA-1-85 mixture with the 3D7 parasite clone, and one experiment for the other combinations. All measurements were done in triplicate. Bars indicate SEM for all replicates. Asterisks indicate the predicted and observed values differed significantly (** P<0.01; ***P<0.001; 2-way ANOVA with Bonferroni post-hoc testing).