STUDIES ON EXOERYTHROCYTIC DEVELOPMENT OF PLASMODIUM FALCIPARUM IN VITRO: DETECTION AND ISOLATION OF PLASMODIUM LIVER STAGES AND ANALYSIS OF CIRCUMSPOROZOITE PROTEIN ANTIGEN PROCESSING by Peter Dumoulin A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland May, 2015 Abstract 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 ii methods can be applicable for the analysis of human host factors limiting development of P. falciparum EEFs. iii Main goals 1. Assess the ability of two forms of Plasmodium falciparum CSP, to serve as targets for MHC class I-restricted CD8+ T cell responses Specific Aims: A. To develop an in vitro system for monitoring the MHC class I processing and presentation of peptide epitopes derived from the P. falciparum CSP antigen B. To assess the ability of targets expressing one of the CSP forms to induce specific CD8+ T cell activation in a MHC class I-restricted and non-restricted manner C. To examine the role of major cellular proteolytic machineries in the processing of two forms of CSP D. To investigate the role of the ubiquitin system in the MHC class I-restricted processing of peptide epitopes from two forms of the CSP antigen 2. To develop an experimental system for detection and isolation of Plasmodium falciparum EEFs in vitro Specific Aims: A. To develop an in vitro hepatocellular model permissive for propagation of P. falciparum EEFs B. To develop flow cytometry-based detection and specific isolation of P. falciparum EEFs C. To compare dynamics of P. falciparum EEF development in vitro using different experimental hepatocellular systems D. To validate the applicability of our experimental model for the analysis of human host factors limiting development of P. falciparum EEFs. iv Table of Contents Abstract .............................................................................................................................. ii Main goals ......................................................................................................................... iv Table of Figures.............................................................................................................. viii Chapter 1 : General Introduction ................................................................................... 1 History of elucidating the Plasmodium life cycle ..................................................................... 5 From skin to merosome ............................................................................................................. 9 Targeting exoerythrocytic forms: whole organism vaccines ................................................ 13 Mechanisms of immune protection ........................................................................................ 17 Biology of CSP .......................................................................................................................... 23 Techniques to study the Plasmodium liver stage ................................................................... 26 Concluding remarks ................................................................................................................ 28 Chapter 2 : Processing and presentation of two forms of CSP on MHC class I ....... 29 Introduction .............................................................................................................................. 30 Presentation of peptides on MHC class I ........................................................................... 30 Targeting Plasmodium: role of the MHC class I antigen processing and presentation pathway ................................................................................................................................. 34 Two forms of CSP as targets of the CD8+ T cell immune response ................................. 38 Results ....................................................................................................................................... 40 Ectopic expression of CSP in hepatocytes .......................................................................... 40 Identification of CSP in traversed and infected hepatocytes during in vitro infection .. 41 Two forms of CSP differentially activate CD8+ T cells..................................................... 43 Targets expressing one of two forms of CSP are differentially lysed by CD8+ T cells... 49 Two forms of CSP do not alter immune phenotypes of targets or overall susceptibility to death stimuli ..................................................................................................................... 51 Half-life of two forms of CSP and dependence of proteasomal degradation on MHC class I presentation ............................................................................................................... 53 CSP-Full forms polyubiquitin chains more efficiently than CSP-Short ......................... 54 Individual lysine residues in the N-terminus of CSP are required for efficient MHC class I-restricted antigen presentation ............................................................................... 56 Discussion ................................................................................................................................. 59 Chapter 3 : Specific detection and isolation of P. falciparum EEFs in vitro .............. 67 v Introduction .............................................................................................................................. 68 Results ....................................................................................................................................... 72 Immortalized human hepatocyte cell lines are permissive for traversal by P. falciparum sporozoites ............................................................................................................................ 72 Human hepatocyte cell lines exhibit differential ability to support development of P. falciparum EEFs ................................................................................................................... 76 P. falciparum-infected HC-04 hepatocytes can be specifically isolated ........................... 81 Primary human hepatocytes support P. falciparum 3D7HT-GFP EEFs in vitro ........... 84 Discussion ................................................................................................................................. 93 Chapter 4 : Flow cytometry based assays of P. falciparum liver stages in vitro: from sporozoite infectivity to EEF development ................................................................. 100 Introduction ............................................................................................................................ 101 Results ..................................................................................................................................... 104 CD81 is required for P. falciparum infection of primary human hepatocytes, but is not essential for the infection of HC-04 .................................................................................. 104 Humanized CSP-specific antibody 2A10 inhibits traversal and reduces the number of P. falciparum EEFs in human hepatocytes in vitro ......................................................... 107 Discussion ............................................................................................................................... 109 Chapter 5 : Future Directions ...................................................................................... 113 Chapter 6 : Materials and Methods ............................................................................ 119 Human hepatocyte