Antibodies against a short region of PfRipr inhibit Plasmodium falciparum merozoite invasion and PfRipr interaction with Rh5 and SEMA7A

Hikaru Nagaoka,Masamitsu Aoki,Bernard N Kanoi,Takafumi Tsuboi,Edward H Ntege,Eizo Takashima,Akihisa Fukushima

doi:10.1038/s41598-020-63611-6

Abstract

Plasmodium falciparum merozoite invasion into erythrocytes is an essential step of the blood-stage cycle, survival of parasites, and malaria pathogenesis. P. falciparum merozoite Rh5 interacting protein (PfRipr) forms a complex with Rh5 and CyRPA in sequential molecular events leading to erythrocyte invasion. Recently we described PfRipr as a conserved protein that induces strain-transcending growth inhibitory antibodies in in vitro assays. However, being a large and complex protein of 1086 amino acids (aa) with 87 cysteine residues, PfRipr is difficult to express in conventional expression systems towards vaccine development. In this study we sought to identify the most potent region of PfRipr that could be developed to overcome difficulties related to protein expression, as well as to elucidate the invasion inhibitory mechanism of anti-PfRipr antibodies. Using the wheat germ cell-free system, Ecto- PfRipr and truncates of approximately 200 aa were expressed as soluble proteins. We demonstrate that antibodies against PfRipr truncate 5 (PfRipr_5: C720-D934), a region within the PfRipr C-terminal EGF-like domains, potently inhibit merozoite invasion. Furthermore, the antibodies strongly block PfRipr/Rh5 interaction, as well as that between PfRipr and its erythrocyte-surface receptor, SEMA7A. Taken together, PfRipr_5 is a potential candidate for further development as a blood-stage malaria vaccine.

Highlights

Plasmodium falciparum malaria remains a serious challenge to global health
Invasion of erythrocytes by P. falciparum merozoites is a fundamental step in malaria pathogenesis and a primary target for vaccine development[16,41]
We recently determined that P. falciparum Rh5-interacting protein (PfRipr) is highly conserved among African isolates, with the region spanning C720-D934 (PfRipr_5) having only a single amino acid substitution at E829Q30

Summary

Introduction

Plasmodium falciparum malaria remains a serious challenge to global health. In 2016, more than 3 billion people were reportedly at risk of infection, with an estimated 200 million cases and more than 400,000 deaths, primarily in young children living in sub-Saharan Africa[1]. Investigations towards receptor-ligand interactions during merozoite invasion suggest that, in addition to merozoite surface proteins (MSPs), two protein ligand families play key roles prior to tight junction formation; namely, P. falciparum reticulocyte-binding protein homologs (PfRhs) and erythrocyte-binding like proteins (EBLs)[11,18,19]. Rh5, CyRPA, and PfRipr are considered to be promising blood-stage vaccine candidates[5,6,7,24,26,27], since the PfRipr/CyRPA/Rh5 complex plays a central role in the sequential molecular events leading to merozoite invasion and the genes have limited sequence polymorphism in P. falciparum[5,27,28]. We investigated the mechanism of anti-PfRipr antibodies in inhibiting merozoite invasion through functional characterization of PfRipr We utilized both wheat germ cell-free system (WGCFS)-expressed recombinant PfRipr protein and anti-PfRipr antibodies to assess GIA activity in vitro. The identified PfRipr region represents a promising candidate for further development as a blood-stage malaria vaccine

Methods

Results

Conclusion