COLLEGE OF BASIC AND APPLIED SCIENCES SCHOOL OF BIOLOGICAL SCIENCES THE ROLE OF AN INTERCELLULAR ADHESION MOLECULE 1 BINDING MOTIF IN PLASMODIUM FALCIPARUM ERYTHROCYTE MEMBRANE PROTEIN 1 AND OTHER DETERMINANTS INVOLVED IN SEVERE MALARIA PATHOGENESIS BY GERTRUDE ECKLU-MENSAH (10051041) THIS THESIS IS SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY, UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY DEGREE JULY, 2017 DECLARATION This thesis was carried out under the supervision of Dr. Michael F. Ofori, Professors Michael D. Wilson, Anja T.R. Jensen and Lars Hviid. I hereby declare that this is the result of my own research work and that other people‟s work has been duly acknowledged by reference. I further declare that that no material presented in this work has been submitted to any other institution for acquiring a degree. The paper published in Cell Host and Microbe (Appendix E) was a collaborative study, in addition to researchers from the University of Ghana and the University of Copenhagen involved different partners from other institutions. I outline below the various contributions made by partners to this study. Method/analysis Responsibility Sample collection, processing and shared storage Bioinformatics partner BOEC culturing sole and shared Crystal- and SPR work partner ELISA sole and shared Expression and purification of sole and shared recombinant proteins Flow cytometry sole and shared Immunofluorescence assay partner Parasite adhesion assays sole and shared Parasite culturing sole and shared Parasite selection sole and shared Real-time Q-PCR sole and shared Thesis preparation sole i ……………………………………………………………… GERTRUDE ECKLU-MENSAH (CANDIDATE) DATE……………………………………. ……………………………………………………………… DR MICHAEL FOKUO OFORI (PRINCIPAL SUPERVISOR) DATE………………………………………. ……………………………………………………………… PROFESSOR MICHAEL DAVID WILSON (SUPERVISOR) DATE……………………………………….. ……………………………………………………………… PROFESSOR ANJA TATIANA RAMSTEDT JENSEN (SUPERVISOR) DATE………………………………………… ii ABSTRACT A central pathogenic feature in Plasmodium falciparum cerebral malaria (CM) is the sequestration of infected erythrocytes (IEs) which results in the obstruction of blood flow, inflammation and damage to the endothelial cell barrier integrity. Adhesion is mediated by a family of approximately 60 highly variant parasite proteins called P. falciparum erythrocyte membrane protein 1 (PfEMP1), which are displayed on the surface of infected cells. All PfEMP1 contain multiple extracellular domains of Duffy binding like (DBL) and cysteine-rich interdomain region (CIDR) proteins that mediate adhesion to various host endothelial receptors such as intercellular adhesion molecule 1 (ICAM-1). An important unanswered question in malaria pathogenesis has always been whether a specific binding phenotype can be linked to any of the different severe malaria phenotypes. The broad objectives of this study were to investigate the involvement of an ICAM-1 binding motif in PfEMP1 in the development of CM, as well as to evaluate the contributions of the immune response and other determinants driving severe malaria complications. A highly conserved motif in DBLβ domain of group A PfEMP1 molecules was identified using sequence information. IEs expressing PfEMP1 containing the motif had a dual binding affinity for endothelial protein C receptor (EPCR) and ICAM-1 when tested under in vitro flow adhesion assays. Quantitative real time PCR (qPCR) showed higher transcription of this motif in parasites isolated from children with CM compared with non-cerebral severe malaria (p=0.020). The sequences defining the motif were exceptionally conserved and thus suggested its ability to induce cross- reactive responses. Broadly reactive cross-inhibitory antibodies against the motif were acquired following P. falciparum natural infections, in addition to being experimentally induced. Such cross-reactive antibodies interfered with ICAM-1 iii binding of recombinant motif-containing DBLβ domains and IEs from paediatric CM patients. These findings make the motif an attractive candidate to be included in a vaccine cocktail that seeks to prevent CM complications. Markers of endothelial activation (sICAM-1, sEPCR, Ang-2, HMGB1) in Ghanaian children with severe or uncomplicated malaria and their levels following resolution of infection were determined using ELISA. The levels were higher in malaria patients in comparison with healthy controls (p < 0.001). These levels declined significantly in convalescent patients except for high mobility group box 1 protein (HMGB1) which remained high to a similar degree as observed during infection (p > 0.05), an observation reported for the first time in malaria. Blood outgrowth endothelial cells (BOECs) from peripheral blood mononuclear cells of Ghanaian malaria patients were isolated and characterised as physiologically relevant cells to potentially study falciparum-infected erythrocyte adhesive interactions. Flow cytometry analysis showed that BOECs expressed several endothelial surface markers including malaria parasite adhesion proteins ICAM-1 and EPCR. Thus, future use of these cells could advance our understanding of cytoadhesion involving both receptors. Very little is known about the frequency of IgM-binding PfEMP1 in the P. falciparum genome, a phenotype that has been associated with severe disease. Quantitative real time PCR was used to assess the var transcript levels of 3D7/NF54 parasites selected for IgM binding. This study uncovered at least six new var/PfEMP1 candidates that could potentially mediate binding to IgM. Overall, this study expands current knowledge on the contributions made by both host and parasite factors to severe malaria pathogenesis. Detailed knowledge of these factors is crucially important for effective management of malaria. iv DEDICATION To all children under five years of age in malaria endemic countries v ACKNOWLEDGEMENTS First to my supervisors, Dr. Michael Ofori, Professors Michael Wilson, Anja Jensen and Lars Hviid for your valuable support and guidance throughout the PhD journey. I am highly indebted to Anja Jensen who welcomed me with both arms into her group and has been a great and patient mentor. Thank you for making me feel at home in Copenhagen, for the get-togethers and good motivation during trying moments. I am very grateful to Lars Hviid for keeping his doors wide open. I could always enter without prior notice and he was ever prepared to have a discussion. Thank you for accepting me as your scientific daughter, for your enthusiasm in my work and frank advice. My PhD study would have been very fruitless without the cooperation of the consented participants, parents and guardians in Hohoe. Thank you very much! To the super smart Target ladies: Anja B, Mette, Rebecca, Soffie, Vanessa and Yvonne, I am very grateful for the contributions made to my project, for good times in and outside the lab. Thanks to Yvonne for introduction into the world of endothelial cells, Anja B for teaching me cytoadhesion assays, Vanessa, for a memorable eight month period in and outside the lab. I do share my award from the Danish Society for Parasitologists spring symposium with you. Thanks to past and present members of the Surface team, for great friendship and amazing collaboration. My sincerest thanks to Pilar and Mary for sharing their rich experiences with me. My gratitude goes to the staff at the CMP, University of Copenhagen for the interactive and multinational environment and even more for the stimulating discussions at scientific updates. Many thanks to Christian and Regis, for giving me a long lasting impression about wonderful Copenhagen and your delightful personalities. Regis, I am very grateful for the timely support. Thanks to the cake club members for divine cakes. My sincerest appreciation to my MAVARECA colleagues: William, Frederica and Betty for the vi scientific discussions and sharing in my many cheers and wails. I highly acknowledge contributions made by the Director of Noguchi Memorial Institute for Medical Research, Prof. Kwadwo Koram and also kind contributions from the Immunology Department particularly Rev. Alex Danso, Kakra Dickson, Eric Kyei-Baffour, Ben Tornyigah and Cecilia Smith. A special thanks to the staff of the Hohoe Municipal Hospital and the Onchocerciasis unit for good collaboration and field support throughout my project. I am thankful to the staff of Biochemistry, Cell and Molecular Biology Department, University of Ghana for their support and valuable advice. I am very grateful to Anja B, Mary, Pilar, Samira, William vanP. and Yvonne for proof reading and constructive comments on my work. Samira and Mary, your useful insights and efforts are highly acknowledged mucho gracias! Special thanks to two smart colleagues Isaac Darko and Sam Aboagye for the lively scientific and social discussions and more importantly for not allowing me to sink into the PhD ocean. Thanks to very loyal friends: Andy, Chantal, Henrietta, Jose P, Simone, Sylvia, Lily, Lydia, Phinna, Richard Appiah Otoo for always believing in me. I am truly humbled. Special thanks to Frs. Ben Ohene, Dominic Ofori and Andrej for your constant prayers and motivation. Fr. Ofori, your frequent calls and rich experiences have been a wonderful blessing. My kindest appreciation to Drs. A. Ghansah and B. Egyir for the incredible advice and encouragement. I am deeply grateful to Augustine Awoyale for your extraordinary support and being my number one enthusiastic cheer leader. To my parents and siblings, for the numerous sacrifices, patience, prayers and unconditional support, thank you is not enough! I owe a lot of gratitude to the Danish ministry of foreign affairs (DANIDA) for generously funding my PhD study. vii TABLE OF CONTENTS DECLARATION ............................................................................................................ i ABSTRACT .................................................................................................................. iii DEDICATION ............................................................................................................... v ACKNOWLEDGEMENTS .......................................................................................... vi LIST OF ABBREVIATIONS .................................................................................... xvii CHAPTER ONE ............................................................................................................ 1 INTRODUCTION ...................................................................................................... 1 1.1 Background .......................................................................................................... 1 1.2 Objective 1: The involvement of an ICAM-1 binding PfEMP-1 motif in CM .... 2 1.3 Objective 2: The involvement of host immune responses in malaria .................. 5 1.4 Objective 3: Establishment of a blood outgrowth endothelial cell in vitro system and its potential use to study P. falciparum cytoadhesion properties ........................ 6 1.5 Objective 4: Identification of PfEMP1 mediated IgM binding variants in P. falciparum genome ..................................................................................................... 7 CHAPTER TWO ........................................................................................................... 8 LITERATURE REVIEW ........................................................................................... 8 2.1 Global burden and strategies ................................................................................ 8 2.1.1 Malaria in Ghana ......................................................................................... 11 2.2 Malaria parasites................................................................................................. 12 2.3 Life cycle of human Plasmodium parasites ........................................................ 13 2.4 Plasmodium falciparum disease manifestations................................................. 17 2.4.1 Severe anaemia ............................................................................................ 18 2.4.2 Metabolic acidosis ....................................................................................... 19 2.4.3 Cerebral malaria .......................................................................................... 19 2.4.4 Pregnancy-associated malaria, PAM ........................................................... 20 2.5 Naturally acquired immunity.............................................................................. 21 2.6 Variant surface antigens (VSA) and PfEMP1 .................................................... 23 2.6.1 Var gene architecture ................................................................................... 26 2.6.2 Var gene transcription and regulation ......................................................... 29 2.6.3 Antigenic variation and var switching......................................................... 35 2.7 Endothelial receptors involved in cytoadhesion ................................................. 37 2.7.1 Intercellular adhesion molecule-1 ............................................................... 38 2.7.2 Endothelial Protein C Receptor ................................................................... 42 viii 2.7.3 Cluster of Differentiation 36........................................................................ 45 2.8 PfEMP1 in disease and immunity ...................................................................... 47 2.9 Pathogenesis of CM............................................................................................ 48 2.9.1 The sequestration model .............................................................................. 48 2.9.2 The inflammation model ............................................................................. 49 2.9.3 Endothelial activation .................................................................................. 52 2.10 PfEMP1-mediated IgM binding and disease severity ...................................... 54 2.11 Blood outgrowth endothelial cells (BOECs) .................................................... 56 CHAPTER THREE...................................................................................................... 60 THE ROLE OF AN ICAM-1 BINDING MOTIF IN PFEMP1 IN THE PATHOGENESIS OF CM ....................................................................................... 60 3.1 Introduction ........................................................................................................ 60 3.2 Hypothesis and specific objectives..................................................................... 66 3.3 Methods .............................................................................................................. 67 3.3.1 Study sites, ethics and sample collection..................................................... 67 3.3.2 Sample processing ....................................................................................... 69 3.3.3 Recombinant proteins .................................................................................. 70 3.3.4 Rat immunisations ....................................................................................... 