Antibodies against thrombospondin-related anonymous protein do not inhibit Plasmodium sporozoite infectivity in vivo

Abstract

Thrombospondin-related anonymous protein (TRAP), a candidate malaria vaccine antigen, is required for Plasmodium sporozoite gliding motility and cell invasion. For the first time, the ability of antibodies against TRAP to inhibit sporozoite infectivity in vivo is evaluated in detail. TRAP contains an A-domain, a well-characterized adhesive motif found in integrins. We modeled here a three-dimensional structure of the TRAP A-domain of Plasmodium yoelii and located regions surrounding the MIDAS (metal ion-dependent adhesion site), the presumed business end of the domain. Mice were immunized with constructs containing these A-domain regions but were not protected from sporozoite challenge. Furthermore, monoclonal and rabbit polyclonal antibodies against the A-domain, the conserved N terminus, and the repeat region of TRAP had no effect on the gliding motility or sporozoite infectivity to mice. TRAP is located in micronemes, secretory organelles of apicomplexan parasites. Accordingly, the antibodies tested here stained cytoplasmic TRAP brightly by immunofluorescence. However, very little TRAP could be detected on the surface of sporozoites. In contrast, a dramatic relocalization of TRAP onto the parasite surface occurred when sporozoites were treated with calcium ionophore. This likely mimics the release of TRAP from micronemes when a sporozoite contacts its target cell in vivo. Contact with hepatoma cells in culture also appeared to induce the release of TRAP onto the surface of sporozoites. If large amounts of TRAP are released in close proximity to its cellular receptor(s), effective competitive inhibition by antibodies may be difficult to achieve.

FIG. 1

The domains of TRAP. (A) Schematic diagram of TRAP: SS, signal sequence; N-10, conserved N-terminal 10 amino acids; A-domain; TSR, thrombospondin type 1 repeat; TM, transmembrane domain; CD, cytoplasmic domain. (B and C) The crystal structure of the CD11b A-domain (22) (B) was used as the template to model the P. yoelii TRAP A-domain (C). Shown in magenta are Mg²⁺ ions coordinated by the MIDAS. The B-cell epitopes of the TRAP A-domain are highlighted as follows: yellow, B1; red, B2; green, B3.

FIG. 2

Confocal images of P. yoelii sporozoites stained for TRAP by IF. (A) Staining pattern seen in all permeabilized sporozoites. (B) Typical faint TRAP surface staining pattern. (C) Bright cap-like TRAP surface staining pattern rarely seen on sporozoites. All sporozoites were stained with monoclonal antibody F3B5. All images were scanned under identical conditions. Scale bar units are micrometers.

FIG. 3

Calcium ionophore A23187 increases the proportion of sporozoites with caps of bright TRAP surface staining. (A) Sporozoites were incubated with the indicated concentrations of A23187 for 2 min before fixation and IF labeling of surface-exposed TRAP. (B) The effect was reversed by pretreatment with BAPTA/AM, a membrane-permeant calcium chelator. For each treatment, 100 sporozoites were counted in blinded fashion. Each bar represents the average of two independent experiments.

FIG. 4

Confocal images of P. yoelii sporozoites incubated with HepG2 cells. Sporozoites were stained for surface-exposed TRAP (green) prior to permeabilization, followed by intracellular TRAP staining (red), using the monoclonal antibody F3B5. The actin of HepG2 cells, but not sporozoites, was stained with phalloidin (red). Thus, the red staining of parasites is due exclusively to TRAP. (A) Sporozoites in contact with cells often displayed bright cap patterns of surface TRAP labeling. (B) Only weak surface TRAP staining was typically detectable on sporozoites that were not in contact with cells. Images were scanned under identical conditions. Scale bar units are micrometers.