Bacterially Expressed Full-Length Recombinant Plasmodium falciparum RH5 Protein Binds Erythrocytes and Elicits Potent Strain-Transcending Parasite-Neutralizing Antibodies

Abstract

Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an essential merozoite ligand that binds with its erythrocyte receptor, basigin. PfRH5 is an attractive malaria vaccine candidate, as it is expressed by a wide number of P. falciparum strains, cannot be genetically disrupted, and exhibits limited sequence polymorphisms. Viral vector-induced PfRH5 antibodies potently inhibited erythrocyte invasion. However, it has been a challenge to generate full-length recombinant PfRH5 in a bacterial-cell-based expression system. In this study, we have produced full-length recombinant PfRH5 in Escherichia coli that exhibits specific erythrocyte binding similar to that of the native PfRH5 parasite protein and also, importantly, elicits potent invasion-inhibitory antibodies against a number of P. falciparum strains. Antibasigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5, further confirming that they bind with basigin. We have thus successfully produced full-length PfRH5 as a functionally active erythrocyte binding recombinant protein with a conformational integrity that mimics that of the native parasite protein and elicits potent strain-transcending parasite-neutralizing antibodies. P. falciparum has the capability to develop immune escape mechanisms, and thus, blood-stage malaria vaccines that target multiple antigens or pathways may prove to be highly efficacious. In this regard, antibody combinations targeting PfRH5 and other key merozoite antigens produced potent additive inhibition against multiple worldwide P. falciparum strains. PfRH5 was immunogenic when immunized with other antigens, eliciting potent invasion-inhibitory antibody responses with no immune interference. Our results strongly support the development of PfRH5 as a component of a combination blood-stage malaria vaccine.

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

“…As demonstrated previously for PfRH5 (12), antibodies against CyRPA-FL exhibited highly potent invasion inhibition, with an EC 50 of ∼3 mg/mL (total IgG) (Fig. 4 A and B).…”

“…We also demonstrated that the three proteins are colocalized on the apical surface of the invading merozoite, of which only CyRPA was shown to be GPI-linked. Importantly, antibodies against full-length CyRPA potently blocked erythrocyte invasion by multiple P. falciparum strains, as observed previously only for PfRH5 antibodies (9)(10)(11)(12). Because CyRPA does not bind with the erythrocyte surface, it…”

“…To identify the unknown component(s) of the 200 kDa complex, we performed coimmunoprecipitation with schizont-stage lysates using functional PfRH5 antibodies described recently (12). Mass spectrometric analysis of the proteins coimmunoprecipitated by the PfRH5 immune antibodies in comparison with those immunoprecipitated by the preimmune control antibodies identified several parasite proteins, of which only three-PfRH5 itself, PfRipr, and a previously unidentified interacting parasite protein, CyRPA (15) (PF3D7_0423800)-were consistently detected with significant high scores between independent experiments (Fig.…”

“…The quest to develop successful blood-stage malaria vaccines that efficiently block this process have focused on essential parasite proteins like merozoite surface protein 1 (MSP-1) and apical membrane antigen 1 (AMA-1); however, these are highly polymorphic, unable to elicit strain-transcending neutralizing antibodies, and have thus failed in field trials (4). Among the large repertoire of invasionrelated proteins, the family of P. falciparum reticulocyte bindinglike homologous (PfRH) proteins have emerged as key determinants of different invasion pathways (2,3), of which PfRH5 is the only essential conserved parasite ligand (5)(6)(7)(8) that elicits potent strain-transcending neutralizing antibodies (9)(10)(11)(12). It is localized in the rhoptry and secreted to the merozoite surface during erythrocyte invasion (6).…”

Evaluating antidisease immunity to malaria and implications for vaccine design

“…Fortunately, promising synergistic inhibitory effects at comparatively lower antibody concentrations are observed when antibodies are used in combination for invasion assays, as seen in PfCyRPA–PfRh5 and PfF2–PfRH5–PfAARP–PfRH2 combinations 70. These in‐vitro findings provided evidence that buttressed the idea of a multicomponent vaccine.…”