71 3.3.5 Antibody purification .................................................................................. 72 3.3.6 ELISA-based binding inhibition assay ........................................................ 72 3.3.7 RNA purification and DNase treatment ...................................................... 73 3.3.8 cDNA synthesis and qPCR .......................................................................... 75 3.3.9 Parasite cultures ........................................................................................... 76 3.3.10 Thawing of frozen parasites ...................................................................... 78 3.3.11 Gelatin purification .................................................................................... 78 3.3.12 Magnetic activated cell sorting (MACS) purification ............................... 79 3.3.13 Parasite selection for surface reactivity ..................................................... 80 3.3.14 IEs Surface reactivity to specific antibodies by flow cytometry ............... 82 3.3.15 Inhibition of IEs in flow-based adhesion assays ....................................... 82 3.3.16 Immunofluorescence assay ........................................................................ 84 3.3.17 Bioinformatics analysis ............................................................................. 84 3.3.18 Data analysis .............................................................................................. 85 3.4 Results ................................................................................................................ 85 ix 3.4.1 Binding to ICAM-1 by DBLβ hybrid protein containing ICAM-1 binding motif...................................................................................................................... 85 3.4.2 Adhesion of IEs expressing motif-containing ICAM-1 binding DBLβ to ICAM-1 ................................................................................................................ 86 3.4.3 Binding of ICAM-1 to recombinant DBLβ containing ICAM-1 binding motif predicted from sequence analysis ............................................................... 87 3.4.4 Phylogenetic analysis of ICAM-1 and non-ICAM-1 binding DBLβ domains .............................................................................................................................. 89 3.4.5 Cross-reactive adhesion inhibition by antibodies raised against motif- containing DBLβ motif-peptides .......................................................................... 90 3.4.6 Motif-encoding var transcript levels in SM patients ................................... 93 3.4.7 Selection of parasites expressing PfEMP1 domains predicted to bind both EPCR and ICAM-1............................................................................................... 95 3.4.8 Adhesion of selected IEs to immobilised recombinant ICAM-1 and EPCR receptors................................................................................................................ 97 3.5 Discussion ........................................................................................................ 103 CHAPTER FOUR ...................................................................................................... 108 ANTIBODY RESPONSES TO PFEMP1 LINKED WITH THE DEVELOPMENT OF PAEDIATRIC CM ........................................................................................... 108 4.1 Introduction ...................................................................................................... 108 4.2 Hypothesis and specific objectives................................................................... 109 4.3 Methods ............................................................................................................ 110 4.3.1 Study demographics .................................................................................. 110 4.3.2 Patient samples and ethics ......................................................................... 111 4.3.3 Recombinant proteins ................................................................................ 111 4.3.4 DBLβ-specific antibodies and purification of motif-specific antibodies .. 112 4.3.5 ELISA ........................................................................................................ 113 4.3.6 Plasmodium falciparum parasite culture ................................................... 115 4.3.7 ICAM-1 Adhesion-Inhibition of IE under physiological flow conditions 116 4.3.8 Immunofluorescence assay ........................................................................ 117 4.3.9 Data analysis. ............................................................................................. 117 4.4 Results .............................................................................................................. 118 4.4.1 Characteristics of patients for the antibody reactivity profile ................... 118 4.4.2 Plasma antibody responses to DBLβ domains of group A PfEMP1 ......... 120 4.4.3 Antibody responses determined by age and disease severity .................... 121 x 4.4.4 ICAM-1 binding-inhibition by antibodies against motif-containing DBLβ domains............................................................................................................... 123 4.4.5 ICAM-1 adhesion-inhibition of recombinant motif domains and P. falciparum isolates by motif-specific peptide antibodies ................................... 127 4.5 Discussion ........................................................................................................ 131 CHAPTER FIVE ....................................................................................................... 136 MARKERS OF INFLAMMATION AND ENDOTHELIAL ACTIVATION IN GHANAIAN CHILDREN WITH MALARIA ...................................................... 136 5.1 Introduction ...................................................................................................... 136 5.2 Hypothesis and specific objectives................................................................... 138 5.3 Methods ............................................................................................................ 138 5.3.1 Study outline .............................................................................................. 138 5.3.2 Sample collection and processing.............................................................. 139 5.3.3 ELISA for markers of Inflammation/endothelial activation...................... 139 5.3.3.1 Quantitative determination of sICAM-1 in human plasma samples ...... 140 5.3.3.2 Quantitative determination of sEPCR in human plasma samples .......... 140 5.3.3.3 Measurement of Ang-2 concentration in human plasma samples .......... 141 5.3.3.4 Measurement of HMGB1 concentration in human plasma samples ...... 142 5.3.4 Data analysis .............................................................................................. 142 5.4 Results .............................................................................................................. 143 5.4.1 Baseline information on study groups ....................................................... 143 5.4.2 Soluble ICAM-1 (sICAM-1) levels ........................................................... 145 5.4.3 Soluble EPCR (sEPCR) levels .................................................................. 146 5.4.4 Plasma Ang-2 levels .................................................................................. 147 5.4.5 Plasma HMGB1 levels .............................................................................. 148 5.5 Discussion ........................................................................................................ 149 CHAPTER SIX .......................................................................................................... 158 CHARACTERISATION OF BLOOD OUTGROWTH ENDOTHELIAL CELL (BOEC) IN VITRO SYSTEM AND ITS POTENTIAL USE TO STUDY THE ADHESION PROPERTIES OF PLASMODIUM FALCIPARUM-INFECTED ERYTHROCYTES ................................................................................................ 158 6.1 Introduction ...................................................................................................... 158 6.2 Hypothesis and specific objectives................................................................... 161 6.3 Methods ............................................................................................................ 162 xi 6.3.1 Sample collection and processing and isolation of peripheral blood mononuclear cells ............................................................................................... 162 6.3.2 Thawing of PBMCs ................................................................................... 163 6.3.3 Preparation of rat tail collagen type I-coated tissue culture surfaces ........ 163 6.3.4 PBMCs and BOEC cultures ...................................................................... 164 6.3.5 Passage of cells .......................................................................................... 164 6.3.6 Freezing of BOECs.................................................................................... 165 6.3.7 Thawing of BOECs ................................................................................... 165 6.3.8 Phenotypic assessment of BOECs ............................................................. 165 6.3.9 Analysis ..................................................................................................... 166 6.4 Results .............................................................................................................. 167 6.4.1 Patients‟ demographics .............................................................................. 167 6.4.2 Success rate of BOEC colony establishment and morphological characterisation ................................................................................................... 167 6.4.3 Phenotypic assessment of resting and activated BOECs ........................... 170 6.5 Discussion ........................................................................................................ 172 CHAPTER SEVEN ................................................................................................... 179 FREQUENCY OF IGM BINDING PFEMP1 VARIANTS IN THE GENOME OF P. FALCIPARUM ................................................................................................... 179 7.1 Introduction ...................................................................................................... 179 7.2 Hypothesis and specific objectives................................................................... 181 7.3 Methods ............................................................................................................ 181 7.3.1 Parasite culture .......................................................................................... 181 7.3.2 IE selection and single-cell cloning of IgM-binding IEs ........................... 182 7.3.3 Repeated selection of IgM positive clones ................................................ 183 7.3.4 TRizol samples and var gene transcription analysis ................................. 184 7.3.5 IgM reactivity to the surface of IEs ........................................................... 184 7.3.6 Data analysis .............................................................................................. 185 7.4 Results .............................................................................................................. 185 7.4.1 var transcription profile of NF54 G6 IEs propagated with or without blasticidin............................................................................................................ 185 7.4.2 IgM selection and var gene profile of IgM positive IEs............................ 187 7.4.3 Binding of IgM positive PfEMP1 IEs ....................................................... 191 7.5 Discussion ........................................................................................................ 192 xii CHAPTER EIGHT .................................................................................................... 196 GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ... 196 8.1 General discussion............................................................................................ 196 8.2 Conclusions ...................................................................................................... 205 8.3 Recommendations ............................................................................................ 206 ReferenceS ................................................................................................................. 208 AppendiCES .............................................................................................................. 255 Appendix A ............................................................................................................ 255 Appendix B............................................................................................................. 256 Appendix C............................................................................................................. 262 Appendix D ............................................................................................................ 263 Appendix E ............................................................................................................. 264 xiii LIST OF FIGURES Figure 2.1: Changes in P. falciparum prevalence in 2-10 year olds in African countries. ...................................................................................................................... 10 Figure 2.2: The life cycle of P. falciparum. ................................................................. 16 Figure 2.3:The link between age and malaria severity. ............................................... 23 Figure 2.4: Cytoadhesion phenotypes in P. falciparum-associated SM pathogenesis. 25 Figure 2.5: Plasmodium falciparum var genes chromosomal organisation and PfEMP1 domain structure........................................................................................................... 29 Figure 2.6: Var gene regulation in P. falciparum. ....................................................... 31 Figure 2.7: A possible mechanism of ICAM-1 and EPCR cooperation in CM pathogenesis. ................................................................................................................ 51 Figure 3.1: Domain structural organisation of PfEMP1 proteins containing DC4. ..... 63 Figure 3.2: Phylogenetic tree of 16 ICAM-1 binding (red) and 19 non-ICAM-1 binding (blue) DBLβ domains. .................................................................................... 65 Figure 3.3: Identification of the ICAM-1 binding region in PFD1235w DBLβ3_D4. 66 Figure 3.4: ICAM-1 binding of predicted DBLβ-containing ICAM-1 binding motif. 88 Figure 3.5: Phylogenetic tree of ICAM-1 binding and non-ICAM-1 binding DBLβ. . 90 Figure 3.6: Binding inhibition of ICAM-1 to motif-containing PfEMP1 by plasma antibodies. .................................................................................................................... 