Antibodies to Plasmodium circumsporozoite protein (CSP) inhibit sporozoite's cell traversal activity

Abstract

Plasmodium sporozoites are deposited in the skin of the mammalian host by Anopheles mosquitoes. To continue the life cycle, the sporozoites have to invade the host's hepatocytes, where they transform into exoerythrocytic forms (EEFs) inside a parasitophorous vacuole. During their route from the skin to the liver, the parasites traverse the capillary epithelium in the dermis to enter the blood circulation, and cross the endothelium of liver sinusoids to enter the parenchyma. Cell traversal by sporozoites is usually measured by quantifying dyes that enter or are released from cells during incubation with salivary gland sporozoites. These methods do not distinguish cell traversal from cell wounding. Here we validate an assay that quantifies cell traversal of sporozoites through monolayers of MDCK cells that form tight junctions. We compared cell traversal of wt sporozoites and of parasites lacking the Type I membrane protein TLP (TRAP-like protein) previously implicated in cell traversal. We provide direct evidence that TLP ko sporozoites are defective in cell traversal and that they are retained inside the MDCK cytoplasm. We then used the MDCK assay to study the effect of a monoclonal antibody (3D11) to the circumsporozoite protein (CSP) on the parasite's cell traversal. We show that 3D11 inhibits cell traversal at nanomolar concentrations. We conclude that antibodies elicited by CSP-based vaccines are likely to inhibit the migration of sporozoites from the skin to the liver.

Introduction

The sporozoites of malaria parasites undergo profound genetic changes while they move from the mosquito oocysts into the salivary glands (Matuschewski, 2006), where they can remain dormant for days (Zhang et al., 2010). After delivery into the skin of the mammalian host by mosquito bite, the parasites start a long journey to the liver, where they enter hepatocytes and develop into the exoerythrocytic forms. During this migration the parasites need to cross the vascular endothelium to enter the blood or lymphatic circulation, and the endothelium of liver sinusoids to enter the parenchyma (Mota et al., 2001, Amino et al., 2008, Ejigiri and Sinnis, 2009). Sporozoite migration and infection of hepatocytes are achieved by the concerted activity of an actin–myosin motor located beneath the parasite's plasma membrane (Kappe et al., 2004, Dowse and Soldati, 2004) and by products of specialized secretory organelles named rhoptries, dense granules and micronemes (Carruthers and Sibley, 1997). Sporozoites do not have flagella, cilia or lamellipodia, nevertheless they use an actin/myosin motor to move very rapidly over solid substrates by gliding motility (Heintzelman, 2006, Tardieux and Ménard, 2008, Daher and Soldati-Favre, 2009, Munter et al., 2009). The first sporozoite membrane protein shown to link the motor to the extracellular substrate was the thrombospondin related anonymous protein (TRAP) (Sultan et al., 1997). In the absence of TRAP sporozoites do not glide. Other members of the TRAP family were identified later (Lacroix and Ménard, 2008), including TLP (that is the subject of this paper; Moreira et al., 2008, Heiss et al., 2008). All members bear a cytoplasmic tail that is acidic and interchangeable. This tail contains a sub-terminal tryptophan that is essential for motility (Kappe et al., 1999). The extracellular domains of TRAP family members consist of two adhesive modules that bind to substrates: the thrombospondin type I repeat and the A domain.

Two methods are generally used to study cell traversal. In the calcein green assay, target cells loaded with a fluorescent dye are incubated with sporozoites and the amounts of fluorescence released into the supernatant are measured (Coppi et al., 2007). This assay was used in the studies of the TLP knockout (Moreira et al., 2008). However, the calcein assay measures cell wounding rather than cell traversal. A second cell traversal assay uses polarized monolayers of MDCK cells seeded between two chambers (Mota et al., 2001). The sporozoites are placed in the upper chamber, and after an appropriate time the parasites in the bottom chamber are counted. As described, this assay does not exclude the possibility that sporozoites migrate between cell junctions as is the case of Toxoplasma tachyzoites (Barragan et al., 2005). Here we ruled this out and used the MDCK assay to confirm the role of TLP in cell traversal. In addition, we studied the effect of antibodies to the sporozoite's circumsporozoite protein (CSP) on their ability to traverse cells.