A Library of Functional Recombinant Cell-surface and Secreted P. falciparum Merozoite Proteins

Abstract

Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world's major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.

Functional Characterization and Comparison of Plasmodium falciparum Proteins as Targets of Transmission-blocking Antibodies

“…Proteins were transiently expressed in non-adherent HEK293E cells and affinity purified from whole supernatant using a HisTrap Excel Ni-Sepharose column. Fifteen out of 22 proteins (68%) showed detectable expression by Western blot using an anti-petahistidine antibody, which is in keeping with previous reports for P. falciparum protein expression in HEK293E cells [22]. Thirteen out of those 15 proteins (87%), including Pfs25, were purified in sufficient quantities for a small-animal vaccination study, i.e.…”

“…We cannot rule out the negative impact of N-linked glycans masking critical epitopes, but on this note the literature is variable. Crosnier et al recently evaluated the relative contributions of codon-optimization, an exogenous signal peptide, and mutation of N-glycosylation sites on expression of functional asexual blood-stage protein PfRH5 in the HEK293E cell system, reporting the relative increases in protein expression to be equivocal, ~3.5-fold, and ~5.5-fold, respectively, compared with the native sequence [22]. By contrast, several other reports exist, albeit in other platforms, wherein retaining native glycans has not made a difference to yield or function [27,[45][46][47][48].…”

“…! have improved success rates in the production of soluble protein for use in immunoepidemiology [43] and receptor-ligand interaction studies [22,44]. We chose to use transiently transfected, non-adherent HEK293E cells for protein expression; 15 of 22 proteins (68%) were expressed with detection by anti-pentahistidine antibody in our study.…”

“…Still, the field has yet to capitalize on the wealth of knowledge accrued over the past decade. While rational screening of protein libraries has been applied to both the pre-erythrocytic [21] and erythrocytic stages [22,23], this approach has never been reported for the identification of new TBV targets.…”

Comparative antibody responses against three antimalarial vaccine candidate antigens from urban and rural exposed individuals in Gabon

“…PfAMA1 and PfRh5 were provided by the Welcome Trust Institute of London. Recombinant P. falciparum proteins were expressed by transient transfection of HEK293 cells from the 3D7 strain using expression plasmids described in Crosnier et al [26]. Recombinant PfRH5 was processed when expressed in HEK293 media supplemented with fetal calf serum; this processing was reduced by using HEK293 cells adapted to serum-free media and was prevented, when necessary, by the addition of 2 to 10 μg/ml aprotinin.…”