92 Figure 3.7: var/PfEMP1 expression of HB3, IT4 and 3D7 IEs. .................................. 96 Figure 3.8: Binding of P. falciparum laboratory isolates to recombinant protein receptors. ...................................................................................................................... 99 Figure 3.9: Binding of P. falciparum laboratory isolates to HBMEC under flow conditions. .................................................................................................................. 102 Figure 4.1: Plasma antibody reactivity to P. falciparum DBLβ domains. ................. 122 Figure 4.2: Inhibition of ICAM-1 binding by antibodies raised against motif- containing DBLβ domains. ........................................................................................ 125 Figure 4.3: Inhibition of ICAM-1 binding to P. falciparum lab isolates by antibodies raised against motif DBLβ domains. ......................................................................... 127 Figure 4.4: Inhibition of ICAM-1 binding by motif peptide-specific antibodies to motif ........................................................................................................................... 128 Figure 4.5: Adhesion inhibition of ICAM-1 binding of P. falciparum parasites by motif peptide-specific antibodies. .............................................................................. 130 Figure 5.1: Median sICAM-1 levels among study groups. ........................................ 145 Figure 5.2: Median sEPCR levels among study groups............................................. 146 Figure 5.3: Median plasma Ang-2 levels among study groups.................................. 147 Figure 5.4: Median plasma HMGB1 levels among study groups. ............................. 148 Figure 6.1: Morphology of expanded BOEC cultures in resting (A) and activated (B) states. .......................................................................................................................... 169 Figure 6.2: Characteristics of cell surface antigens. .................................................. 171 Figure 7.1: var gene transcription profile of 3D7/NF54 with or without blasticidin. 187 Figure 7.2: var gene profiling of IgM positive P. falciparum NF54 following subcloning and in vitro expansion. ............................................................................ 189 xiv Figure 7.3: Schematic of the extracellular domain organisation of the PfEMP1 proteins encoded by major var gene transcripts in identified subclonal IEs. ............ 190 xv LIST OF TABLES Table 3.1: Data on the study participants used for P. falciparum var gene expression experiments .................................................................................................................. 94 Table 3.2: Var subtypes transcript levels in children admitted with malaria............... 94 Table 4.1: General characteristics of patients related to figure 4.1............................ 119 Table 5.1: Baseline clinical profile and the levels of soluble markers of inflammation and endothelial activation. ......................................................................................... 144 Table 6.1: Patients profile and success rate of BOEC colony generation.................. 169 Table 6.2: Flow cytometry assessment of BOEC cells .............................................. 172 xvi LIST OF ABBREVIATIONS Abs Antibodies ACT Artemisinin-based combination therapy Ang Angiopoietin ANOVA Analysis of variance APC Activated protein C BCS Blantyre coma score BOEC Blood outgrowth endothelial cells BSA Bovine serum albumin BSD Blasticidin S deaminase CC Community controls CD Cluster of differentiation cDNA Complementary deoxyribonucleic acid CHO Chinese hamster ovary CIDR Cysteine-rich interdomain region CM Cerebral malaria CSA Chondroitin sulphate A Ct Cycle threshold DAPI 4‟,6- diamidino-2-phenylindole dihydrochloride DBL Duffy-binding like DC Domain cassette DEPC Diethylpyrocarbonate DPBS Dulbecco‟s phosphate buffered saline E. coli Escherichia coli EC Endothelial cell ECFC Endothelial colony forming cells EDTA Ethylenediaminetetracetic acid EGM-2 Endothelial cell growth medium-2 xvii ELISA Enzyme -inked immunosorbent assay EPCR Endothelial protein C receptor FBS Foetal bovine serum 2 % FBS 2 % foetal bovine serum in phosphate buffered saline Fc Fragment crystallisable (tail) region of an immunoglobulin FC Febrile controls FITC Fluorescein isothiocyanate GLURP Glutamate-rich protein GLURP-R2 C-terminal repeat region-2 of GLURP H Hour (s) H2O Water H2O2 Hydrogen peroxide HRP Horse radish peroxidase H2SO4 Sulphuric acid H3K4me2/3 Histone 3 lysine 4 bi and tri-methylation Hb Haemoglobin HBEC5i Human brain endothelial cells HBMEC Human brain microvascular endothelial Cells HCl Hydrochloric acid HDMEC Human eermal microvascular endothelial cells HEK Human embryonic kidney His Histidine HMGB1 High mobility group box 1 ICAM-1 Intercellular adhesion molecule-1 125I Iodine-125 IE Infected erythrocytes IFNγ Interferon gamma IgG Immunoglobulin G IgG-HRP Horseradish peroxidase conjugated to Immunoglobulin G xviii IgM Immunoglobulin M IL Interleukin IPTp Intermittent preventive therapy in pregnancy LFA-1 Lymphocyte function-associated antigen-1 LLIN Long-lasting insecticide-treated nets Mac1 Macrophage-1 antigen MACS Magnetic activated cell sorting MFI Mean fluorescent intensity Min Minute MOH Ministry of Health MSP2 Merozoite surface protein-2 NaCl Sodium chloride NTS N-terminal segment OD Optical density OM Other malaria OPD Out-patients department PAM Pregnancy-associated malaria PAR1 Protease-activated receptor 1 PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline PECAM-1 Platelet endothelial cell adhesion molecule 1 PfEMP1 Plasmodium falciparum erythrocyte membrane protein 1 PfHP1 P. falciparum heterochromatin protein 1 PfRNAse II P. falciparum exoribonuclease II PfSETvs P. falciparum variant-silencing SET gene qPCR Quantitative real-time polymerase chain reaction rCD36 Recombinant cluster of differentiation 36 rEPCR Recombinant endothelial protein C receptor rICAM-1 Recombinant intercellular adhesion molecule-1 xix RDT Rapid diagnostic test RIF/RIFIN Repetitive interspersed family RNA Ribonucleic acid pol II Polymerase enzyme II RPMI Roswell Park Memorial Institute S Second (s) SA Severe anaemia SD Standard deviation SEM Standard error of mean sEPCR soluble Endothelial protein C receptor sICAM-1 soluble Intercellular adhesion molecule-1 SIR2 Silent Information Regulator 2 SM Severe malaria STEVOR Sub-telomeric variable open reading frame Temp Temperature TM Thrombomodulin TNF-α Tumour necrosis factor alpha Tris Tris(hydroxymethyl)aminomethane Tu Transcript units UK United Kingdom UM Uncomplicated malaria Ups Upstream promoter sequence USA United States of America VSA Variant surface antigens ` vWF von Willebrand factor WHO World Health Organisation Wk Week xx CHAPTER ONE INTRODUCTION 1.1 Background Plasmodium falciparum causing malaria is largely responsible for most childhood malaria-related deaths and morbidity. Severe falciparum malaria manifests as three overlapping syndromes of severe anaemia (SA), cerebral malaria (CM) and respiratory distress. By far, CM is the most lethal disease outcome with a fatality rate of about 20 % even after adherence to effective anti-malarial treatments (Birbeck et al., 2010). A minority of CM survivours are also known to sustain variable degrees of neurocognitive disorders (reviewed in Hviid and Jensen, 2015; Birbeck et al., 2010). The mechanisms underlying the development of CM remain poorly elucidated but are believed to involve cytoadhesion of the P. falciparum virulent protein, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) to human endothelial receptors such as intercellular adhesion molecule-1 (ICAM-1) and endothelial protein C receptor (EPCR), alterations in inflammatory cytokines and endothelial activation (reviewed in Storm and Craig, 2014; Cunnington et al., 2013b). The general objectives of this study were to investigate the role of an ICAM-1 binding motif in PfEMP1, in the development of CM, as well as evaluate the contributions of the immune response and other determinants involved in severe disease. 1 1.2 Objective 1: The involvement of an ICAM-1 binding PfEMP-1 motif in CM Cytoadhesion of infected erythrocytes (IEs) to receptors on the host endothelium is a prominently recognised pathological feature in CM development. The parasite protein thought to facilitate this adhesion process is PfEMP1, which is coded by var genes in a mutually exclusive order, resulting in the exposure of one or few PfEMP1 molecules at a time. Their expression can be altered at any given time to avoid immune recognition and enhance parasite survival (reviewed in Hviid and Jensen, 2015). PfEMP1 variants are generally classified into groups A-E and two hybrid groups (B/A, B/C) depending on their chromosomal location or into domain cassettes (DC) which involve particular arrangements of the adhesion domains of Duffy-binding like proteins (DBL) and cysteine-rich interdomain region proteins [CIDR] (Rask et al., 2010; Lavstsen et al., 2003). There is strong evidence that links the expression of group A and group B/A PfEMP1, including those that contain DC4, DC8 and DC13 to SM, while group C PfEMP1 is largely confined to uncomplicated malaria [UM] (Bengtsson et al., 2013b; Lavstsen et al., 2012; Rottmann et al., 2006; Jensen et al., 2004). With respect to the human host, a variety of receptors are well known to interact with PfEMP1, although there is no clear-cut evidence of the involvement of particular receptor(s) in CM. ICAM-1 expressed on endothelial cells in the microvasculature is believed to serve as a receptor for adhesion of IEs during CM pathogenesis. Immunopathological evaluations of autopsy samples of CM patients reported increased endothelial ICAM-1 expression together with retained IEs in the brain microvasculature (Armah et al., 2005; Turner et al., 1998; Turner et al., 1994). However, the role of ICAM-1 in CM has not always been consistent. Notably, IEs that bind to ICAM-1 express DBLβ domains of groups B and C PfEMP1 that can also mediate CD36 binding which are 2 associated with UM; in addition, group A PfEMP1 expressing IEs linked to CM are also known to bind to ICAM-1. Moreover, IEs selected on human brain endothelial cells expressing PfEMP1 of DC8 and DC13 do not bind ICAM-1 but EPCR through their CIDRα domain (Turner et al., 2013). Altogether suggesting that, group A PfEMP1 involved in CM may not preferentially bind ICAM-1 providing a weak evidence for the role of ICAM-1 in the development of CM. Thus Bengtsson et al (2013) sought to clarify the role of group A PfEMP1 ICAM-1 binding in CM. The authors identified a group of 3D7 group A PfEMP1 PFD1235w (PlasmoDB: PF3D7_0425800) orthologous proteins in parasite isolates from falciparum-infected Ghanaian children. This group of proteins possessed a highly similar N-terminal three tandem domain (DBLα1.1/1.4-CIDRα1.6- DBLβ3) which was identified as domain cassette 4 (DC4). IEs selected to express DC4 PfEMP1 adhered to ICAM-1 and not CD36, a phenotype linked to UM and the binding capacity mapped to the C-terminal portion of the DBLβ3 domain. Indeed, DBLβ3 domain-specific antibodies acquired during natural infections interfered with ICAM-1 binding as well as cross-reactive of ICAM-1 binding across different parasite genomes containing DC4. Further experiments resulted in the characterisation of a conserved ICAM-1 binding motif within the DBLβ3 region. The CIDRα subtype in DC4 was also found to be present in the EPCR binding PfEMP1 of DC13, suggesting the potential of IEs with such domains to bind two different endothelial receptors. Although Bengtsson et al (2013b) used laboratory-adapted parasite lines and very few field isolates, the findings demonstrated that parasites harbouring this ICAM-1 binding motif may be relevant for vaccine development that inhibits malaria parasite sequestration in the brain. Therefore, it is imperative to investigate the widespread occurrence of parasites expressing PfEMP1 containing the ICAM-1 binding motif 3 predicted to cause CM in field isolates, demonstrate their ability to bind to ICAM-1 and link this binding to clinical disease. In addition, it is important to show the ability of motif-specific antibodies to inhibit ICAM-1 binding, which offers clinical protection. The aim of this study was to dissect further the molecular details characterising ICAM-1 binding by group A PfEMP1 by using sequence information to define a sequence motif that can be used to identify ICAM-1-binding DBLβ domains. Additionally, the relationship between the expression of DBLβ domains involved in ICAM-1 binding and CM development was investigated. Hypothesis 1: Plasmodium falciparum IEs associated with CM express a particular subset of group A PfEMP1 that bind to both ICAM-1 and EPCR and contain specific amino acid sequences (motif) that mediates the binding. Specific objective 1.1: To assess the transcription levels of motif-encoding var genes and EPCR binding CIDRα domains in parasites obtained from defined categories of malaria patients. Specific objective 1.2: To assess the binding of P. falciparum IE to ICAM-1 and EPCR under physiological flow conditions Specific objective 1.3: To measure motif-specific IgG in plasma of children with acute malaria. 4 1.3 Objective 2: The involvement of host immune responses in malaria There is overwhelming data showing that a robust immune response is evoked during P. falciparum infection. The immune response involves a delicate compromise between pro- and anti-inflammatory cytokines which are important for the control of parasite multiplication and ultimate clearance of infection (reviewed in Crompton et al., 2014). The collapse of this balance has been consistently linked to the events that characterise SM pathogenesis. Indeed many studies report elevated levels of pro- inflammatory cytokines in children with SM relative to UM patients or an increase in the proportion of pro- to anti-inflammatory cytokines (Dodoo et al., 2002; Kurtzhals et al., 1998; Kwiatkowski et al., 1990). The joint actions of parasite sequestration and excessive host inflammatory response are involved in the self-amplification of SM pathology resulting from widespread activation of the endothelium and release of activation markers. Endothelial activation and inflammation result in the upregulation of constitutively expressed endothelial ligands, which in-turn promotes massive sequestration leading to a compromise in the vascular integrity (Abdi et al., 2014). In this study, biomarkers of inflammation and endothelial activation were measured in paediatric malaria patients and their levels following disease resolution were evaluated. Hypothesis 2: Higher levels of endothelial markers of activation are associated with SM Specific objective 2.1: To assess plasma for biomarkers of activation and inflammation in acute and convalescent children with either UM or SM. 5 1.4 Objective 3: Establishment of a blood outgrowth endothelial cell in vitro system and its potential use to study P. falciparum cytoadhesion properties Studies on the interactions between malaria parasites and receptors on the host endothelium have largely relied on postmortem samples, murine or in vitro models of non-human origin. Although these studies have provided incredible insights, they have peculiar challenges (reviewed in White et al., 2010; Andrews et al., 2005). Thus, there is an ever-growing interest to establish more in vitro models to unravel the molecular details of cytoadhesion which may offer anti-adhesion therapies to complement existing treatment. Blood outgrowth endothelial cells (BOECs) represent an alternate in vitro tool to study parasite binding characteristics since it has the capacity to express diverse malaria parasite adhesion receptors. Moreover, they are an easily accessible source of material present in peripheral blood mononuclear cells (PBMCs) and are culture adaptable with a very high proliferative potential (Lin et al., 2000). They can be derived from malaria patients and hence serve as physiologically relevant cells to study parasite adhesion obtained from patients. Hypothesis 3: Cultures of blood outgrowth endothelial cells (BOEC) can be established from small volumes of blood from children. Specific objective 3.1: To isolate and characterise BOECs obtained from PBMCs of malaria patients. 