PLASTICITY AND DIVERSITY OF THE PLASMODIUM FALCIPARUM PLACENTAL MALARIA ANTIGEN VAR2CSA by ELDIN TALUNDZIC (Under the Direction of David S. Peterson) ABSTRACT As long as humans have walked this earth, malaria may have stalked them. Malaria is a disease of poverty and underdeveloped countries. Today, half of the world population lives in regions at risk for developing malaria and more than half a million deaths occur annually due this disease. Malaria is unforgiving, as most of the morbidity and mortality occurs in pregnant women and children under the age of five years. The majority of malaria- related deaths occur in Sub-Saharan Africa because there is limited access to effective vaccine and treatment. The malaria parasite evades host immune defenses through antigenic variation of proteins called Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). These proteins are expressed on the surface of infected erythrocytes, which allow them to bind to various host tissues. The proteins are encoded by a highly polymorphic var (variant) gene family, consisting of ~60 genes. While polymorphic, one of these genes, var2csa, is the most conserved PfEMP1 identified to date. The genes ancient origin and evolutionary significance is indicated by the fact that an ortholog is found in the genome of chimpanzee malaria P. reichenowi. VAR2CSA is only expressed during pregnancy. In pregnant women, Plasmodium falciparum is able to bind to a unique low sulfated form of chondroitin in the placenta by expressing VAR2CSA on the surface of the infected erythrocyte. The massive sequestration of these erythrocytes in the placenta results in pregnancy complications, including low-birth weight (LBW), maternal and neonatal death. While women in their first and second pregnancies are at highest risk for developing placental malaria (PM), partial immunity is achieved after multiple pregnancies through protective antibodies. This suggests that a vaccine to protect against placental malaria (PM) may be feasible. The dissertation herein provides detailed information on the genetic complexity of PM and methods of identifying and constructing representative allelic variants of VAR2CSA that may be used as a vaccine to protect mothers and their fetuses from placental malaria. INDEX WORDS: Malaria, VAR2CSA, Chondroitin Sulfate A, Placenta, Pregnancy PLASTICITY AND DIVERSITY OF THE PLASMODIUM FALCIPARUM PLACENTAL MALARIA ANTIGEN VAR2CSA by ELDIN TALUNDZIC B.A., The University of Georgia, USA, 2008 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2013 © 2013 ELDIN TALUNDZIC All Rights Reserved PLASTICITY AND DIVERSITY OF THE PLASMODIUM FALCIPARUM PLACENTAL MALARIA ANTIGEN VAR2CSA by ELDIN TALUNDZIC Major Professor: David S. Peterson Committee: Julie M. Moore Carl Bergmann Jeffery R. Hogan Roberto Docampo Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2013 iv DEDICATION I would like to dedicate this work to my family. I could have not accomplished this work without the continued support of my parents, Jasmina and Hasan Talundzic, and my brother Emir Talundzic. We have had a long and difficult journey. Throughout the years, you sacrificed so much to ensure that Emir and I would have a good life filled with many opportunities. Mom and dad, you are my true heroes. Emir, thank you for always supporting me and for being so patient with your big brother. To my beloved grandmother, Ismeta who helped raise me and teach me to follow my passion in ,(و س لم ع ل يه هللا ص لى) Strikovic life, thank you for everything. Lastly, to the rest of my family, including those we lost during .(و س لم ع ل يه هللا ص لى) our journey .(ال صال ح ين م سار ف ي ل ي هللا وف ق نا) v ACKNOWLEDGEMENTS The completion of this work involved the effort and support of many people. First and outmost, I want to sincerely thank my major professor, David Peterson for guiding me throughout the years and providing me with the support and environment to grow both intellectually and professionally. For always believing in me and encouraging me to think outside the box. Thank you. You are an amazing mentor and friend. To Julie Moore, whose passion and dedication to research inspired me to choose this career path. Thank you for exposing me to this amazing line of work as an undergraduate and recognizing my passion for research and encouraging me to follow it. I thank you for always challenging me and believing in me. I would not be here today if it wasn’t for you. I thank my committee members, for their advice, assistance, and intellectual support. Thank you for taking time to meet with me individually and for encouraging me and challenging me to do my best. I could have not asked for a better committee. Thank you for your criticism and suggestions. To my friends and colleagues who helped with the smiles and hugs. To the undergraduates who were eager to learn and help with the experiments. Thank you! Lastly, to all the courageous mothers who provided us with placental blood samples. This project would have not been possible if it were not for your donation(s). vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ...................................................................................................... v LIST OF TABLES ................................................................................................................... ix LIST OF FIGURES ................................................................................................................... x CHAPTER 1 INTRODUCTTION ................................................................................................ 1 Significance ...................................................................................................... 3 Hypothesis and specific aim(s) ......................................................................... 5 References ....................................................................................................... 8 2 LITERATURE REVIEW ..................................................................................... 11 Early history of malaria .................................................................................. 11 Economic and social burden of malaria ......................................................... 13 Malaria drug resistance ................................................................................. 14 Malaria life cycle ............................................................................................ 16 Antigenic variation, host cell hijacking and pathogenesis ............................. 19 Malaria and the immune system ..................................................................... 23 Malaria in pregnancy ...................................................................................... 30 Treatment for placental malaria ..................................................................... 31 Placental malaria antigen and ligand: VAR-2-CSA ...................................... 32 Placental pathology during a malaria infection .............................................. 35 Placental malaria immune responses .............................................................. 37 vii Evolutionary importance of var2csa and placental malaria ........................... 40 Summary and gaps in knowledge ................................................................... 41 References ...................................................................................................... 46 3 SEQUENCE POLYMORPHISM, SEGMENTAL RECOMBINATION AND TOGGLING AMINO ACID RESIDUES WITHIN THE DBL3X DOMAIN OF THE VAR2CSA PLACENTAL MALARIA ANTIGEN ..................................... 74 Abstract .......................................................................................................... 75 Introduction .................................................................................................... 76 Results ............................................................................................................ 78 Discussion(s) .................................................................................................. 84 Materials and Methods ................................................................................... 85 References ...................................................................................................... 90 4 GRAVIDITY ASSOCIATED SELECTION DRIVES ANTIGENIC VARIATION IN PLACENTAL MALARIA ............................................... 104 Abstract ........................................................................................................ 105 Introduction .................................................................................................. 106 Results .......................................................................................................... 107 Discussion(s) ................................................................................................ 110 Materials and Methods ................................................................................. 112 References .................................................................................................... 116 5 HIGH THROUGHPUT DETECTION OF SPECIES-SPECIFIC MALARIA INCLUDING SUBMICROSCOPIC INFECTIONS USING A MULTIPLEX REAL-TIME PHOTO-INDUCED ELECTRON TRANSFER PCR .......... 133 Abstract .......................................................................................................