Antibodies against Thrombospondin-Related Anonymous Protein Do Not InhibitPlasmodiumSporozoite 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 wellcharacterized 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 Adomain 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.

A long and winding road: The Plasmodium sporozoite's journey in the mammalian host

“…This is likely because the controlled release of AMA-1 results in a high local concentration of the protein, making it difficult for antibodies to compete with AMA-1 binding to its receptor or binding partner, which has yet to be identified. A similar situation is observed with anti-TRAP antibodies which poorly inhibit hepatocyte invasion despite the fact that TRAP is clearly essential for this process [57]. AMA-1 is proteolytically processed similarly in sporozoites and merozoites [69,74]; with a loss of an NH 2 -terminal fragment prior to secretion onto the surface and a carboxy-terminal cleavage, sensitive to a subset of serine protease inhibitors, that sheds the extracellular domain from the sporozoite surface.…”

“…Additionally, it has a cytoplasmic tail that links the surface of the sporozoite to the motility machinery of the parasite [51]. TRAP is normally present in small amounts on the sporozoite surface, however, contact with hepatocytes triggers the release of large amounts onto the zoite surface, suggesting a role in hepatocyte invasion [57]. This has been definitively shown with the generation of sporozoites expressing TRAP with alterations in the A-domain or TSR that would be predicted to abolish the adhesive interactions of the respective domain [67,68].…”

“…Exocytosis of these organelles is stimulated by the mobilization of parasite intracellular calcium [54]. These events have been elucidated primarily using Toxoplasma tachyzoites (reviewed in [55,56]), however, a few studies have shown that when Plasmodium sporozoites contact hepatocytes, there is an upregulation of microneme secretion through the mobilization of intracellular calcium [32,57]. Many of the proteins found in micronemes contain cell-adhesive domains that function in zoite-host cell interactions required for invasion (reviewed in [56]).…”

The Relevance of Structural Biology in Studying Molecules Involved in Parasite–Host Interactions: Potential for Designing New Interventions

“…[81] Toxoplasma gondii, Plasmodium Structures of the human thrombospondin-1 type 1 repeats (PDB accession code 1lsl) and the TSR domain of a Plasmodium TRAP (2bbx) are known. [82][83][84][85] Ancylostoma ceylanicum Ace-MIF Cytokine ortholog CD74 The macrophage migration inhibiting factor (MIF) acts as a lymphocyte chemo-attractant, similar to the human orthologue and possesses tautomerase activity. Vaccination provided partial protection from disease.…”

Efficacy model for antibody-mediated pre-erythrocytic malaria vaccines

“…TRAP is found primarily within the sporozoite's micronemes and on the sporozoite surface [16]. Antibodies to TRAP inhibit sporozoite gliding motility [17] and hepatocyte invasion [18]; however, there is some evidence to suggest that TRAP antibodies do not inhibit sporozoite infectivity in vivo [19]. LSA-1 is expressed soon after the sporozoite invades the hepatocyte in the liver [20].…”