P113 is a merozoite surface protein that binds the N terminus of Plasmodium falciparum RH5

Abstract

Invasion of erythrocytes by Plasmodium falciparum merozoites is necessary for malaria pathogenesis and is therefore a primary target for vaccine development. RH5 is a leading subunit vaccine candidate because anti-RH5 antibodies inhibit parasite growth and the interaction with its erythrocyte receptor basigin is essential for invasion. RH5 is secreted, complexes with other parasite proteins including CyRPA and RIPR, and contains a conserved N-terminal region (RH5Nt) of unknown function that is cleaved from the native protein. Here, we identify P113 as a merozoite surface protein that directly interacts with RH5Nt. Using recombinant proteins and a sensitive protein interaction assay, we establish the binding interdependencies of all the other known RH5 complex components and conclude that the RH5Nt-P113 interaction provides a releasable mechanism for anchoring RH5 to the merozoite surface. We exploit these findings to design a chemically synthesized peptide corresponding to RH5Nt, which could contribute to a cost-effective malaria vaccine.

Human vaccination against RH5 induces neutralizing antimalarial antibodies that inhibit RH5 invasion complex interactions

“…Quantitative assessment by ELISA and CFCA using the structured RH5ΔNL protein (which lacks the disordered N-terminus and internal loop) suggested that most of vaccine-induced IgG recognize this region of RH5 and that antibodies against RH5Nt constitute only a minor, but consistent, proportion of the total anti-RH5_FL response. Subsequent testing by AVEXIS confirmed the sera from vaccinees could block the interaction of RH5_FL with P113, CyRPA, and basigin, consistent with known information relating to the binding of these molecules (29,49,56,68) and the measurable antibody responses against both RH5Nt and RH5ΔNL. Interestingly, blocking activity for all 3 interactions correlated with the anti-RH5_FL IgG response, suggesting qualitatively similar responses were induced in all vaccinees.…”

“…Recombinant RH5_FL and RH5ΔNL proteins were generated using Drosophila melanogaster Schneider 2 (S2) polyclonal stable cell lines (ExpreS 2 platform, ExpreS 2 ion Biotechnologies) (69), while RH5Nt protein was produced using HEK293-6E cells as previously described (29).…”

“…Further analysis of the peptide data highlighted peptide 28 -against which the sera from all 16 volunteers reacted-which is also recognized by the nonneutralizing mouse mAb 4BA7 (56), as well as peptide 5 in RH5Nt, which is recognized by the nonneutralizing mouse mAb RB3 (56). Other peptides of interest include peptides 1-3 within RH5Nt, given that the P113-binding region was previously mapped to a 19-mer sequence running from aa K33-K51 (29). These peptide reagents included the N38Q aa substitution in the vaccine construct to remove a site of potential N-linked glycosylation.…”

“…Somewhat surprisingly, RH5 appears to be under relatively low-level immune pressure following natural infection (12,(23)(24)(25)(26), with functional constraints also linked to basigin binding and host RBC tropism (21,27,28) -both of these factors potentially explain its relatively high degree of sequence conservation. Recently, the N-terminal region of RH5 (RH5Nt) has been shown to bind the essential glycosylphosphatidylinositol-anchored (GPI-anchored) merozoite protein P113, providing a mechanism for anchoring RH5 to the merozoite surface (29). When bound by P113, RH5_FL is able to bind basigin and CyRPA, but not Ripr.…”

Neutralising antibodies block the function of Rh5/Ripr/CyRPA complex during invasion of Plasmodium falciparum into human erythrocytes

“…This may arise from the fact that the protein used in this study, despite its ability to bind to basigin (Chen et al, ) and form a ternary complex with CyRPA and Ripr, was produced as a truncated protein, lacking the 15 kDa N‐terminal portion. Note that the N‐terminal region of PfRh5 is cleaved from the protein and not required for basigin binding although antibodies against this specific domain were inhibitory in GIA assays (Galaway et al, ). Furthermore, it is difficult to directly compare GIA results from different studies using different antibody concentrations and different assay measurements.…”