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.

Fig. 1. PbTLP ko sporozoites are impaired in cell-passage activity

Wild type and PbTLP ko salivary gland sporozoites were added to filter inset and incubated for 1 h at 37°C. (A) The percentage of sporozoites that cross the MDCK monolayer and are present in the bottom of the well was counted. (B) Sporozoites that cross the MDCK monolayer and develop into EEFs inside HepG2 cells seeded in the bottom chamber were quantified by real time PCR. (C) Occludin staining shows that MDCK cells formed tight junctions. (D) TER was relatively constant during sporozoites migration. Data (mean ± SD) are from quadruplicates analyzed by unpaired two-tailed Student’s t-test.

Fig. 2. Greater number of PbTLP ko sporozoites are retained in the cytoplasm of MDCK cells compared to wt

(A) The number of wt and PbTLP ko sporozoites found in the cytoplasm of MDCK cells. (B) The number of wt and PbTLP ko sporozoites present in the MDCK cells estimated by inside/outside staining. (C) Photograph of wt MDCK filter section. (D) Photograph of PbTLP ko MDCK filter section. Data (mean ± SD) are from quadruplicates analyzed by unpaired two-tailed Student’s t-test.

Fig. 3. Monoclonal antibody 3D11 inhibits cell traversal and gliding motility of sporozoite

(A) 3D11 significantly inhibits cell traversal (p = 0.003). (B) Gliding motility was also decreased significantly when comparing the number of >10 circles of treated and nontreated sporozoites (p = 0.02). Total number of circles is presented on top of the bar. Data (mean ± SD) are from duplicates.