Oral or Poster Presentation
Concurrent Session 1B - Placental Fetal Physiology
Introduction: Placental malaria results in low birth weight and fetal morbidity. It develops when iRBCs adhere to chondroitin sulfate A (CSA) glycosylated syndecan-1 (SDC-1) on placental syncytiotrophoblasts (ST) via VAR2CSA, a parasite protein localized to the iRBC surface. Vaccine candidates are being investigated and the protective antibodies generated will need to be assayed for blocking efficiency. Current in vitro assays use plated CSA and are non-physiological. Our aim is to optimize a physiological cell-based assay to test adhesion-blocking activity of antibodies using cultured human ST. We hypothesized that iRBC would bind to ST and that this would be blocked by removing CSA from ST or blocking VAR2CSA on iRBC.
Methods: SDC-1 expression on ST in human term placental sections was measured using immunohistochemistry. Primary human term trophoblasts were cultured with Br-cAMP (100μM) to induce ST formation. Blocking conditions used chondroitinase (10U/mL), soluble CSA (200μg/mL), or decorin (200ng/mL). Infected or uninfected RBC were added to fixed ST. After washing, bound RBC were fixed, stained for glycophorin A and quantified using brightfield microscopy with a polarized filter.
Results: SDC-1 was localized to the apical membrane of ST (n=6). Chondroitinase treatment of ST to remove CSA reduced bound iRBC by 40±0.15% (n=3) or 56±0.15% (n=3) depending on washing technique used. Preincubating iRBCs with decorin, a proteoglycan that binds VAR2CSA, reduced iRBC binding by 35±0.19% (n=5) or 38±0.27% (n=3) depending on washing technique used. Preincubating iRBCs with soluble CSA reduced binding by 42±0.32% (n=4).
Conclusion: Specific iRBC binding to ST was reduced after removal of CSA or preincubation of iRBC with decorin or soluble CSA. This physiological assay using cultured human ST will be a useful tool to test the blocking efficiency of antibodies produced during vaccine development for placental malaria.