6 1.5 Objective 4: Identification of PfEMP1 mediated IgM binding variants in P. falciparum genome Apart from mediating adhesion to endothelial receptors, PfEMP1 variants are known to interact with components found in human serum, particularly non-immune IgM. This particular interaction has been observed in some IEs involved in rosetting and IEs binding to chondroitin sulphate A (CSA) in pregnancy-associated malaria (PAM), both phenotypes being linked with SM development (Creasey et al., 2003; Rowe et al., 2002). Although the physiological relevance of IgM binding is not well understood, it is believed that this interaction together with other serum factors reinforces the relatively weak interactions between IEs expressing PfEMP1 and their receptors on erythrocytes, thereby promoting disease severity (reviewed in Pleass et al., 2016). Recent data show that multiple PfEMP1 variants in a given parasite genome have the capacity to bind IgM. Indeed, Jeppesen et al (2015) provided evidence of at least five IgM-binding PfEMP1 subtypes in the genome of P. falciparum NF54. There remains a possibility of identifying more IgM-binding PfEMP1 within a parasite genome and it is pertinent to investigate this given the clinical importance of IgM binding. Hypothesis 4: The P. falciparum NF54/3D7 genome encodes several uncharacterised IgM-binding PfEMP1 variants. Specific objective 4.1: To identify PfEMP1 variants involved in IgM binding in the NF54/3D7 parasite. 7 CHAPTER TWO LITERATURE REVIEW 2.1 Global burden and strategies Malaria burden is difficult to estimate, notably in low-income countries where data collection and reporting quality are significant challenges. Nevertheless, the World Health Organisation (WHO) reported that about 3.2 billion people in 91 countries worldwide were in danger of being infected with malaria parasites while 1.2 billion of them were at high risk of developing malaria in the year 2015 (WHO, 2016). Approximately 200 million people were infected with malaria parasites globally, of which about 429,000 of them died. The vast majority of deaths (92 %) occurred in Sub-Saharan Africa, with the heaviest toll being children under the age of five and pregnant women (WHO, 2016), portraying malaria as one of the world‟s greatest human scourge. Despite these alarming figures, incredible efforts have been made to reduce the global burden of malaria and this has resulted in an approximately 21 % significant reduction in malaria cases between 2010 and 2015 (WHO, 2016). In Africa alone, the number of malaria-infected cases in children between two and 10 years old has reduced by approximately 50 %, from 2000 to 2015 (Bhatt et al., 2015; Figure 2.1). These interventions have included rapid diagnostic tests (RDT), artemisinin-based combination therapy (ACT), intermittent preventative treatment (IPT) and long-lasting insecticide-treated bed nets (LLIN) (reviewed in Alonso and Tanner, 2013; Flaxman et al., 2010). More recently, the WHO has introduced a new initiative, entitled “T3: Test, Treat and Track” a programme which entreats malaria endemic countries to provide widespread access to diagnostic testing and treat people only after they have 8 been confirmed malaria positive in order to establish a robust monitoring and control system (Bhutta et al., 2014). Although evidence shows that the implementation of these interventions are effective and proving successful, the parasites, as well as their mosquito hosts, have developed formidable strategies to thwart malaria control efforts. For instance, drug resistance to artemisinin, the only potent antimalarial available has emerged in the Greater Mekong region in Southeast Asia (Dondorp et al., 2009; Noedl et al., 2008), with significant threats to India (Tun et al., 2015); with most researchers concerned that it might spread to Africa and other endemic countries soon. Meanwhile, the transmitting Anopheles mosquitoes have developed resistance to pyrethroid insecticides (Knox et al., 2014). In addition, a switch in mosquito behaviour has been reported in Tanzania where mosquitoes have altered their feeding habits from night to day, smartly evading the protection offered by insecticide-treated bed nets (Russell et al., 2011). Indeed, the overwhelming burden caused by malaria for individuals and governments of endemic countries cannot be overemphasized as the disease is closely linked to hampered socio-economic development and poverty. Some notable factors accounting for lower rates of economic growth include effects of the disease on fertility, population growth, worker productivity, absenteeism, premature death and medical costs. Together, these factors worsen the plight of these endemic countries that are already considered as low income, further deepening their gap with developed countries (Nonvignon et al., 2016; Orem et al., 2012; Sachs and Malaney, 2002). It is therefore obvious that additional tools such as more effective drugs and vaccines to complement existing interventions will remain vital for sustainable malaria control and a global health priority. Massive efforts have been made for several decades to develop a malaria vaccine but the promise of a highly effective vaccine has remained 9 elusive. The only vaccine, RTS,S which is yet to be deployed is only partially effective, conferring a fleeting protection against clinical malaria. Significantly, when trials were conducted in differing malaria transmission settings in Africa among children between 5-17 months, the vaccine reduced clinical malaria by about 39 %, with absolutely no protection conferred against SM among children who did not receive the fourth dosage of the vaccine (RTS,S, 2015). Thus, it does not represent the true „gold standard‟ vaccine against malaria. Clearly, an in-depth understanding of the intricate immunity to malaria will undoubtedly be critical to the development of a vaccine, even if it offers protection against severe disease. Figure 2.1: Changes in P. falciparum prevalence in 2-10 year olds in African countries. (a) The prevalence of P. falciparum in 2000 (b) P. falciparum prevalence in 2015. Gray regions represent P. falciparum free regions and blue zones areas with a low prevalence. Red spots are regions with the highest prevalence (Modified from Bhatt et al., 2015). 10 2.1.1 Malaria in Ghana Ghana lies within the coastal belt of West Africa, bordered by Burkina Faso to the north, the Atlantic Ocean to the south, Cote d‟Ivoire and Togo to the west and east respectively. In Ghana, everyone is at risk of getting infected with malaria parasites. The public health and economic burden of malaria in Ghana cannot be overemphasized as it remains one of the leading causes of morbidity and mortality. In 2013, malaria accounted for approximately 44 % of outpatient visits and 22.3 % of under-five mortality (MOH, 2014a). Reports show that it remains one of the most important causes of total work days lost and an estimated total cost of malaria to businesses is about US$ 6.5 million, with direct costs accounting for 90 % (Nonvignon et al., 2016). Malaria transmission in Ghana is intense, with peaks in the major wet season (April to July). Plasmodium falciparum is the predominant cause of malaria, transmitted by four major mosquito vectors: Anopheles gambiae (sensu stricto), A. melas, A. arabiensis and A. funestus (sensu stricto) (Duah et al., 2016). With regards to disease interventions, Ghana officially adopted the use of ACTs as the first choice of antimalarial drugs for the treatment of uncomplicated malaria in 2005 (Koram et al., 2005). Currently, the national malaria control programme of Ghana recommends artesunate–amodiaquine as the first line of treatment based on its efficacy, cost effectiveness and impact on the local industry, with artemether- lumefantrine and dihydroartemisinin-piperaquine as alternatives for those who cannot tolerate the reactions sometimes associated with artesunate–amodiaquine (Quashie et al., 2013; Koram et al., 2005). Complementary vector control strategies include LLINs and indoor residual spraying (MOH, 2014b). 11 2.2 Malaria parasites Malaria is an infectious disease caused by the eukaryotic intracellular protozoan parasite belonging the kingdom Protista, phylum Apicomplexa, class Sporozoa, order Eucoccidia and genus Plasmodium. Plasmodium contains three genomes: a 23 Mb nuclear genome made up of 14 linear chromosomes, a very small linear mitochondrial genome and a 35 kb circular genome of red-algal ancestry found in the apicoplast (reviewed in Antinori et al., 2012). Sequence data reveal about 5,365 genes present on the 14 chromosomes, of which known functions have been assigned to approximately 1,800 genes (Gardner et al., 2002). Over 100 Plasmodium species infecting a diverse range of definitive hosts including reptiles, birds and mammals have been identified and out of these, four distinct parasite species are well adapted and known to traditionally infect humans: Plasmodium falciparum, P. malariae, P. ovale and P. vivax (reviewed in Miller et al., 2002). Either by natural or accidental means human beings occasionally can be infected with parasites from simian species such as P. knowlesi and P. cynomolgi. Plasmodium knowlesi has however emerged as an important cause of human malaria in South-East Asia and has therefore been considered a fifth human malaria parasite (reviewed in Antinori et al., 2012). The current geographical distribution of Plasmodium species infecting humans shows P. falciparum as being endemic in tropical Africa, while P. vivax is more prevalent in South America compared to P. falciparum infections. Both P. falciparum and P. vivax are predominant in south-eastern Asia and western Pacific. Plasmodium malariae may occur in all malaria endemic areas however, its distribution is generally low. Mixed infections of P. falciparum and P. malariae are sometimes reported in the tropics of Africa. Plasmodium ovale is also infrequently observed in tropical Africa while P. knowlesi infection is concentrated in some forest zones of South-East Asia (reviewed 12 in Autino et al., 2012). Plasmodium falciparum remains the most lethal of all the infecting species accounting for more than 90 % of the world‟s malaria mortality (WHO, 2016; Snow et al., 2005) as such, most research has obviously focused more on P. falciparum. In recent times, P. vivax research has attracted a lot of interest largely due to its widespread geographic nature, and also increasing awareness of its involvement in severe disease and death which was previously thought to be benign (Naing et al., 2014; Baird, 2013). 2.3 Life cycle of human Plasmodium parasites Malaria is transmitted from person to person through the bite of infected female Anopheline mosquitoes. There are over 435 identified species of Anopheles mosquitoes, of which 30-40 are vectors of major importance transmitting malaria parasites. In sub-Saharan Africa, Anopheles gambiae, A. funestus and A. arabiensis predominantly transmit malaria (reviewed in Cholewinski et al., 2015; Sinka et al., 2010). The human malaria parasite naturally inhabits two different hosts for successful completion of its life cycle. One-half of the cycle takes place in the female Anopheles mosquito, depicted by a sexual multiplication phase (sporogony) while the other half, characterised by an asexual phase (schizogony) occurs in the mammalian host (reviewed in Antinori et al., 2012). A schematic representation of the life cycle is shown in figure 2.2. Human infection begins when Plasmodium sporozoites resident in the salivary gland of the infected Anopheles mosquito are injected into the skin of the human host along with anticoagulant-containing saliva to facilitate ingestion of blood. A proportion of sporozoites deposited in the skin vigorously migrate until they encounter a blood 13 vessel and enter the blood circulation. They rapidly invade hepatocytes and reside in a parasitophorous vacuole. This marks the onset of the parasite‟s liver-stage (pre- erythrocytic schizogony) development, lasting between two and 16 days depending on the species and also usually asymptomatic in patients. Sporozoites differentiate into liver stage schizonts within the parasitophorous vacuole, followed by further growth and multiple asexual nuclei divisions into thousands of newly formed merozoites. In P. falciparum, thousands of daughter merozoites can be produced within a week in a hepatic schizont; by contrast, P. vivax and P. ovale parasites can persist for a long period in the liver as dormant hypnozoites without any clinical presentation, which can be reactivated after weeks or even years later to cause relapses by invading the blood stream (Siciliano and Alano, 2015; Mota and Rodriguez, 2008; Silvie et al., 2008; Amino et al., 2006). Once the hepatic schizonts burst, released merozoites in circulation initiate the erythrocytic stage by infecting erythrocytes which are the primary sites of infection in malaria. The duration of the erythrocytic stage varies, depending on the transmitted species; it takes 48 h for P. falciparum, P. vivax and P. ovale, 72 h for P. malariae and 24 h for P. knowlesi (White et al., 2014). Asexual division commences with parasites resident in a parasitophorous vacuole in erythrocytes. They transform from the ring stage followed by a growth phase leading to the formation of a trophozoite. There are multiple rounds of nuclear divisions resulting in the formation of schizonts, the final stage of asexual development in the human host. When schizonts mature, they rupture the host erythrocyte, releasing merozoites that rapidly invade new erythrocytes in circulation, continuing the asexual life cycle. The cyclic invasion and synchronous lysis of erythrocytes result in the release of parasites and erythrocytic material into circulation which triggers the production of pro-inflammatory cytokines which are responsible for the majority of 14 the clinical manifestations observed in a malaria patient (Tuteja, 2007). A small proportion of the merozoites diverge from the schizogony pathway to an alternate route of sexual commitment. Thus, these merozoites differentiate to produce male (micro) and female (macro) gametocytes, an action triggered by poorly elucidated mechanisms. These sexual erythrocytic stages have no further activity within the human host and are essential for transmitting the infection to Anopheles mosquitoes. A mosquito taking a blood meal on an infected individual may ingest these gametocytes into its midgut, where macrogametocytes form macrogametes and exflagellation of microgametocytes produce microgametes. These gametes fuse to form a diploid zygote that further transforms into a motile ookinete and penetrates the cell wall in the midgut to develop into an oocyst. Several mitotic divisions within the oocyst produce many sporozoites and when the oocyst ruptures, the released sporozoites migrate and localise in the salivary glands waiting to be transmitted to another host during the next blood meal (Tuteja, 2007). 