Passive Immunoprotection of Plasmodium falciparum-Infected Mice Designates the CyRPA as Candidate Malaria Vaccine Antigen

Abstract

An effective malaria vaccine could prove to be the most cost-effective and efficacious means of preventing severe disease and death from malaria. In an endeavor to identify novel vaccine targets, we tested predicted Plasmodium falciparum open reading frames for proteins that elicit parasite-inhibitory Abs. This has led to the identification of the cysteine-rich protective Ag (CyRPA). CyRPA is a cysteine-rich protein harboring a predicted signal sequence. The stage-specific expression of CyRPA in late schizonts resembles that of proteins known to be involved in merozoite invasion. Immunofluorescence staining localized CyRPA at the apex of merozoites. The entire protein is conserved as shown by sequencing of the CyRPA encoding gene from a diverse range of P. falciparum isolates. CyRPA-specific mAbs substantially inhibited parasite growth in vitro as well as in a P. falciparum animal model based on NOD-scid IL2Rγnull mice engrafted with human erythrocytes. In contrast to other P. falciparum mouse models, this system generated very consistent results and evinced a dose-response relationship and therefore represents an unprecedented in vivo model for quantitative comparison of the functional potencies of malaria-specific Abs. Our data suggest a role for CyRPA in erythrocyte invasion by the merozoite. Inhibition of merozoite invasion by CyRPA-specific mAbs in vitro and in vivo renders this protein a promising malaria asexual blood-stage vaccine candidate Ag.

Multiprotein complex between the GPI-anchored CyRPA with PfRH5 and PfRipr is crucial for Plasmodium falciparum erythrocyte invasion

“…Our data substantiate a previous study that found CyRPA monoclonal antibodies raised against a cell surface expressed recombinant protein to inhibit in vivo P. falciparum growth in a passive immunoprotection animal model (15). Our work has proved CyRPA to be GPI anchored and has identified its physiological role to secure PfRH5, a key parasite ligand that lacks any membrane anchorage moiety, to the merozoite surface.…”

“…CyRPA is highly conserved (single polymorphism among 18 P. falciparum strains) (15) and like PfRH5 is not under immune pressure (9,13). The CyRPA gene could not be genetically knocked out, and its indispensible nature suggests that its interaction with PfRH5 and PfRipr is imperative for erythrocyte invasion.…”

“…Using these specific antibodies we performed coimmunoprecipitation experiments in which the PfRH5 antibodies, in comparison with its preimmune control antibodies, pulled down PfRipr as well as another previously unidentified interacting partner, CyRPA (15). CyRPA is a ∼35-kDa protein and thus we have identified the third component of the 200-kDa high molecular weight complex that comprised PfRH5 and PfRipr.…”

“…Here we show that PfRH5 and PfRipr interact with a GPIlinked parasite protein, CyRPA (Cysteine-rich protective antigen) (15) to form an essential complex on the surface of an invading merozoite. Individual antibodies against each of the three proteins successfully coimmunoprecipitated all three proteins, confirming their presence as a multiprotein complex.…”

Recent Developments in Malaria Vaccinology

“…Promising immuno-epidemiological studies (Osier et al, 2014b) and preclinical studies have now highlighted combinations of P. falciparum merozoite antigens including other EBA-and RH-family members (PfEBA175, PfRH2, PfRH4) and apical asparagine-rich protein (PfAARP) (Healer et al, 2013;Pandey et al, 2013), schizont egress antigen 1 (PfSEA-1) (Raj et al, 2014), as well as improved formulations of PfAMA1 that improve the qualitative antibody response (Srinivasan et al, 2014). Also, two cysteine-rich merozoite proteins that interact with PfRH5, the P. falciparum RH5 interacting protein (PfRipr) (Chen et al, 2011) and the cysteine-rich protective antigen (CyRPA) (Dreyer et al, 2012;Reddy et al, 2015), have recently been identified and define the components of the PfRH5 adhesion complex, which is essential for erythrocyte invasion. Antibodies to both, PfRipr as well as CyRPA, have been shown to inhibit merozoite invasion into erythrocytes.…”