Browsing School, Graduate by Subject "var genes"
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Identification of Parasite Erythrocyte Membrane Antigens Specific to Cerebral Malaria and Severe Malarial Anemia PathogenesisPlasmodium 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.
New tools for var gene assembly and analysis of genetic diversity of var2csa, the locus encoding a placental malaria vaccine candidate antigen: vaccine development implicationsEncoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes (iEs), resulting in immune evasion and severe malaria disease. Sequencing and assembling the 40-60 var gene complement for individual infections has been notoriously difficult, impeding molecular epidemiological studies and the assessment of var-encoded proteins as subunit vaccine candidates. VAR2CSA, a member of this family, mediates the binding of iEs to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally-acquired immunity and, as such, is a leading vaccine candidate against placental malaria. The gene encoding the VAR2CSA is technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time-consuming and costly. We developed and validated novel tools to assemble var gene sequences from clinical samples, with a focus on var2csa. These tools include Exon-Targeted Hybrid Assembly (ETHA), which is based on a combination of Pacific Biosciences (PacBio) and Illumina data, an amplicon sequencing of var2csa using PacBio, and a pipeline to reconstruct var2csa from short sequence reads. Using ETHA, we characterized the repertoire of var genes in samples from uncomplicated malaria infections in children from a single Malian village and showed them to be as genetically diverse as vars from isolates from around the globe. The amplicon sequencing allowed us to recover a segment encoding the first four domains in the 5' end of VAR2CSA (~5kb), and the full-length of the VAR2CSA's extracellular region from clinical malaria samples. The reconstruction of var2csa from short reads permitted us to generate a worldwide set of var2csa sequences. We used the VAR2CSA sequences to show that despite the extreme sequence variation, VAR2CSA from around the globe shared conserved motifs supporting the feasibility of VAR2CSA-based vaccines. The findings presented in this work will aid the understanding of malaria pathogenesis and inform the design of malaria vaccines based on PfEMP1, in particular VAR2CSA-based vaccines.