STUDIES ON EXOERYTHROCYTIC DEVELOPMENT OF PLASMODIUM FALCIPARUM IN VITRO: DETECTION AND ISOLATION OF PLASMODIUM LIVER STAGES AND ANALYSIS OF CIRCUMSPOROZOITE PROTEIN ANTIGEN PROCESSING

Plasmodium falciparum;antigen processing;liver stage;not listed

Dumoulin, Peter C.Groopman, John D. ;

Johns Hopkins University

关键词: Plasmodium falciparum;    antigen processing;    liver stage;    not listed;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39387/DUMOULIN-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Malaria, the disease caused by Plasmodium parasites, remains a major global health burden despite efforts to eradicate the parasite. Infection is initiated when a mosquito deposits sporozoites into the dermis that then actively invade hepatocytes. Sporozoites and the resulting liver stage of Plasmodium falciparum infection are leading targets for generation of a protective vaccine. The circumsporozoite protein is the major surface protein on sporozoites and is the target of vaccines aimed at preventing blood stage infection. During the process of invasion, sporozoites cleave the N-terminus of CSP and consequently two forms of CSP are potentially exposed to the immune system. We utilized ectopic expression of two natural form of CSP to quantify their abilities to serve as targets for MHC class I-restricted CD8+ T cells. We determined that presentation of both forms of CSP on MHC class I depends on proteasomal activity, however, presentation of full length CSP is more efficient and is conferred by the presence of N-terminal lysine residues absent in the cleaved form of CSP. To evaluate the presence of these two forms during in vitro infection, we developed methods to allow for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. We showed that exoerythrocytic forms can be detected and isolated from in vitro infected cultures of the HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Furthermore, we present an experimental evidence, that our methods can be applicable for the analysis of human host factors limiting development of P. falciparum EEFs.