15 Figure 2.2: The life cycle of P. falciparum. The asexual part of the cycle occurs in humans and it involves a liver stage and a blood stage. The sexual stage occurs in the mosquito. Injected sporozoites from the Anopheles mosquito invade hepatocytes and develop into merozoites which are released into the bloodstream to invade erythrocytes. Development in the erythrocytes results in schizonts which are released to invade new erythrocytes. Some merozoites develop into sexual gametocytes which are picked up by the mosquito. Fusion of gametes forms a zygote which finally produces many sporozoites to continue the life cycle (Adapted from Cowman et al., 2012). 16 2.4 Plasmodium falciparum disease manifestations The clinical presentations of malaria are diverse and overlapping, with a wide spectrum of disease severity which includes asymptomatic infection, mild, uncomplicated malaria and severe, life-threatening malaria. Clinical presentation and outcome appear to be determined by a vast number of factors including host immunity, virulence of the parasite, host age, as well as geographical and social factors (Crawley et al., 2010; Miller et al., 2002). Asymptomatic infection is highly prevalent and mostly associated with immune individuals. Such individuals have persistently low parasite densities with no clinical symptoms observed (Hamad et al., 2000). Disease pathogenesis is largely due to the multiplication of the asexual blood stage parasites since the liver stage is well known to be asymptomatic. Initial clinical signs of infection are nonspecific and typically mimic many of the diseases that are fever or flu related. They include fever, chills, headaches, nausea and malaise, which begin to appear about a week after inoculation of parasite sporozoites into the host. These symptoms generally coincide with the periodic lysis of infected erythrocytes containing matured parasites into circulation. These early classical symptoms are mostly observed in uncomplicated infections which is a common clinical syndrome in a majority of people and they usually resolve rapidly with the aid of a robust host immune system (reviewed in Storm and Craig, 2014). However, a relatively few number of infections (about 1.0 %) if not timely treated do progress to severe, life-threatening disease as well as death through unexplained mechanisms (reviewed in Storm and Craig, 2014). SM (SM) is notably manifested in non-immune patients, young children from highly endemic locations, adults in low endemic regions and people with limited acquired immunity like primigravida (Storm and Craig, 2014; Hviid, 2010). Children under five years of age are most susceptible 17 to SM. Indeed, about 10-20 % of children are highly likely to suffer from severe malaria before the inception of acquired immunity in intense P. falciparum transmission areas and approximately 70 % of children may die due to malaria infection (WHO, 2016; Goncalves et al., 2014). It is a complex disease that may affect multiple organs and encompasses one or more of the following complications: SA, metabolic acidosis and CM, thus hampering exact diagnosis and disease classification (Goncalves et al., 2014; Storm and Craig, 2014; Idro et al., 2005; Mackintosh et al., 2004). 2.4.1 Severe anaemia Severe malaria anaemia is largely responsible for most malaria-associated hospitalisations and deaths in children particularly from holoendemic transmission regions (Obonyo et al., 2007). It is further worsened by co-infections with bacteria, helminths and viruses which are very common in endemic regions, leading to high fatality particularly in children with secondary infections (Calis et al., 2008). The World Health Organisation (WHO) defines SA as having haemoglobin concentration < 5 g/dL (in children) partnered with P. falciparum parasites. Several factors are believed to contribute to the pathological processes of SA including the destruction of IEs accompanied by clearance of uninfected erythrocytes and decreased erythrocyte production (Awah et al., 2009; Lamikanra et al., 2007). 18 2.4.2 Metabolic acidosis Metabolic acidosis and respiratory distress are often characterised by an unusual deep breathing and have been shown to be strong predictors in SM. Although the pathogenic mechanisms are not well defined, metabolic acidosis is enhanced by low oxygen levels in tissues as a consequence of anaemia, obstruction of microvascular blood flow caused by sequestration of IEs and rosetting. Accumulation of lactic acid resulting from the reduced clearance of the parasites by the liver has also been observed (reviewed in Marsh et al., 1996). 2.4.3 Cerebral malaria CM represents one of the most lethal complications of malaria accounting for approximately 20 % of fatal cases in malaria endemic populations. CM is clinically defined as a state of unarousable coma with detectable falciparum parasites in peripheral blood and most of the times retinopathy, with the exemption of other potential causes of coma (Beare et al., 2011; Idro et al., 2005). Coma is assessed by a Blantyre score (1 or 2 out of 5) for children and Glasgow score (<11 on a scale of 15) for adults (Beare et al., 2011; Molyneux, 1989; Teasdale and Jennett, 1974). CM can be treated and the recovery is relatively fast, however, some survivours are known to unfortunately suffer some residual brain-associated disabilities such as language disorder, motor deficits and cognitive loss (Birbeck et al., 2010) and there is no adjunctive therapy to aid their recovery. Although the molecular mechanisms leading to the development of CM and more precisely why some patients are able to recover while others suffer fatal outcomes remain difficult to explain, it is well recognised that the pathological processes differ between adults and children (Hawkes 19 et al., 2013; Dondorp, 2008). In addition, considerable insights from post-mortem observations reveal sequestered parasites localised in brain cells with wide-spread vascular pathology and disruption of blood brain barrier function (Dorovini-Zis et al., 2011). Consistent with that, CM in children has been placed in two subclasses based on the observed histopathological data. Children are classified as belonging to subgroup 1 (CM1) if they have sequestered parasites only while subgroup 2 (CM2) have sequestered parasites in addition to perivascular ring haemorrhages, fibrin platelet deposits and monocytes (Milner, Jr. et al., 2014; Moxon et al., 2013; Dorovini-Zis et al., 2011; Seydel et al., 2006). It is well acknowledged that a cocktail of factors involving the parasite and host drive CM pathogenesis (detailed in section 2.9). 2.4.4 Pregnancy-associated malaria, PAM Pregnant women notably primigravids from endemic countries become vulnerable to severe falciparum malaria irrespective of their previous exposure. IEs adhere to CSA on syncytiotrophoblasts sequestering them in the placenta, which restricts blood flow as well as nutrients to the growing foetus. Also, excessive parasite sequestration stimulates the activation of host immune responses leading to placental inflammation (Conroy et al., 2013; Khattab et al., 2013; Suguitan, Jr. et al., 2003). PAM results in increased risk of maternal anaemia, newborn anaemia, low birth weight, still -birth and pre-term delivery (Steketee et al., 2001). 20 2.5 Naturally-acquired immunity After several years of encountering the parasites repeatedly, it is observed that there is an age-related decline in febrile malaria and parasite density in individuals living in endemic regions (Figure 2.3). This acquired immunity is variable depending on the transmission dynamics as it has been observed that residents in hyperendemic regions acquire clinical immunity relatively faster than people living in low transmission regions (reviewed in Fowkes et al., 2016; Langhorne et al., 2008). Generally, the building of natural immunity is a slow or gradual process which passes through different phases with immunity achieved relatively quicker against severe and deadly malaria. Children in the first few months of their life rarely suffer from severe forms of malaria disease as it is widely believed that they benefit from maternally protective antibodies (reviewed in Hviid, 2005). Conversely, severe disease is confined to toddlers generally beyond six months with immunity to non-cerebral severe disease rapidly acquired after one or two clinical episodes and complete immunity to severe disease and death attained generally by age of five years notably in areas of high malaria transmission (Griffin et al., 2015; Goncalves et al., 2014; Reyburn et al., 2005; Gupta et al., 1999). As children grow or develop towards the adolescent age, they develop mild disease and thereafter, they progressively become refractory to symptomatic infections. In essence, adults constantly encounter and harbour low level parasites but are asymptomatic, altogether suggesting that complete protection from febrile malaria as observed with other pathogens is remarkably less efficient and that sterilising protection is an impossible feat to achieve in natural populations (reviewed in Langhorne et al., 2008; Amanna et al., 2007; Hviid, 2005). First time pregnant women living in malaria-endemic regions, however, do not enjoy the protected immunity 21 acquired from prior and persistent exposure as the presence of a placenta makes them susceptible to certain malaria parasite subsets that preferentially sequester in the placenta leading to severe consequences for both the mother and the foetus. Immunity is, however, built progressively to severe disease with increasing parity (reviewed in Hviid, 2010). The importance of naturally-acquired immunity from P. falciparum malaria has long been recognised. It is believed that antibodies (IgG) are vital components in this response and are directed towards the blood stage parasites where clinical symptoms are observed (reviewed in Fowkes et al., 2016; Hviid, 2005; Sabchareon et al., 1991; McGregor et al., 1963). Significantly, the transfer of purified total IgG from immune Gambian adults to children very ill from malaria rapidly improved the clinical outcome of infection (Cohen et al., 1961). The general notion is that non-sterile development of immunity occurs slowly and also fleeting and that ongoing exposure to low-level parasites is an essential requirement to sustain or reinforce immunity, complicating efforts to determine the precise correlates of protection. The slow rate has been attributed to the extensive genetic and antigenic diversity of the parasite (reviewed in Takala et al., 2009; Scherf et al., 2008) effectively avoiding an immune attack, always a step ahead of the immune system. Thus, many years of frequent exposure to different isolates are required in order to build up a broad spectrum of protective antibodies. Recent accumulating data show that the remarkably slow development is orchestrated by the parasite where it sabotages B cell function, resulting in the generation of the so-called atypical memory B cells that produce ineffective and transient antibodies (Muellenbeck et al., 2013; Portugal et al., 2013; Weiss et al., 2011). Despite these findings, other studies demonstrate that long lasting protective antibody responses which had no bearing on exposure were detectable even 22 up to 40 years after infection (reviewed in Fowkes et al., 2016). Clearly, more research is needed to resolve the issue of the longevity of protective antibody and memory B cell responses as they remain critical in the quest for a potent malaria vaccine. Figure 2.3:The link between age and malaria severity. Acquisition of immunity to the different clinical forms of malaria with increasing age in an area with transmission of P. falciparum. (Adapted from Cowman et al., 2016). 2.6 Variant surface antigens (VSA) and PfEMP1 Several hours after merozoite invasion of erythrocytes, parasite variable antigens are exported and localised on the surface of infected IEs, presenting the possibility of immune recognition and subsequent attack. These variant surface antigens (VSAs) are encoded by large multigene families concentrated at the telomeres and subtelomeres 23 and they include repetitive interspersed family (RIFIN) proteins and subtelomeric variable open reading frame (STEVOR) proteins, both of which account for the largest number of genes (~190 genes), P. falciparum erythrocyte membrane protein 1 (PfEMP1, 60 genes), surface-associated interspersed gene family (10 genes) proteins and P. falciparum Maurer‟s cleft two transmembrane (12 genes) proteins (reviewed in Chan et al., 2014). These VSAs have been implicated in several mechanisms or strategies relating to immune evasion since they undergo antigenic variation, thus allowing parasites to cause persistent infections. Considerable interest has been generated with PfEMP1 presumably resulting from its uniqueness to P. falciparum, the species that causes the severe manifestations of malaria, hence it has been the most intensely investigated. However, recent emerging data seem to suggest that the other VSA, particularly RIFIN may have important roles in disease pathology than previously known (Goel et al., 2015; Chan et al., 2014). Radioactive labelling of a matured IE with 125I provided proof of the surface location of PfEMP1 on IE and not on the surface of uninfected erythrocytes (Leech et al., 1984b). The var encoded PfEMP1 is a large, variable molecular weight protein of about 200-350 kDa, sensitive to trypsin treatment which rapidly removes the protein from the surface of IEs (Howard et al., 1988; Leech et al., 1984b) and it is soluble in sodium dodecyl sulphate implicating its association with the cytoskeleton (Aley et al., 1984). Furthermore, PfEMP1 is known to be highly immunogenic with a wide array of antigenic epitopes. Notably, immunoprecipitation experiments demonstrated that sera collected from monkeys that had been infected with a homologous strain agglutinated IEs of the specific strain only and these strain-specific sera were able to interfere with cytoadhesion of the homologous IEs (Howard et al., 1988). Expression of PfEMP1 allows infected parasites to be sequestered in various tissues such as the 24 brain by cytoadhering to diverse host endothelial receptors. It is also involved in rosetting through its interaction with uninfected erythrocytes (Carlson et al., 1990) and autoagglutination which results from the platelet-mediated clumping of infected IEs (Pain et al., 2001; Roberts et al., 2000) [Figure 2.4], thus allowing these parasites to occlude blood vessels and cause local inflammation linked with severity of disease (reviewed in Rowe et al., 2009). Cytoadhesion also serves as a strategy that allows the parasite to leave the peripheral circulation and hence avoid spleen mediated killing. More importantly, the ability to express different forms of PfEMP1 provides a smart mechanism to avoid detection by antibodies elicited towards the previous PfEMP1 variant, contributing to the pathogenic nature of P. falciparum (Baruch et al., 1995). a b c Figure 2.4: Cytoadhesion phenotypes in P. falciparum-associated SM pathogenesis. a) Cytoadherence results from the binding of IEs to host endothelial cells. b) Rosetting occurs when IEs bind to uninfected erythrocytes. c) Platelet-mediated clumping is the process where IEs agglutinate and it is mediated by platelets. Images are observed under a light microscope after Gimesa staining (Adapted from Rowe et al., 2009). 