University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School January 2015 Immunological Characterization Of Duffy Binding Protein Of Plasmodium vivax Miriam Thankam George University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the Molecular Biology Commons, and the Public Health Commons Scholar Commons Citation George, Miriam Thankam, "Immunological Characterization Of Duffy Binding Protein Of Plasmodium vivax" (2015). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/5689 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Immunological Characterization of Duffy Binding Protein of Plasmodium vivax by Miriam Thankam George A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Global Health College of Public Health University of South Florida Major Professor: John H. Adams, Ph.D. Francis B. Ntumngia, Ph.D. Ricardo Izurieta, MD, Dr.PH Wei Wang, Ph.D. Date of Approval: July 8, 2015 Keywords: Epitope mapping, phage display, malaria Copyright © 2015, Miriam Thankam George DEDICATION To my parents, family, friends and colleagues all over the world who have shown me that this is not just a quest to solve a problem or even doing what is morally right but a gentle voice glorifying God. ACKNOWLEDGMENTS Completion of this dissertation has been made possible through the support of my doctoral committee, past and present members of the Adams’ laboratory our collaborators, my family and friends. I would like to especially thank the following people: My mentor, Dr. John Adams, for guiding me and encouraging me to explore various research endeavors. Dr. Francis Ntumngia who has been a strong voice of scientific reason and mentoring me through each step. Dr. Ricardo Izurieta and Dr. Wei Wang, who brought their multidisciplinary experience as they served on my doctoral committee; their valuable input helped tie various aspects of this project together. Dr. Jesse Schloegel for his invaluable insight and for all the work he did towards this project. Our collaborators, Per-Åke Nygren and Johan Seijsing, at KTH Royal Institute (Sweden) for providing me with a solid foundation that is necessary for the advancement of this field and for allowing me to use their facilities. Our collaborators, Dr. Mick Foley and Dr. Joanne Casey, at Latrobe University (Australia) for carrying out a part of the experiments. Finally, the members of the Adams’ laboratory, IDRB 3rd floor family, COPH faculty and staff who have helped me in many big and small ways during my doctoral research. TABLE OF CONTENTS List of Tables ................................................................................................................... iii List of Figures .................................................................................................................. iv Abstract ........................................................................................................................... vi Chapter One: Introduction ................................................................................................ 1 Worldwide Malaria ................................................................................................. 1 Malaria caused by P. vivax ......................................................................... 2 Malaria Life Cycle .................................................................................................. 3 Immune response to malaria ...................................................................... 6 Malaria Vaccines ................................................................................................... 9 P. vivax asexual blood stage vaccines ..................................................... 10 The Duffy binding protein .......................................................................... 11 Phage Display And Biopanning ........................................................................... 16 Phage display library ................................................................................ 21 Tables And Figures ............................................................................................. 23 References .......................................................................................................... 27 Chapter Two: Defining The Immunoreactive Surface Of Plasmodium Vivax Duffy Binding Protein Region II .................................................. 40 Abstract ................................................................................................................ 40 Introduction .......................................................................................................... 41 Materials And Methods ........................................................................................ 45 Generation of phage displayed DBPII gene fragment libraries ................. 45 Panning of phage displayed DBPII gene fragment library ........................ 46 Panning a random peptide library on mAb-3D10 ...................................... 47 ELISA of phage displayed rounds of panning ........................................... 48 Immunoblots ............................................................................................. 48 Immunizations ........................................................................................... 49 Measurement of antibody titers ................................................................ 50 Measurement of functional inhibition of DBP-Erythrocyte binding ............ 50 Immunofluorescence assay of COS7-surface expressed DBPII .............. 51 Results ................................................................................................................. 52 Discussion ........................................................................................................... 56 Tables And Figures ............................................................................................. 62 Acknowledgments ............................................................................................... 76 References .......................................................................................................... 77 i Chapter Three: Randomized Helical Scaffold Affibody Library To Map Conformational Epitopes On Plasmodium Vivax Duffy Binding Protein ................... 84 Abstract ................................................................................................................ 84 Introduction .......................................................................................................... 84 Materials And Methods ........................................................................................ 88 Biotinylation of antibodies ......................................................................... 88 Affiibody selections ................................................................................... 89 ELISA screen of clones ............................................................................ 90 Sequencing ............................................................................................... 91 Results ................................................................................................................. 91 Discussion ........................................................................................................... 92 Tables And Figures ............................................................................................. 96 Acknowledgments ............................................................................................. 100 References ........................................................................................................ 100 Chapter Four: Summary ............................................................................................... 105 References ........................................................................................................ 109 Appendix 1: IACUC Approval Letter ............................................................................. 112 Appendix 2: Copyright Approval Letters ....................................................................... 113 ii LIST OF TABLES Table 2.1: Peptides identified through biopanning used for immunizations ................ 72 iii LIST OF FIGURES Figure 1.1: Countries with ongoing transmission of malaria in 2013 .......................... 23 Figure 1.2: Spatial distribution of P.vivax malaria endemicity in 2010 ........................ 23 Figure 1.3: Malaria life cycle ....................................................................................... 24 Figure 1.4: Merozoite Invasion of Erythrocytes .......................................................... 25 Figure 1.5: Schematic drawing of the important structural domains of DBP. .............. 26 Figure 1.6: Crystal structure of PvDBPII ..................................................................... 26 Figure 1.7: ‘DEK’ epitope and putative sulfotyrosine-binding pocket on PvDBPII crystal structure ......................................................................................... 27 Figure 2.1: Schematic