Identification of a potent combination of key Plasmodium falciparum merozoite antigens that elicit strain-transcending parasite-neutralizing antibodies

Abstract

Blood-stage malaria vaccines that target single Plasmodium falciparum antigens involved in erythrocyte invasion have not induced optimal protection in field trials. Blood-stage malaria vaccine development has faced two major hurdles, antigenic polymorphisms and molecular redundancy, which have led to an inability to demonstrate potent, strain-transcending, invasion-inhibitory antibodies. Vaccines that target multiple invasion-related parasite proteins may inhibit erythrocyte invasion more efficiently. Our approach is to develop a receptor-blocking blood-stage vaccine against P. falciparum that targets the erythrocyte binding domains of multiple parasite adhesins, blocking their interaction with their receptors and thus inhibiting erythrocyte invasion. However, with numerous invasion ligands, the challenge is to identify combinations that elicit potent strain-transcending invasion inhibition. We evaluated the invasion-inhibitory activities of 20 different triple combinations of antibodies mixed in vitro against a diverse set of six key merozoite ligands, including the novel ligands P. falciparum apical asparagine-rich protein (PfAARP), EBA-175 (PfF2), P. falciparum reticulocyte binding-like homologous protein 1 (PfRH1), PfRH2, PfRH4, and Plasmodium thrombospondin apical merozoite protein (PTRAMP), which are localized in different apical organelles and are translocated to the merozoite surface at different time points during invasion. They bind erythrocytes with different specificities and are thus involved in distinct invasion pathways. The antibody combination of EBA-175 (PfF2), PfRH2, and PfAARP produced the most efficacious strain-transcending inhibition of erythrocyte invasion against diverse P. falciparum clones. This potent antigen combination was selected for coimmunization as a mixture that induced balanced antibody responses against each antigen and inhibited erythrocyte invasion efficiently. We have thus demonstrated a novel two-step screening approach to identify a potent antigen combination that elicits strong strain-transcending invasion inhibition, supporting its development as a receptor-blocking malaria vaccine.

Fig 1

Subcellular localizations of the six P. falciparum merozoite proteins and their erythrocyte binding characteristics. A diagram of a merozoite invading an erythrocyte is depicted, highlighting the localizations of the six antigens in the different apical organelles (rhoptries and micronemes). The erythrocyte binding characteristics of the six antigens are tabulated with respect to the identity of their erythrocyte receptor, the type of the receptor with respect to its sialic acid content, and sensitivity to enzymes such as trypsin. Our antigens are localized in different apical compartments and are believed to be released at different time points during invasion and in addition bind erythrocytes with different specificities, implying an involvement in different invasion pathways. Localization data for PfRH1 and PfRH4 are based only on confocal immunofluorescence microscopy, and no immunoelectron microscopy data are reported for these two proteins. S. No., serial number.

Fig 2

Functional domains of the six P. falciparum merozoite proteins expressed as recombinant proteins. Shown is a schematic diagram of the six merozoite proteins involved in erythrocyte invasion, and the erythrocyte binding domains against which the recombinant proteins have been produced are highlighted (gray).

Fig 3

Erythrocyte invasion-inhibitory activity of PfRH antibodies in combination against P. falciparum clone 3D7. Total IgGs purified from rabbit sera raised against the receptor binding domains of RH1, RH2, and RH4 were tested for their invasion-inhibitory activities individually (2.5 to 10 mg/ml) and in combination. Combinations of two RH IgGs were assessed at two concentrations (2.5 and 2.5 mg/ml, and 5.0 and 5.0 mg/ml), and combinations of three RH IgGs were tested at 3.3 mg/ml each. AMA-1 IgG (5 mg/ml) was used as a positive control. The negative control is the average of the inhibition of purified IgG from preimmune rabbit sera and a control rabbit immunized with a nonrelated peptide. Three independent assays were performed in duplicate. The error bars show the standard errors of the means.

Fig 4

Invasion-inhibitory activities of antibody combinations against P. falciparum. Total IgGs purified from rabbit sera against the six proteins (PfRH1, PfRH2, PfRH4, PfF2, PfAARP, and PTRAMP) were assayed individually at a concentration of 3.3 mg/ml and in combinations of three IgGs (3.3 plus 3.3 plus 3.3 mg/ml) against sialic acid-independent clone 3D7 (A) and sialic acid-dependent clone Dd2 (B). Three independent assays were performed in duplicate. The error bars show the standard errors of the means. P values were calculated by using the Student t test.

Fig 5

Strain-transcending activities of three triple-antibody combinations against five diverse P. falciparum clones. The invasion inhibitions of three antibody combinations (PfF2+RH2+AARP, RH1+RH2+RH4, and RH1+RH2+PfF2) were assayed with five P. falciparum clones: 3D7, 7G8, HB3, Dd2, and MCamp (MC). 3D7, 7G8, and HB3 are sialic acid-independent clones; Dd2, MCamp, are sialic acid-dependent clones. Three independent assays were performed in duplicate. The error bars show the standard errors of the means.

Fig 6

Immunogenicity of the antigens in mice when used alone and in combination. Antigen combinations (PfF2+RH2+AARP and RH1+RH2+RH4) as well as the individual corresponding antigens were used to immunize mice. Day 70 sera from the immunized animals were probed against the respective antigens to determine their immunogenicity. (A) Immunogenicity of antibodies against PfF2+RH2+AARP (purple) compared with that of the sera raised against the individual antigens (green). (B) Immunogenicity of antibodies against RH1+RH2+RH4 sera (purple) compared with that of antibodies against sera raised against the individual corresponding antigens (green). For ELISAs, each individual antigen was coated separately onto 96-well plates for both the coimmunized as well as the individual antigens. Preimmune or prebleed (PB) sera were used as controls. The data points represent average values for the six mice included in each group. Two independent experiments were done in duplicate. The error bars represent the standard errors of the means.

Fig 7

Invasion-inhibitory activities of antibodies raised against the two coimmunized antigen formulations. Total IgGs from mouse sera raised against the immunogens (RH1, RH2, RH4, PfF2, AARP, RH1+RH2+RH4, and PfF2+RH2+AARP) were evaluated for their invasion-inhibitory activities (at concentrations of 1, 3.3, 5, and 10 mg/ml) against sialic acid-independent clone 3D7 (A) and sialic acid-dependent clone Dd2 (B). Two independent assays were performed in duplicate. The error bars show the standard errors of the means.

Fig 8

Strain-transcending activities of mouse antibodies raised against the coimmunized triple-antigen combinations. The strain-transcending neutralizing activities of purified total IgGs raised against the coimmunized PfF2+RH2+AARP and RH1+RH2+RH4 antigen formulations were evaluated against three sialic acid-independent (3D7, 7G8, and HB3) and two sialic acid-dependent (Dd2 and MCamp) clones at a concentration of 10 mg/ml. Two independent assays were performed in duplicate. The error bars show the standard errors of the means.