25 2.6.1 Var gene architecture The first complete view and considerable insight into var gene architecture and chromosomal organisation was obtained from genomic sequences of the 3D7 reference isolate (Gardner et al., 2002). Subsequently, comparative analysis of var genes from the IT4 and HB3 genomes with the 3D7 genome revealed a similar number of approximately 60 protein-coding genes per haploid genome (Kraemer et al., 2007; Gardner et al., 2002). Var genes are present on nearly all the parasite chromosome except for chromosome 14, which contains a pseudogene, and they are followed by other multigene families such as RIFIN and STEVOR. Approximately 65 % of the var genes are concentrated at the subtelomeric chromosomal regions, reflecting their importance in the survival of the parasite in terms of genetic diversity and variant expression since high recombination events occur at these subtelomeric sites (Gardner et al., 2002). These subtelomeric var genes are found adjacent to the non-coding telomere repeat elements (1-6) with a considerable majority existing in a tail-to-tail orientation with one or more members of the RIF gene family in between them, while the rest assume or adopt a head-to-head or head-to-tail configuration (reviewed in Scherf et al., 2008). The remaining 35 % of var genes are located or resident in central clusters on chromosomes 4, 7, 8 and 12 and are frequently found in tandem arrays in a head-to-tail orientation (Gardner et al., 2002). With reference to upstream promoter sequences which are strongly linked to specific chromosomal location and direction of transcription, almost all var genes fall into four main groups namely: UpsA, UpsB, UpsC and UpsE (Figure 2.5). In addition, there are some subsets falling into intermediates [B/A, B/C] (Kraemer et al., 2003; Kraemer and Smith, 2003; Lavstsen et al., 2003). Based on the 3D7 parasite genome, there are 10 group A var genes (10 genes), all of which are located in the subtelomeric sites and 26 transcribed toward the telomere (Figure 2.5) in the opposite direction of group B var genes (22 genes), which are situated near the telomere and transcribed towards the centromere. Group C var genes (13 genes) can be found near the centre of the chromosome. The intermediate groups B/A (4 genes) and B/C (10 genes) have Ups B- like promoters preceding the coding regions of group A or C respectively and are mapped to either subtelomeric or central chromosomal locations. Group A var genes are more conserved and larger in size when compared with Groups B and C (reviewed in Hviid and Jensen, 2015; Petter and Duffy, 2015; Lavstsen et al., 2003). UpsE consists of a single var gene coding for VAR2CSA which is involved in binding to receptors on the placenta during pregnancy-associated malaria (Salanti et al., 2003). They are also subtelomeric var genes but are transcribed in the opposite orientation to UpsB var genes (Rask et al., 2010; Kraemer and Smith, 2003; Lavstsen et al., 2003; Gardner et al., 2002). Overall, var gene organisation appears to be very much conserved between parasites of genetically distinct backgrounds (Kraemer et al., 2007), and recombination tends to occur between var genes from the same 5‟ flanking sequence and gene orientation, hence maintaining this genetic diversity (Bull et al., 2008; Gardner et al., 2002). Var genes share a common architecture encoded in two exon structures and are connected together by an intron. The large hypervariable exon 1 (4-10 kb) consists of a short N-terminal segment (NTS, 75-100 amino acid), multiple Duffy-binding like domains, (DBL, 100-250 amino acid), a cysteine-rich interdomain (CIDR, 70-200 amino acid) and a transmembrane region. Exon II, with a size of about 1.0-1.5 kb, codes for a more conserved intracellular region of the acidic terminal segment which interacts with proteins on the erythrocyte cytoskeleton as well as parasi te proteins (Gardner et al., 2002; Kyes et al., 2001). The intron joining the two exons has a high 27 adenine and thymine content, a conserved nucleotide sequence and a size of about 0.8- 1.2 kb. The DBL and CIDR are highly variable extracellular region with multiple distinct domain structures which have the propensity to bind to several red blood cell adhesion receptors. These adhesion domains have been classified according to conserved residues into various groups that signify distinct binding specificities and phenotypes. DBL domains are categorised into seven types namely α, β, γ, δ, ε, δ and x. While CIDR domains are grouped into four distinct classes α, β, δ and γ (Rask et al., 2010; Smith et al., 2000). The DBLα domain is the most abundant and is present in nearly all var genes immediately after the N-terminal segment and also harbours most of the conserved var sequences found in exon 1 (Kraemer et al., 2007; Kyes et al., 2007). Although studies have shown that extreme diversity is found within and between var genes of P. falciparum isolates (Chen et al., 2011; Barry et al., 2007; Trimnell et al., 2006; Bull et al., 2005; Fowler et al., 2002), the arrangement order of var domains is not random. Thus, a conserved head structure has been defined or mapped out and that is composed of NTS-DBLα-CIDR1 domains for most var genes except for VAR2CSA and type 3 var genes (Kraemer and Smith, 2006; Kyes et al., 2001). Var genes can also be classified based on the domain cassette (DC) system which consists of combinations of specific DBL and CIDR domain structures occurring in tandem in a PfEMP1 protein (Figure 2.5), thus providing some measure for defining associations with disease phenotypes. Indeed, there is evidence showing that the domain cassette classification of var genes has functional relevance with respect to malaria pathogenesis (Warimwe et al., 2012). 28 Figure 2.5: Plasmodium falciparum var genes chromosomal organisation and PfEMP1 domain structure. (a) Group A and B var genes occupy subtelomeric chromosomal sites and are transcribed in opposite directions. Group C var genes are located in chromosomal central sites. (b) PfEMP1 proteins are made up of a conserved non-random head structure that is composed of NTS-DBLα-CIDR1 domains. (c) P. falciparum genomes encode tandem DBL CIDR domains (domain cassettes; DC). DCs are linked to specific binding phenotypes. For instance, DC4 is involved in ICAM- 1 binding. TM, transmembrane; ATS, acidic terminal sequence; UPS, upstream promoter sequence (Adapted from Hviid and Jensen, 2015). 2.6.2 Var gene transcription and regulation Each var gene possesses two distinct promoters, one upstream of exon 1 that mediates the expression of a functional messenger ribonucleic acid (mRNA) and is active in the early developmental stages, from 12 to 18 h after invasion. The second is a bidirectional promoter found within the intron and it is responsible for the transcription of non-coding (sterile) RNA mainly by the late-stage parasites and is also 29 believed to regulate gene expression (Epp et al., 2009; Frank et al., 2006; Voss et al., 2006; Calderwood et al., 2003; Deitsch et al., 2001b). It is widely accepted that var genes are transcribed in the so-called mutually exclusive manner, which allows only one member of the multigene family to be expressed on the surface of an IE while keeping the rest of the members in a state of transcriptional dormancy (Chen et al., 1998; Scherf et al., 1998). However, one study has demonstrated the capability of dual PfEMP1 transcripts to be expressed on the surface of a single IE (Joergensen et al., 2010). Var gene transcription or synthesis begins relatively early at the ring stage soon after merozoite invasion of erythrocytes, peaks around 18 h post invasion and further declines to low levels within 20-24 h (Dahlback et al., 2007; Kyes et al., 2000). Although numerous var transcripts have been reportedly identified in the ring stages of malaria parasites, the consensus is that only a single and dominant locus is translated into a functional PfEMP1 protein in matured trophozoites, transported and deposited on the surface of IEs (Chen et al., 1998; Scherf et al., 1998). Transport of PfEMP1 through several membranes or compartments occurs via complex and poorly understood mechanisms that involve conserved sequences in the N-terminal, the transmembrane and cytoplasmic domains of PfEMP1 and other partner proteins (reviewed in Przyborski et al., 2016; Hviid and Jensen, 2015; Melcher et al., 2010). Significantly, PfEMP1 is transported across the parasitophorous vacuole membrane supported by a Plasmodium export element (Marti et al., 2004), while the parasitophorous vacuole membrane resident Plasmodium translocon complex is responsible for translocation into the cytoplasm of the host cell. Trafficking of PfEMP1 is halted around 30-36 h as they are no longer observed on the surface of IEs. The regulation of var gene expression is stringent, highly organised and occurs at the transcriptional level in the absence of DNA rearrangements (reviewed in Ralph and 30 Scherf, 2005; Scherf et al., 1998). Regulation of var genes at various stages (silent, poised or active) is believed to be orchestrated by tightly interconnected epigenetic- dependent mechanisms owing to the availability of relatively few eukaryotic transcription factors in the Plasmodium genome (Figure 2.6). These multilayered epigenetic mechanisms include reversible histone alterations, promoter–intron interaction and sub-nuclear localisation processes (reviewed in Hviid and Jensen, 2015; Guizetti and Scherf, 2013). Deciphering the mechanisms defining var regulation may lead to the identification of factors crucially involved in immune evasion and that would provide drug target opportunities. Figure 2.6: Var gene regulation in P. falciparum. (A).Transcription of a single var gene begins at the ring stage following the invasion of erythrocytes by merozoites. Transcription of var gene is halted in matured trophozoites and schizonts but remains in a poised state, prepared for activation in the next cycle of erythrocyte invasion. (B). All var genes irrespective of their chromosomal position occur in clusters at the periphery of the nucleus (blue). Meanwhile, silent var genes group together at perinuclear repressive sites (gray) while the single active var gene (green) is found at a dedicated site surrounded by components of euchromatin that will permit its transcription. The mechanism controlling the shift from an active to a poised state is not well elucidated (Adapted from Guizetti and Scherf, 2013). 31 The nuclear arrangement of var genes around the periphery appears to be a fundamental feature of all silenced var genes and that occurs regardless of their chromosomal location (Scherf et al., 2008). These transcriptionally silent var genes are generally found clustered (Figure 2.6) and packaged in condensed chromatin states and known components of repressive heterochromatin seem to be necessary for their maintenance (Lopez-Rubio et al., 2009; Ralph et al., 2005). Although the molecular details of this perinuclear localisation are largely unknown, Zhang et al (2011a) showed that the conserved var intron contains a short 18 bp motif which is able to recruit an actin-containing complex to the nuclear periphery with silenced var genes (Zhang et al., 2011a). Reversible histone alterations play pivotal roles in var gene silencing (Lopez-Rubio et al., 2007; Freitas-Junior et al., 2005). The process involves the removal of acetyl groups on histones, propagated by the Nicotinamide Adenine Dinucleotide (NAD+) dependent Silent Information Regulator 2 (SIR2) family of proteins. Notably, chromatin immunoprecipitation experiments specifically showed the colocalisation of PfSIR2A at a subtelomeric var promoter with a silenced var gene. Moreover, genetic disruption of SIR2A in parasites resulted in a loss of silencing and subsequent activation of subtelomeric UpsA and UpsB var genes (Duraisingh et al., 2005). The repressing activity of UpsC var genes, however, has been linked to SIR2B suggesting the differential regulation of var groups (Tonkin et al., 2009). Altogether, SIR2 proteins exert their transcriptional silencing activity possibly by placing a conserved heterochromatin Histone 3 lysine 9 trimethylated (H3K9me3) mark in the promoter regions of repressed var genes (Chookajorn et al., 2007; Lopez-Rubio et al., 2007), which recruits P. falciparum heterochromatin protein 1 (PfHP1) and that results in heterochromatin formation and decreased accessibility 32 for transcription (Coleman et al., 2012; Perez-Toledo et al., 2009; Duraisingh et al., 2005). Gene silencing involving the single intron connecting exons 1 and 2 in the conserved genetic structure of var genes has also been implicated. The intron confers the ability to silence via pairing with its respective upstream promoter (Swamy et al., 2011; Calderwood et al., 2003; Deitsch et al., 2001b), and with the aid of RNA pol II and PfSETvs enzymes which have specificity for the Histone 3 lysine 36 trimethylated (H3K36me3), gene silencing marks are introduced into the two exons (Ukaegbu et al., 2014; Jiang et al., 2013). Conversely, blocking the interaction or incorporating an unpaired promoter into a silent var gene cluster leads to its activation (Avraham et al., 2012; Dzikowski et al., 2006; Deitsch et al., 2001b). Epp et al (2009) have also provided evidence of the involvement of non-coding RNA in var silencing. The var intron promoter mediates bidirectional transcription resulting in long transcripts of sense and antisense non-coding RNA (Epp et al., 2009). The expression of the var antisense non-coding RNA which are associated with chromatin at telomeric sites stimulates the activation of a silent var gene (Amit-Avraham et al., 2015). An additional layer of regulation apparently exists at least for specific var genes. With reference to that, Zhang et al. (2014) recently identified PfRNAse II, a chromatin- associated exoribonuclease known to degrade nascent or short RNA to be enriched at silenced var genes. PfRNAse II was shown to silence transcription of group A var genes and more importantly, depletion of PfRNase II correlated with elevated expression of group A var genes, permitting