Immunoscreening of Plasmodium falciparum proteins expressed in a wheat germ cell-free system reveals a novel malaria vaccine candidate

Abstract

The number of malaria vaccine candidates in preclinical and clinical development is limited. To identify novel blood-stage malaria vaccine candidates, we constructed a library of 1,827P. falciparum proteins prepared using the wheat germ cell-free system (WGCFS). Also, a high-throughput AlphaScreen procedure was developed to measure antibody reactivity to the recombinant products. Purified IgGs from residents in malaria endemic areas have shown functional activity against blood-stage parasites as judged by an in vitro parasite Growth Inhibition Assay (GIA). Therefore, we evaluated the GIA activity of 51 plasma samples prepared from Malian adults living in a malaria endemic area against the WGCFS library. Using the AlphaScreen-based immunoreactivity measurements, antibody reactivity against 3 proteins was positively associated with GIA activity. Since anti-LSA3-C responses showed the strongest correlation with GIA activity, this protein was investigated further. Anti-LSA3-C-specific antibody purified from Malian adult plasmas showed GIA activity, and expression of LSA3 in blood-stage parasites was confirmed by western blotting. Taken together, we identified LSA3 as a novel blood-stage vaccine candidate, and we propose that this system will be useful for future vaccine candidate discovery.

Figure 1. Reactivity of Malian adult IgGs to recombinant proteins produced in the WGCFS.

Proteins were sorted by their mean ASC, and the cut-off value was determined as mean plus 3SD of negative control antibodies (dashed line). Approximately 49% of proteins were considered reactive to the Malian IgG samples.

Figure 2. Schematic representation of the P. falciparum LSA3 protein.

The design of the recombinant proteins expressed; repeat sequence regions (R1, R2, R3), PEXEL motif, and predicted transmembrane domains (in yellow) are shown. The numbers are amino acid positions. The boundaries of the repeat regions were defined by comparison of the primary structure in LSA3 between 3D7 and K1 strain.

Figure 3. Characterization of LSA3 in blood stage parasites.

(a) Human anti-LSA3-C-specific IgG has significant GIA activity. Human anti-LSA3-C specific IgG was tested at 0.48 mg/ml. Rabbit anti-His-GST IgG was tested at 20 mg/ml as a negative control. Three independent GIA experiments in triplicate were performed, and the mean and SEM are shown. (b) LSA3 was expressed in blood-stage parasites. Western blot was performed with human, mouse or rabbit anti-LSA3-C antibodies. Parasite lysate was obtained from 1 × 10⁴ P. falciparum infected red cells at mixed developmental stages. Molecular weight marker (Lane 1), purified 0.5 ng of C-terminal His-tagged recombinant LSA3-C protein (Lane 2) and the blood-stage parasite lysate (Lane 3) were separated by SDS-PAGE under reducing conditions. (c) IFA of schizont-stage parasites. Human anti-LSA3-C antibody was used for the IFA. Mouse anti-AMA1, -RAP1, -RON2, or -RESA antibodies were used for counter-staining to determine subcellular localization of microneme, rhoptry body, rhoptry neck or dense granule, respectively. Parasite nuclei were stained with DAPI. Scale bars indicate 5 μm.

Figure 4. Immunoelectron microscopy observations of LSA3 in merozoites of schizont-infected erythrocytes.

Rabbit anti-LSA3-C antibody was used for primary antibody. The gold particles were detected specifically at dense granules. Scale bars indicate 200 nm.