• Designing Next Generation Genomics and Serological Tools for Surveillance of Plasmodium vivax Malaria to Guide Elimination Efforts in Southeast Asia

      Agrawal, Sonia; Plowe, Christopher V.; 0000-0003-4484-7433 (2019)
      Malaria is a major global health problem caused by mosquito-borne, protozoan parasites belonging to the genus Plasmodium. Plasmodium vivax, the human malaria parasite with the widest global distribution, accounts for majority of the total malaria cases outside sub-Saharan Africa. The inability to establish long term in vitro culture system and low parasite densities, combined with high levels of human genomic DNA isolated from patient samples with P. vivax infections, makes it difficult to obtain sufficient amounts of parasite DNA for whole genome sequencing (WGS). New, reliable, highly sensitive and specific methods are needed to produce high quality P. vivax WGS data. Genome-wide analyses of the parasite, using WGS, have the potential to improve our understanding of parasite population dynamics and help identifying locations that serve as possible transmission sources and sinks. Additionally, protein microarrays that can simultaneously measure human antibody responses to a large number of Plasmodium antigens have the potential to identify P. vivax specific biomarkers to detect not only current but also past malaria infections, providing a more sensitive surveillance tool for identifying human populations at risk. To address these needs, using Roche/NimbleGen SeqCap EZ whole genome capture technology, high quality WGS data was generated from P. vivax clinical samples collected from the China-Myanmar border. This new genome-wide data along with publicly available WGS from circulating isolates in Southeast Asia were utilized to characterize parasite genetic diversity and relatedness, population structure, complexity of infection, and distinguish locally transmitted infections from imported P. vivax infections revealing clonal parasite population on the China-Myanmar border. Using protein microarray analyses, several P. vivax specific serologic markers during active infection were identified that may serve as useful biomarkers of current or recent P. vivax infection supporting the possibility of serology as a tool for estimating species-specific malaria exposure to P. vivax in heterogeneous malaria transmission settings. The combination of next generation tools attempted to be designed as part of this dissertation will help improve the understanding of the genomic epidemiology and estimates of transmission patterns of this human malaria parasite, thus, guiding rational P. vivax malaria control and elimination policies in Southeast Asia.
    • Identification of Parasite Erythrocyte Membrane Antigens Specific to Cerebral Malaria and Severe Malarial Anemia Pathogenesis

      Stucke, Emily Marie; Travassos, Mark A.; Takala-Harrison, Shannon; 0000-0002-8256-3290 (2022)
      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.