many group A var genes to circumvent silencing (Zhang et al., 2014). At any given time, only a single var gene is selectively transcribed for the first 16 h of the parasite‟s life cycle and mostly maintained in successive generations through 33 mechanisms not well elucidated (Figure 2.6). Activation of the selected var gene involves the replacement of repressive histone marks and loss of PfHP1, which now renders it permissive for transcription (Perez-Toledo et al., 2009; Lopez-Rubio et al., 2007) . Thus, the promoter region of the active var gene is now enriched with loosely packed chromatin which is associated with reversible alterations of histone marks, including Histone 3 lysine 4 bi and tri-methylation (H3K4me2/3) and acetylated Histone 3 lysine 9 (H3K9ac) (Lopez-Rubio et al., 2007). An additional modification involved in transcriptional activation include the histone variant H2A.Z. Petter et al (2011) showed that H2A.Z is concentrated at the transcriptional start site of active var genes and mediates elevated transcriptional activities during the ring stages, and are depleted later in matured stages (Petter et al., 2011). Further, H2A.Z-mediated var gene activation is antagonised by PfSIR2A silencing factor which results in the removal of PfH2A.Z from the var promoter. Again var gene activation encompasses the dissociation from silent clusters and relocation of the activated var gene to a dedicated nuclear compartment that promotes transcription (Lopez-Rubio et al., 2009; Voss et al., 2006; Ralph et al., 2005) The transcription of the selected var gene locus peaks up around 12 to 16 h post- invasion and then is transiently suppressed during matured schizonts stages (Figure 2.6). The transcribed var gene, however, tends to be retained in the so-called poised state and subsequently carried over or inherited in the next cycle of erythrocyte invasion unless a var switch occurs. Although the mechanism involving this transition from poised to active state is not well understood, data from chromatin immunoprecipitation experiments indicate that the gene activation mark H3K4me2 which is catalysed by the histone methyltransferase, PfSET10 co-localises with the poised var gene post ring stages, presumably to retain the euchromatic epigenetic 34 memory that will ensure reactivation in the successive cell cycle (Volz et al., 2012). It seems that a sophisticated system has been put in place to achieve a regulated var gene expression. Nevertheless, some questions remain unanswered and that includes the factors that modulate switching between genes and also what informs or controls the choice and activation of a specific var gene. 2.6.3 Antigenic variation and var switching As a means of survival in their host as well as to promote lifelong perpetuation, most pathogenic organisms have evolved a wide range of defense strategies and P. falciparum is no exception. Antigenic variation, a process which involves the expression of new subpopulations of clonally variant proteins at regular intervals happens to be one crucially important mechanism employed by parasites to prolong the duration of infection. The first inkling of antigenic variation was observed in P. knowlesi, and was then subsequently reported in P. chabaudi and sometime later in P. falciparum (reviewed in Kyes et al., 2001). The parasite‟s ability to avoid successful destruction by the immune system has been credited to its capacity to generate and sustain diversity through the process of antigenic variation. Malaria parasites accomplish such variation through antigenic alternations between different mutually expressed var encoded proteins over the period of an infection (Recker et al., 2011; Scherf et al., 2008). Frequent alterations of antigenic forms of PfEMP1 result in a switch in receptor usage that enables the parasite to smartly avoid immune destruction. Consistent with that, antibodies elicited to a specific antigen earlier on in the infection or to previous exposures will be ineffective against another variant 35 expressed in case a parasite switch occurs. This process is responsible for the protracted nature of the infection and it is translated into the classical parasitaemia waves and recrudescences often associated with malaria infections (Petter and Duffy, 2015; Scherf et al., 2008; Miller et al., 1994). Antigenic variation has been blamed for thwarting the efforts to develop an effective vaccine (Ferreira et al., 2007). During the process of transcriptional switching, a new gene must be selected for activation and that coincides with the simultaneous suppression of the previously active gene. This process appears to be highly organised among members of the var family to limit unnecessary immune exposure as well as prevent exhaustion of the entire var gene reserves (Ukaegbu et al., 2015). Understanding the molecular nuances of var gene switching has been extremely difficult because information on epigenetic processes that control antigenic variation is still fragmentary and limited. Despite these challenges, studies from long-term in vitro culturing of laboratory parasites and human volunteers infected with parasites have provided significant snapshots on the var switching network or dynamics. Var gene switching rates reported from in vitro falciparum cultures occur at an approximately 2 % per parasite generation, irrespective of their chromosomal or promoter occupation (Fastman et al., 2012; Frank et al., 2007; Roberts et al., 1992). By contrast, switching experiments replicated in human volunteers inoculated with malaria parasites reported a high rate of about 16 % per generation (Peters et al., 2002). It appears that different switching rates are associated with each var gene. Thus, switching is not a random event but rather an organised pattern exists in the choice of which genes are expressed in the event of a switch (Horrocks et al., 2004). Furthermore, mathematical models have confirmed the non-randomness of var switching which results from a compromise between parasite intrinsic switching and 36 immune-dependent selection (Recker et al., 2011). It is not entirely clear whether external factors or signals can influence var switching pattern owing to the dearth of data. Notwithstanding, the relatively few studies suggest that in vitro stimuli such as starvation, stress, high temperature or lactate levels can modulate rate and pattern of transcriptional switching (Merrick et al., 2012; Rosenberg et al., 2009). There is also evidence credited to the immune system in var gene alterations. Several investigations have shown that group A var genes are more commonly expressed in patients but subsequently change in a random manner to express groups B and C var genes when cultured in vitro, implicating the role of host factors or immune pressure (Bachmann et al., 2011; Zhang et al., 2011b; Blomqvist et al., 2010; Warimwe et al., 2009). 2.7 Endothelial receptors involved in cytoadhesion A very wide range of host endothelial molecules are known to interact with the extracellular domains of PfEMP1 and currently, about 21 distinct receptors have been reported, reflecting the high variability in PfEMP1 binding behaviour (Esser et al., 2014). ICAM-1, CD36 and CSA (Berendt et al., 1989; Oquendo et al., 1989; Rogerson et al., 1995) are among the most extensively characterised and have been shown to be the most widely utilised by falciparum malaria parasites. Some receptors are preferentially enriched in some tissues and organs and that has an influence on the type of PfEMP1 expressed to specifically interact with these receptors, leading to the so-called organ-specific syndrome. A classic example is observed in PAM where IE binding to CSA on placental syncytiotrophoblasts has been associated with specific PfEMP1 variants (reviewed in Fried and Duffy, 2015; Salanti et al., 2004). In CM, it is believed that specific variants of PfEMP1 bind receptors that are highly expressed 37 in brain endothelial cells (reviewed in Wassmer and Grau, 2017), although not always consistent. Recent data suggest that individual cytoadhesion domains (domain cassettes) within one PfEMP1 molecule found on the surface of IEs can bind to specific host receptors (reviewed in Hviid and Jensen, 2015). Hence, a complex binding process probably exists where one PfEMP1 is capable of engaging multiple host receptors and interaction with these receptors could result in a synergistic or additive impact or effect between cytoadhesive molecules and this may correlate with disease severity (Adams et al., 2014; Aird et al., 2014; Yipp et al., 2007; Heddini et al., 2001). 2.7.1 Intercellular adhesion molecule-1 ICAM-1 is a transmembrane glycoprotein containing five extracellular Ig-like domains and a short cytoplasmic tail (Brown et al., 2013; Chakravorty and Craig, 2005; van de Stolpe and van der Saag, 1996). Depending on the level of post- translational glycosylation and cell type, ICAM-1 has a variable molecular weight ranging from 90–115 kDa. It is widely distributed at low levels on several cells including immune cells and endothelial cells. Its expression is significantly increased particularly in brain endothelial cells in response to many proinflammatory cytokines including tumour necrosis factor α (TNF-α) and interleukin-1β (IL-1β) (Amodu et al., 2005; Chakravorty and Craig, 2005; Berendt et al., 1992). ICAM-1 is well known for stabilising cell–cell interactions and transmigration of leucocytes to sites of inflammation by binding to lymphocyte function-associated antigen (LFA)-1 and macrophage 1 antigen (Mac 1) (van de Stolpe and van der Saag, 1996; Marlin and Springer, 1987). Nevertheless, several ligands have been identified including plasma 38 fibrinogen, human rhinoviruses and Plasmodium-IEs (van de Stolpe and van der Saag, 1996; Languino et al., 1993; Berendt et al., 1989; Staunton et al., 1989). In particular, IEs bind to the N-terminal Ig-like domain of ICAM-1, which is distinct but also overlaps with the LFA-1 binding region (Tse et al., 2004; Berendt et al., 1992; Ockenhouse et al., 1992). Previous studies from humans and mice have provided strong arguments for the critical involvement of ICAM-1 in SM pathology (Armah et al., 2005; Hearn et al., 2000). Notably, histological studies by Turner et al., (1994) reported increased localisation of IEs and pronounced ICAM-1 expression in brain sections from patients who died from CM. Binding assays reported the adhesion of IEs to ICAM-1 to be significantly higher in CM or other forms of SM in general than in controls, although a direct link to severe disease had been sometimes inconsistent (Turner et al., 2013; Ochola et al., 2011; Rogerson et al., 1999; Newbold et al., 1997; Ockenhouse et al., 1991). Also, an identified non-synonymous polymorphism in ICAM-1 (ICAM-1kilifi) with high frequency in malaria endemic populations that had differential binding capacity to IEs under flow conditions would have been thought to be protective against severe disease; however, that was not the case as data gathered so far have been conflicting (Fry et al., 2008; Jenkins et al., 2005; Adams et al., 2000; Craig et al., 2000; Kun et al., 1999; Bellamy et al., 1998; Fernandez-Reyes et al., 1997). Also, in vitro selection of IEs adhering to human brain endothelial cells highly expressed DC 8 and 13 PfEMP1 (Avril et al., 2012; Claessens et al., 2012) that failed to bind ICAM-1. On the parasite side, available data suggest that ICAM-1 binding has evolved into two binding lineages. Group A DBLβ associated with severe disease, with a particular interest in the discovery of DC4, a group A PfEMP1 that binds to ICAM-1 (Bengtsson 39 et al., 2013b; Oleinikov et al., 2009). DC4 was discovered through orthologic search of the 3D7 var gene PFD1235w in parasite isolates from Ghanaians with malaria (Bengtsson et al., 2013b) and it comprises three domains (DBLα1.1/1.4-CIDRα1.6- DBLβ3). IEs selected in vitro to express DC4 mediated ICAM-1 binding but not binding to CD36 and experiments using chimeric protein constructs revealed the ICAM-1 interaction site in DBLβ3, a region located in the C-terminal portion (Bengtsson et al., 2013b). DC4 DBLβ3 antibodies are acquired naturally in early age and they have been shown to be cross-reactive and cross-inhibitory of DC4 containing ICAM-1 binders across different falciparum genomes (Bengtsson et al., 2013b). Furthermore, Lennartz et al (2015) characterised a monoclonal antibody 24E9 that targeted conserved epitopes of DC4 DBLβ3 parasites from different parasite genomes by blocking or interrupting ICAM-1 interaction site on DBLβ domain. Thus, the accumulated data provided the feasibility of considering these DBLβ proteins in the design of a PfEMP1 based vaccine. The other lineage involves DBLβ PfEMP1 in groups B and C and this forms a group of ICAM-1 binders that have the capacity to synergise with CD36 to enhance firm endothelial adhesion, a binding phenotype linked with UM (Janes et al., 2011; Ochola et al., 2011; Howell et al., 2008; Yipp et al., 2000; McCormick et al., 1997; Cooke et al., 1994). Altogether, these data suggest that ICAM-1 binding alone may not be sufficient in causing CM. Indeed, we recently provided more evidence using bioinformatics and structural biology in identifying a conserved sequence (motif) in PfEMP1 that allowed us to predict ICAM-1 binding (chapter three). Comparative analysis of many parasite genomes revealed that these PfEMP1 that mediated ICAM-1 binding possessed also an EPCR-binding CIDRα1 domain suggesting that PfEMP1 with this motif could synergistically engage both receptors (Lennartz et al., 2017). 40 Although, it has previously been reported in only one study that some groups of PfEMP1 contain domains that can interact both with EPCR via CIDRα1 and ICAM-1 via DBLβ domain (Avril et al., 2016), we showed in more detail the dual and simultaneous binding ability of these PfEMP1 constructs containing both ICAM-1 and EPCR binding domains with high affinity in surface plasmon resonance experiments (Lennartz et al., 2017). Furthermore, IEs expressing PfEMP1 predicted to bind to both receptors were selected in vitro and binding assays under flow conditions provided evidence for increased adhesion in the presence of both receptors in comparison with either receptor alone (Lennartz et al., 2017; Chapter three). Quantitative real-time PCR (qPCR) data showed significantly high transcription of motif-containing ICAM-1 binders in CM compared with SA (SA) and other forms of SM (Lennartz et al., 2017; Chapter three). Thus, this study provides evidence for the first time of the ability to predict group A ICAM-1 binders based on sequence information and also showed that parasites expressing this PfEMP1 motif interacted with both ICAM-1 and EPCR and had a link with CM development (Lennartz et al., 2017; Chapter three). Membrane-associated ICAM-1 is believed to be cleaved from cellular surfaces into circulation since soluble forms consist of only the extracellular portion of ICAM-1. Soluble ICAM-1 (sICAM-1) has been quantified in plasma samples and the physiological concentration falls between 200-300 ng/mL. However, these levels have been found to be markedly high in several disease manifestations including malaria (Chakravorty and Craig, 2005; Gearing and Newman, 1993). Several studies have examined the levels of sICAM-1 in falciparum infected patients and the consensus is that the levels are higher in SM compared with UM or controls but the levels do not significantly discriminate between the different types of SM syndromes (Cserti- 41 Gazdewich et al., 2010; Tchinda et al., 2007; McGuire et al., 1996; Jakobsen et al., 1994; Hviid et al., 1993) [Chapter five]. High levels of sICAM-1 in plasma of malaria patients may signify the combined efforts of excessive inflammatory response, increased activation of endothelial cells and decreased removal of sICAM-1 from the circulatory system (Cserti-Gazdewich et al., 2010). 