Identification of Parasite Erythrocyte Membrane Antigens Specific to Cerebral Malaria and Severe Malarial Anemia Pathogenesis

Abstract

Plasmodium falciparum is responsible for the most severe forms of malarial disease, including cerebral malaria and severe malarial anemia. In cerebral malaria, infected erythrocytes are sequestered in the blood vessels of the brain, leading to endothelial activation and inflammation in the brain. Sequestration of infected erythrocytes is mediated by parasite variant surface antigens (VSAs) that facilitate cytoadhesion, whereby VSAs bind endothelial receptors in the host vasculature. P. falciparum erythrocyte membrane protein-1 antigens (PfEMP1s) are the most well-known VSA. PfEMP1s are encoded by the var gene family, and there are ~60 var genes per parasite genome. Only one PfEMP1 is expressed on the surface of each infected erythrocyte. These proteins exhibit extreme genetic diversity, with less than 50 percent shared amino acid identity. Clearance of infected erythrocytes is prevented when VSAs such as PfEMP1s bind to host endothelial receptors, including intercellular adhesion molecule-1 (ICAM-1), cluster of differentiation 36 (CD36), and endothelial protein C receptor (EPCR). Using a custom capture array to enrich for P. falciparum RNA, and RNA from loci encoding VSAs in particular, we successfully sequenced and profiled var gene expression from clinical infections without the need for extensive processing in the field at the time of collection. Capture methods were effective for samples with low parasitemia, and de novo assembly of var gene transcripts was validated by comparison to whole genome sequence data. We then applied these methods to a case-control study of severe malaria in Mali, West Africa, to measure var gene expression associated with severe malaria compared to uncomplicated malaria controls. PfEMP1s encoded by de novo-assembled transcripts were classified to determine domain subtypes and predict potential binding target in the human host. Transcripts encoding EPCR-binding PfEMP1s were not associated with severe cases of malaria compared to uncomplicated malaria controls. However, transcripts encoding both an EPCR-binding domain and an ICAM-1-binding motif were associated with severe cases of malaria in comparison to uncomplicated malaria controls. These “dual-binding” PfEMP1s may be a promising target for development of vaccines and treatments for severe malarial disease.