Plasmodium falciparum reticulocyte-binding homologues are targets of human inhibitory antibodies and play a role in immune evasion

Abstract

Introduction: Antibodies targeting the blood-stage of Plasmodium falciparum play a critical role in naturally acquired immunity to malaria by limiting blood-stage parasitemia. One mode of action of antibodies is the direct inhibition of merozoite invasion of erythrocytes through targeting invasion ligands. However, evasion of inhibitory antibodies may be mediated in P. falciparum by switching between various ligand-mediated merozoite invasion pathways. Here, we investigated the potential roles of invasion ligands PfRH1, PfRH2a and PfRH2b in immune evasion through phenotypic variation, and their importance as targets of human invasion-inhibitory antibodies.

Methods: Serum samples from malaria-exposed children and adults in Kenya were examined for their ability to inhibit P. falciparum invasion, using parasites with disrupted pfrh1, pfrh2a or pfrh2b genes.

Results and discussion: The loss of PfRH1 and PfRH2b substantially impacted on susceptibility to inhibitory antibodies, suggesting that variation in the use of these ligands contributes to immune evasion. The effect was less prominent with loss of PfRH2a. Differential inhibition of the knockout and parental lines points to PfRH1 and PfRH2b as targets of acquired growth inhibitory antibodies whereas PfRH2a appeared to be a minor target. There was limited relatedness of the inhibitory responses between different isolates or compared to parasites with deletions of erythrocyte-binding antigens. This further suggests that there is a substantial amount of antigenic diversity in invasion pathways to facilitate immune evasion. These findings provide evidence that PfRH1 and PfRH2b are significant targets of inhibitory antibodies and variation in their expression may facilitate immune evasion. Targeting of multiple invasion ligands in vaccine design is likely to be required to achieve potent inhibitory antibodies and protective efficacy against malaria.

Keywords: P. falciparum; RH proteins; immune evasion; inhibitory antibodies; phenotypic variation; reticulocyte binding homologues.

Figure 1

Antibodies to PfRh1 and PfRh2 in the study population. Prevalence of antibodies against PfRH1 (A, C) and PfRH2 (B, D) in Cohort 1 by ELISA (n=148). Responses have been classified by age (A, B) or parasitemic status (C, D) and show the median OD at 405nm and interquartile ranges of age groups 2–5, 6–14, and 18–81 years (yrs) (A, B), or by parasitemic status by PCR (pos, positive; neg, negative; C, D). P-values indicate the significance of differences across the groups. Statistical significance was determined by the Kruskal–Wallis test (across age groups) and by the Wilcoxon rank sum (Mann–Whitney) test (by parasitemic status). The PfRH2 antigen used corresponded to a conserved region of PfRH2a and 2b. ns, non-significant.

Figure 2

Relationship between PfRH1-specific antibodies and antibodies to other invasion ligands. Correlation between the presence of PfRH1-specific antibodies to different invasion ligands in Cohort 1. Values indicate Spearman’s r (rho) correlation (p<0.01 for all). RIII–V: regions III–V of EBA175 and EBA140, respectively. RH2-2030 covers a fragment in the common region of PfRH2a and PfRH2b. PfRH4.9: erythrocyte-binding region of PfRH4. PfRH1: erythrocyte-binding region.

Figure 3

Differential inhibition of 3D7ΔRH1 vs wild-type (WT) parasites by naturally-acquired antibodies. (A) Proportion of samples that differentially inhibited the wild-type and knockout (KO) lines. Samples have been classified according to their inhibitory activity. Red, wild-type > KO: the wild-type more inhibited than knockout. Yellow, KO > wild-type: knockout more inhibited than wild-type. Blue, no difference: no differential inhibition between the two lines compared. (B) Inhibition of the wild-type and knockout P. falciparum lines by invasion profile. Samples were classified as inhibitors or non-inhibitors according to their inhibition of the wild-type, compared to 3D7ΔRH1 where the inhibitors inhibited the wild-type more than the knockouts with 25% difference or more in relative growth (indicating the presence of PfRH-specific antibodies), and the non-inhibitors showed 10% or less differential inhibition in either direction. Median growth of all categories was expressed as the percentage growth compared to a PBS control. Error bars show the interquartile range and p-values indicate levels of differences between the different lines tested (ns: not significant) as determined by paired t-tests.

Figure 4

Differential inhibition of 3D7ΔRH2a and 3D7ΔRH2b vs wild-type (WT) parasites by naturally-acquired antibodies. (A) Proportion of samples that differentially inhibited the wild-type and knockout (KO) lines. Samples have been classified according to their inhibitory activity. Red, wild-type > KO: wild-type more inhibited than knockout. Yellow, KO > wild-type: knockout more inhibited than the wild-type. Blue, no difference: no differential inhibition between the two lines compared. (B, C) Inhibition of the wild-type and knockout P. falciparum lines by invasion profile. Samples were classified according to their inhibition of the wild-type compared to 3D7ΔRH2a (B) and 3D7ΔRH2b (C). Samples were classified as inhibitors and non-inhibitors, where the inhibitors inhibited the wild-type more than the knockouts with 25% difference or more in relative growth (indicating the presence of PfRH-specific antibodies), and the non-inhibitors showed 10% or less differential inhibition in either direction. Median growth of all categories was expressed as the percentage growth compared to a PBS control. Error bars show the interquartile range and p-values indicate levels of differences between the different lines tested (ns: not significant) as determined by paired t-tests.

Figure 5

Relatedness in the inhibitory activity against different P. falciparum lines. Proportion of differentially inhibitory samples that inhibited PfRH1 and PfRH2a- or PfRH2b-knockout lines (A), or PfRH1 and EBA140- or EBA175-knockout lines (B) compared to the parental line (both directions of differential inhibition have been included: the wild-type more inhibited than knockout, and knockout more inhibited than the wild-type). The proportion is expressed as the percentage of the total number of samples that were tested against the two lines being compared as well as the wild-type parasites. The values in the overlaps show the proportion of all samples tested that differentially inhibited the compared lines in the same direction.

Figure 6

Proportion of samples with broader inhibitory activity. Samples that inhibited the wild-type more than 3D7ΔRH1 (A), or more than 3D7ΔRH2b (B), were assessed for whether they also inhibited the wild-type more than other knockout lines, including 3D7ΔEBA140, 3D7ΔEBA175, 3D7ΔEBA181, or 3D7ΔRh2a. The number of samples that inhibited the wild-type more than 3D7ΔRH1 (A) or 3D7ΔRH2b (B) and an additional knockout line (as shown on x-axis) is expressed as the percentage of all samples that inhibited the wild-type more than the 3D7ΔRh1 (A) or 3D7RH2b knockouts (B). The proportion of samples that inhibited the wild-type more than EBA140-KO parasites was significantly higher (p<0.001) than observed with other KO parasite lines in (A, B).