Discovery of GAMA, a Plasmodium falciparum merozoite micronemal protein, as a novel blood-stage vaccine candidate antigen

Abstract

One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.

Fig. 1.

Structure and recombinant proteins of GAMA. (A) Schematic of the primary structure of GAMA. The GAMA protein consists of 738 amino acids, with a calculated molecular mass of 85.24 kDa. Indicated are the predicted signal peptide (SP; residues 1 to 24), asparagine-rich region (residues 356 to 485), and C-terminal transmembrane domain (TM; residues 715 to 738). FL GAMA (residues 1 to 738) and three regions of GAMA (Tr1 [residues 25 to 337], Tr3 [residues 500 to 714], and ECTO [residues 25 to 714]) were expressed in recombinant form and used to raise specific antisera. (B) SDS-PAGE of recombinant proteins of GAMA. All the recombinant proteins were synthesized using a wheat germ cell-free protein expression system. The fractions of purified ECTO (79.82 kDa), Tr1 (36.8 kDa), Tr3 (24 kDa), and FL (85.24 kDa) proteins resolved in an SDS-PAGE gel and stained with Coomassie brilliant blue R-250 are shown. M represents a molecular weight marker. E1 and E2 represent the first and the second fractions of purified proteins eluted from affinity purification columns, respectively. Arrows indicate specific bands.

Fig. 2.

Processing and shedding of GAMA. (A) Detection of GAMA in schizont lysate. Total schizont material was examined by Western blotting under reducing (R) and nonreducing (N) conditions, using the rabbit anti-FL, mouse anti-Tr1, and rabbit anti-Tr3 antisera. (B) Detection of GAMA in culture supernatant. Culture supernatant was analyzed by Western blotting under reducing and nonreducing conditions using the rabbit anti-FL.

Fig. 3.

Localization of GAMA in asexual blood-stage parasites. (A) GAMA localization using an immunofluorescence assay. Acetone-fixed P. falciparum 3D7 mature schizonts (top panel) and free merozoites (bottom panel) were probed with rabbit anti-FL (green) and mouse anti-PfAMA1 (microneme marker) (red). Parasite nuclei were stained with DAPI (blue). Scale bars represent 2 μm. (B) GAMA localization using immunoelectron microscopy. The two sections of merozoites in schizont-infected erythrocytes were probed with purified rabbit anti-Tr3 antibody and subsequently by secondary antibody conjugated with gold particles. The arrows indicate the micronemal localization of signals from gold particles. Bars represent 200 nm. Arrows mark micronemes. R's mark rhoptries.

Fig. 4.

Anti-FL antibody inhibits parasite invasion in vitro. (A) Anti-FL antibodies have invasion-inhibitory activity in vitro. The ability of the anti-FL antibodies to inhibit the parasite invasion into erythrocytes was tested in a one-cycle growth inhibition assay. Anti-AMA1 and anti-GST antibodies were used as positive and negative controls, respectively. The error bars represent the standard deviations of the means of the three independent experiments. One-way ANOVA was performed (P < 0.001) and followed by Bonferroni's pairwise multiple-comparison tests to compare anti-GST and anti-FL. (B) GAMA plays a role in the SA-independent invasion pathway. The ability of the anti-FL antibody to inhibit parasite invasion into neuraminidase-treated erythrocytes was tested in a one-cycle growth inhibition assay. Anti-GST antibody was used as a negative control. The bars represent the standard deviations of the means of the three independent experiments. (C) Anti-FL antibodies inhibit parasite invasion in a dose-dependent manner in vitro. The graph shows that the anti-FL antibodies, both total and antigen-specific IgGs, inhibited the invasion and/or growth in a dose-dependent manner in a one-cycle growth inhibition assay, as determined by measuring parasite LDH. The ELISA unit value was assigned as the reciprocal of the dilution giving an optical density (OD) at 415 nm equal to 1 in a standardized assay.

Fig. 5.

Erythrocyte binding assay with native and recombinant GAMA. (A) Erythrocyte binding activities of native and recombinant GAMA proteins. The native GAMA protein in the culture supernatant or recombinant proteins (ECTO, Tr1, and Tr3) were incubated with human erythrocytes. The bound proteins were eluted with 0.5 M NaCl in PBS, pH 7.4, either directly from the incubated erythrocytes (E) or from the erythrocytes washed once with iRPMI (W). The eluted protein was detected by Western blotting either with rabbit anti-FL antibody (for GAMA) or with anti-penta-His antibodies (for ECTO, Tr1, and Tr3). For each experiment, the intact protein (without incubation or elution) was also detected by Western blotting as a control (C) (arrowheads). (Given that Tr3 has a cysteine residue, Tr3 forms an artificial homodimer [marked by an asterisk] with erythrocyte binding capacity.) (B) Tr3 competes for binding to a receptor(s) against native GAMA. The bands in lanes 1 through 5 and bands in lanes 6 and 7 refer to Tr3 and native GAMA, respectively, present in the blot of erythrocyte-bound proteins eluted from either controls or different treatments used in the binding assay (described above the lanes; also refer to Materials and Methods). Tr3 and native GAMA in the blot were detected with anti-penta-His and anti-FL antibodies, respectively. (C) GAMA binds erythrocytes in a receptor-specific manner. The erythrocyte binding abilities of native GAMA (present in the culture supernatant) and recombinant Tr3 were tested by incubation with untreated (U), neuraminidase-treated (N), trypsin-treated (T), and chymotrypsin-treated (C) erythrocytes, then elution with 0.5 M NaCl in PBS, pH 7.4, and detection by Western blotting with anti-penta-His (for Tr3) or anti-FL (for GAMA) antibodies. As a control for erythrocyte treatment, native EBA175 in the identical culture supernatant was also examined and detected by anti-EBA175 (regions 3 to 5) antibody. The values indicate percent changes in signal intensity of the relevant band relative to the band in lane U, calculated using Image J.

Fig. 6.

Additive blocking of invasion. Antibodies, either separately or in combinations, were tested for inhibition of parasite invasion into erythrocytes in a one-cycle growth inhibition assay. Anti-GST antibody was used as a negative control (for concentrations of IgGs, refer to Results). The error bars represent the standard deviations of the means of the three independent experiments. One-way ANOVA was performed (P < 0.001) and followed by Bonferroni's pairwise multiple-comparison tests to compare each experimental group. Statistical significance between other groups was not tested.

Fig. 7.

Human sera from areas of malaria endemicity in Mali and Thai recognize GAMA in an ELISA. Probing of FL with sera from immune adults of Mali (Immune) and naive, nonexposed U.S. adults (Normal) (A) and with sera from P. falciparum-infected asymptomatic adults of Thai (Asymptomatic) and naive, nonexposed Thai adults (Normal) (B). The P values were calculated by Mann-Whitney U test. n indicates the number of sera analyzed. OD, optical density; dil, dilution.