Vaccination with conserved regions of erythrocyte-binding antigens induces neutralizing antibodies against multiple strains of Plasmodium falciparum

Abstract

Background: A highly effective vaccine against Plasmodium falciparum malaria should induce potent, strain transcending immunity that broadly protects against the diverse population of parasites circulating globally. We aimed to identify vaccine candidates that fulfill the criteria.

Methods: We have measured growth inhibitory activity of antibodies raised to a range of antigens to identify those that can efficiently block merozoite invasion for geographically diverse strains of P. falciparum.

Results: This has shown that the conserved Region III-V, of the P. falciparum erythrocyte-binding antigen (EBA)-175 was able to induce antibodies that potently inhibit merozoite invasion across diverse parasite strains, including those reliant on invasion pathways independent of EBA-175 function. Additionally, the conserved RIII-V domain of EBA-140 also induced antibodies with strong in vitro parasite growth inhibitory activity.

Conclusion: We identify an alternative, highly conserved region (RIV-V) of EBA-175, present in all EBA proteins, that is the target of potent, strain transcending neutralizing antibodies, that represents a strong candidate for development as a component in a malaria vaccine.

Figure 1. Antibodies raised in rabbits against recombinant P. falciparum invasion ligands specifically recognize native parasite antigens.

A: Schematic of P. falciparum invasion ligands used to generate antigens for immunization. Black bars below the schematic structures indicate the regions of expressed recombinant proteins. Numbers indicate the lanes on the coomassie gel shown in fig. 1B. B: Antigenic fragments of invasion-related proteins were expressed and purified for rabbit immunization. Immunogens are shown here on SDS-PAGE. (1) Rh2 N-terminal fragment (2) Rh2 C-terminal fragment (3) EBA-175 Region 2 (4) EBA-175 Region 3-5 3D7 (5) EBA-175 RIII-V W2mef (6) EBA-175 RIV-V (7) EBA-175 F2-R5 (8) EBA-140 RIII-V. Proteins were stained with coomassie blue. C: Recombinant Region II of EBA-175 binds to erythrocytes in a sialic-dependent manner. Recombinant proteins EBA-175 F2-RV and RII were tested for binding to erythrocytes. Lanes 1 & 4 show protein pre-assay; 2 & 5, binding to untreated erythrocytes; Lanes 3 & 6, binding to neuraminidase-treated erythrocytes. D: Rabbit antibodies raised to recombinant antigens specifically recognize native parasite proteins. 1–8 correspond with antigens as described in Fig. 1A. Lanes as follows: 3D7 wild type (a), 3D7Δ175 KO (b), W2mef (c), W2mefΔ175 KO (d), FCR3 wild type (e), 3D7Δ140 KO (f).

Figure 2. Epitopes of highly inhibitory, strain-transcending antibodies are located within the conserved region of EBA-175.

(A) GIA against the homologous 3D7 parasite strain. Rabbits were immunized with 3D7 EBA-175 region III-V. IgG purified from rabbit serum was serially diluted from a starting concentration of 2 mg/ml. Data points indicate mean values of triplicate wells ± standard error of the mean (SEM) in a single assay relative to pre-immune IgG, calculated from a 1-cycle FACS-based assay. The dashed red line indicates the IC50 value of IgG (9 μg/ml). (B) Rabbit IgG against EBA-175 region III-V (3D7) – GIA against homologous and heterologous parasite strains. IgG from a single rabbit were tested against 3D7, W2mef, FCR3 and 3D7Δ175 KO parasite strains. Data points represent mean values of triplicates from 3 independent replicate experiments ± SEM in a 2-cycle assay. The antibody is completely specific to EBA-175 as there is no inhibition of the 175-KO parasites. (C) Rabbit IgG against EBA-175 region III-V (W2mef) – GIA against homologous and heterologous parasite strains. IgG from a single rabbit were tested against 3D7, W2mef, FCR3 and 3D7Δ175 KO parasite strains (2 cycle assay). (D) Anti- EBA-175 RIII-V directly inhibit invasion of purified, viable, D10-GFP merozoites. Results shown are mean + standard error of three independent experiments. (E) Mouse serum against EBA-175 region III-V (3D7) – GIA against homologous and heterologous parasite strains. Three individual mouse sera were pooled and tested against 3D7, W2mef, FCR3 and 3D7Δ175 KO parasite strains (1 cycle assay). (F) GIA patterns of Ig raised against both dimorphic alleles of EBA-175 against a panel of laboratory parasite strains are highly correlated. GIA was performed in a one cycle assay using anti-EBA175RIII-V (3D7) IgG at a concentration of 1 mg/ml. Blue histograms indicate parasite strains that invade predominantly via sialic-acid dependent pathway, red indicates sialic acid-independent pathway , grey bars where invasion preference is not known. Parasite strains belonging to the C-type group are shown in blue font whereas those belonging to the F-type are shown in red font. (G) GIA as for (F) but using anti-EBA175RIII-V (W2mef) IgG. (H) GIA using rabbit anti-EBA175 RIV-V (3D7) IgG (One-cycle assay).

Figure 3. EBA-175 inhibitory antibodies block merozoite invasion through receptors other than Glycophorin A.

(A) Western blot of post-invasion culture supernatants from W2mef and W2mef175-Cterm parasites probed with monoclonal antibody 9C4. This specifically recognizes EBA-175 region III-V and shows the truncated form of the protein that includes RIII-V was present in W2mef175-Cterm parasites. (B) EBA-175 requires motifs downstream of region 5 for correct trafficking within merozoites prior to invasion. Late segmented schizonts (ETS preparations) were co-stained with antiserum against EBA-140 to detect micronemes (green), along with inhibitory mAb targeting EBA-175 (red). Indirect immuofluorescence and phase contrast micrographs show correct co-localization within micronemes in wild type parasites (panels 1 &2) and mis-localization of truncated EBA-175 protein in transgenic parasites, often with aberrant, early secretion (panels 3&4). Free and invading merozoites, co-stained with the same W2mef-specific EBA-175 mAb (red) and RON4 to mark the tight junction (green), show that EBA-175 is present during merozoite invasion in both wild type and transgenic parasites. In all cases nuclei are stained with DAPI (blue) and scale bars show 2 μm. (C) W2mef and W2mef175-Cterm parasites were tested in GIA with IgG against EBA-175 RIII-V (3D7). Both parasite lines are inhibited equally efficiently by this IgG, whereas no inhibition is seen against EBA-175 KO parasites (One-cycle assay).

Figure 4. Antibodies targeting region 3–5 of EBA-140 also mediate cross-strain protection against P. falciparum

IgG purified from immune rabbit serum was tested in GIA against 3D7, W2mef and FCR3 strains. No inhibition was observed against EBA-140 knock out parasites. Highest GIA levels were against the homologous strain at 2 mg/ml. Error bars represent SEM (One cycle assay).

Figure 5. Summary of GIA data for antibodies tested against three strains of P. falciparum.

Data is represented as box and whiskers plot; line represents the median inhibition of a particular antibody against the three strains 3D7, FCR3 and W2mef; box, interquartile range; and whiskers GIA minimum and maximum values. Data included in the analysis are derived from triplicate wells in 3 independent, 2-cycle assays with IgG at a concentration of 2 mg/ml. Anti-EBA-175 values in blue, anti-EBA-140 in purple and anti-Rh2 antibodies in brown. Error bars represent SEM.

Figure 6. Data from independent testing of rabbit IgG for GIA activity.

Data points indicate mean values of triplicate wells from a single-cycle pfLDH-based assay using 3D7 parasites. % GIA was calculated relative to pre-immune IgG.