2.7.2 Endothelial Protein C Receptor The human endothelial protein C receptor, EPCR, also known as activated protein C (APC) receptor and encoded by the proc gene is a 46 kDa type 1 transmembrane protein that is structurally related to the major histocompatibility class 1/CD1 family of proteins (reviewed in Rao et al., 2014). EPCR expression is found on diverse cells including immune cells and endothelial cells, with particularly low expression on the brain endothelium or microvascular cells (Laszik et al., 1997). The endothelial cell- bound EPCR functions as a protein C receptor which results in protein C activation via thrombin-thrombomodulin complex signalling. APC interacts with Protease- activated receptor 1 (PAR1) which is involved in regulation of inflammation, coagulation and neuroprotective processes (Rao et al., 2014; Griffin et al., 2012; Stearns-Kurosawa et al., 1996; Fukudome and Esmon, 1994). EPCR is the most recently discovered receptor implicated in SM pathology and evidence to support this role was provided by two independent studies. IEs selected to bind brain endothelial cells expressed PfEMP1 encoding DC8 and DC13 (Avril et al., 2012; Claessens et al., 2012) and were observed to adhere to EPCR (Turner et al., 2013). Moxon et al (2013), while examining samples of children who died from CM showed the shedding of the normally low levels of membrane-associated EPCR coupled with 42 fibrin deposits localised with sequestered IEs in brain microvascular sections. More studies have clearly shown that parasite isolates from adults and children suffering from SM had high transcription levels of DC8 and DC13 (Mkumbaye et al., 2017; Bernabeu et al., 2016; Almelli et al., 2014; Bertin et al., 2013; Lavstsen et al., 2012) with differential binding characteristics or ability to bind EPCR. EPCR interacts with high affinity with CIDRα1.1 and the α1.4-1.8 domains of PfEMP1 and indeed, Jespersen et al., (2016) analysed almost full-length sequences of PfEMP1 from parasites obtained from paediatric SM cases from Tanzania and found the predominance of EPCR-binding CIDRα1 transcripts. More importantly, antibodies targeting CIDRα1.1 regions have been reported to be highly acquired in residents from malarious countries at an early age, further buttressing the key involvement of EPCR in SM (Turner et al., 2015). Recently, the molecular determinants for PfEMP1 interaction with EPCR were elucidated. Using co-crystals of soluble EPCR sEPCR bound to PfEMP1 CIDRα1 domains, the binding site of PfEMP1 was revealed and that region had a considerable overlap with that of the protein C interaction site on EPCR (Turner et al., 2013; Lau et al., 2015). Altogether, the data suggest that in the presence of PfEMP1 parasites, the anti-coagulant state of the endothelium via activation of protein C signalling may be competitively disrupted (Turner et al., 2013; Lau et al., 2015). Indeed, in vitro studies demonstrated that PfEMP1 interaction with EPCR attenuated the activation of protein C as well as the downstream signaling of EPCR-dependent PAR1 activation, resulting in PAR1-dependent compromise of the vascular barrier integrity. This provides a plausible mechanism connecting sequestration, coagulation defects and endothelial activation to SM pathogenesis (Gillrie et al., 2015; Petersen et al., 2015). 43 A plasma soluble form (sEPCR) which results from metalloproteinase-mediated cleavage of the membrane bound EPCR is also present in healthy individuals at very low concentration (Qu et al., 2007; Xu et al., 2000; Kurosawa et al., 1997). Soluble EPCR competitively binds to protein C with similar affinity (Fukudome et al., 1996; Regan et al., 1996). The levels of sEPCR in plasma are believed to be influenced by variations in the genetic sequence of EPCR. Significantly, an exon 4 variant (rs867186-G), with a serine-to-glycine substitution at position 219 appears to be associated with increased shedding of the membrane-bound form into circulation (Uitte de et al., 2004). Many studies have assessed the levels of sEPCR as well as the rs867186-GG variant in an attempt to link these levels with severity of many infectious diseases such as venous thrombosis (Medina et al., 2005; Medina et al., 2004). In malaria, the data shown so far are not clear cut presumably because few studies have been carried out on the relatively new receptor. Moussiliou et al (2015) reported elevated levels of sEPCR in Beninese children diagnosed with CM compared with controls. However, other studies showed reduced levels of sEPCR in SM compared with UM or community controls (Shabani et al., 2016; Hansson et al., 2015) and surprisingly higher levels recorded in convalescent samples in one study (Hansson et al., 2015). In this thesis, significantly elevated sEPCR levels were observed in malaria patients in general compared with healthy community controls (Chapter five). With regard to genetic studies, data from one study in Africa found that the rs867186-GG genotype was associated with increased levels of sEPCR and these levels mediated protection from SM (Shabani et al., 2016), whereas two other African studies found no link between the rs867186-GG genotype and protection from severe disease (Hansson et al., 2015; Schuldt et al., 2014). The only study conducted in Asia seems to suggest 44 that this particular EPCR genetic variant offer protection against SM (Naka et al., 2014). 2.7.3 Cluster of Differentiation 36 The class B scavenger receptor member, CD36, is an 88 kDa transmembrane glycoprotein widely expressed on endothelial and epithelial cells, macrophages, monocytes, platelets and adipocytes. It is keenly involved in fatty acid metabolism, innate immunity and angiogenesis (Febbraio et al., 2001; Greenwalt et al., 1992). Of all receptors recognised to bind to IE, CD36 binding seems to be the most frequently observed in almost all P. falciparum isolates spanning the various clinical categories or presentations and is perhaps advantageous to parasite survival (reviewed in Rowe et al., 2009; Serghides et al., 2003; Oquendo et al., 1989). Besides endothelial sequestration, IE binding to CD36 has been linked to both macrophage-mediated phagocytosis necessary to control parasite density (McGilvray et al., 2000) and also regulating dendritic cell function, in order to hamper immune response (Urban et al., 1999). Despite being one of the most extensively studied, its role in malaria pathogenesis remains highly unresolved. On one hand, studies from Africa explicitly show that CD36 binding in parasites obtained from SM is similar to parasites from UM (Rogerson et al., 1999; Newbold et al., 1997) and moreover results from human population genetics show no association (Fry et al., 2009). Reports from Asia on the other hand, are very conflicting since some data report a link to SM (Ockenhouse et al., 1991) and that CD36 mutations confer protection against severe disease (Omi et al., 2003). Since there is no clear data showing the direct involvement of CD36 in SM or protective mechanisms against severe disease, it is mostly accepted that they are more relevant in uncomplicated and asymptomatic malaria. Indeed, preferentially high 45 transcription of groups B and C var genes coding for CD36 binding phenotypes have been observed from parasite isolates of non-SM (Lavstsen et al., 2012; Kyriacou et al., 2006). Binding to CD36 occurs via CIDRα2-6 domains of groups B and C PfEMP1 and often times partnered by ICAM-1 (Ochola et al., 2011; Janes et al., 2011; Robinson et al., 2003; Baruch et al., 1997). Moreover, CD36 is hardly detected in the brain microvasculature, as IEs that adhere to HBEC have no affinity for CD36 binding, thus making it impossible for CD36 to be a key culprit in cerebral sequestration (Avril et al., 2012; Claessens et al., 2012; Turner et al., 1994). These data do not, however, offer any explanations on the ubiquitous CD36 binding observed in the vascular beds of many non-vital tissues including the skin, adipose tissue and muscle of individuals suffering from non-SM. Recently, the key structural requirements that define the widespread binding nature of IEs to CD36 were elucidated. Based on structural and biophysical analyses of CIDR sequences, Hsieh et al., (2016) revealed that a critical phenylalanine residue that extends from the surface of CD36 fits into a conserved hydrophobic pocket in PfEMP1. The binding cavity or pocket which is concave shaped and also highly unnoticeable or shielded suggests that it may be a difficult target for antibodies. It appears that the conserved phenylalanine residue is important for CD36 to carry out its biological function in fatty acid metabolism. Hence, it is nearly impossible to disrupt or alter this targeted region, since that may have detrimental or grave consequences on host metabolism (Hsieh et al., 2016). Altogether, it shows how smartly the parasite has capitalised on a physiologically relevant host protein by using its polymorphic PfEMP1 protein in order to promote its survival by eluding immune destruction. This complex mechanism to some extent explains the reason behind CD36 being the most frequently encountered or preferred parasite receptor in non- 46 vital organs. The process may facilitate parasite perpetuation as well as reduce host morbidity and mortality which are often associated with non-SM (Hsieh et al., 2016; Cabrera et al., 2014). 2.8 PfEMP1 in disease and immunity It is largely agreed in the literature that the PfEMP1 proteins are crucially important targets of immunity (Bull and Abdi, 2016; Hviid, 2010; Lusingu et al., 2006; Ofori et al., 2002; Bull et al., 1998). Parasite isolates from SM patients with low immunity seem to express a restricted subset of groups A and B/A PfEMP1 and these are more widely recognised by antibodies from older children who have experienced frequent bouts of P. falciparum malaria. Conversely, high transcription of PfEMP1 from group C have been observed from parasites isolated from older children with UM and asymptomatic infections (Warimwe et al., 2009; Kaestli et al., 2006; Kyriacou et al., 2006; Rottmann et al., 2006; Jensen et al., 2004; Nielsen et al., 2002; Ofori et al., 2002). More importantly, antibodies to recombinant PfEMP1 domains are recognised in a particular manner which is a non-random order, of which group A PfEMP1 variants associated with severe disease are firstly recognised (Cham et al., 2010; Cham et al., 2009). Thus, it can be inferred from these studies that some particular subsets of PfEMP1 molecules are preferentially transcribed by parasites causing SM. Indeed, recently accumulating data show that DC 4, 5, 8 and 13 which are members of group A or B/A PfEMP1 are linked with SM. Antibodies against these recombinant DCs are acquired early in life and appear to correlate with protection from severe disease (Turner et al., 2015; Bengtsson et al., 2013b; Bertin et al., 2013; Lavstsen et al., 2012; Magistrado et al., 2007). Together, these highlight the possibility of 47 developing a PfEMP1 based vaccine aimed at reducing SM associated mortality and morbidity in endemic areas. 2.9 Pathogenesis of CM It is well recognised that CM pathology is complex and is thought to be related to several factors including parasite cytoadhesion accompanied by a blockage in the cerebral microvasculature, imbalanced inflammatory immune responses and endothelial activation. Cytoadhesion is mediated by variant parasite surface proteins to multiple host proteins, while host cells including leucocytes, endothelial cells and platelets mediate the inflammatory response (Storm and Craig, 2014; Miller et al., 2013; Wassmer et al., 2011; Berendt et al., 1994; Clark and Rockett, 1994). The combined efforts of sequestration and inflammation are thought to drive endothelial activation resulting in vascular leakage, a compromise of brain barrier integrity and brain swelling leading to death (Seydel et al., 2015; Grau and Craig, 2012). It is entirely unknown which of the factors initiate the pathological processes. Also, the relative involvement or contribution of each of these events has been impossible to investigate and that has frustrated efforts to develop some form of supportive therapy and vaccine. 2.9.1 The sequestration model A prominent observation reported from detailed analysis of CM autopsy samples is an increased mass of sequestered IEs in brain cells (Milner, Jr et al., 2014; Taylor et al., 2004; Silamut et al., 1999; MacPherson et al., 1985), thus providing a link between 48 sequestration and CM. Sequestration, however, appears not to be exclusive to CM as it has also been observed in other organs including the heart and the small intestines with no pronounced pathology (Pongponratn et al., 2003). Sequestration results from the cytoadhesion of particular PfEMP1 variants including DC4, DC8 and DC13 to host receptors such as ICAM-1 and EPCR (Figure 2.7) on brain endothelial cells and that impedes the flow of blood, causes hypoxia and reduced clearance of toxic lactic acid (Hviid and Jensen, 2015; Dondorp, 2008; van der Heyde et al., 2006). Indeed, significantly increased levels of blood lactate concentrations correlate with CM development (Marsh et al., 1995). Also, there is a reduction in the deformability of IEs and uninfected erythrocytes leading to capillary plugging and that process has been linked to poor disease outcome (Dondorp et al., 2000). A number of important observations cannot be accounted for by the sequestration model. Information on the molecular details of CM pathogenesis is usually obtained from studies in P. berghei. Data from these studies show that cerebral IE sequestration is not associated with CM development but rather inflammatory processes are the key players (reviewed in Craig et al., 2012; Cabrales et al., 2010; de Souza et al., 2010). The brain-associated impairments reported in CM survivours are low compared to other brain-associated pathologies that can result in brain anoxia (reviewed in van der Heyde et al., 2006; Clark and Cowden, 2003). 2.9.2 The inflammation model During the asexual blood stage of malaria, parasites or their toxic waste products (haemozoin and glycophospatidyl inositol) which are liberated during the lysis of erythrocytes provoke cells of the immune system as well as endothelial cells to secrete 49 both pro- and anti-inflammatory cytokines such as IL-1, IL-6, IL-10, TNF-α and neuro-active mediators including nitric oxide (Schofield and Grau, 2005; Gazzinelli and Denkers, 2006). The inflammatory cascade involving activated immune cells and secreted cytokines, in turn, mobilise CD4+ and CD8+ T cells, all in an attempt to contain the infection. Notably, most of the malaria symptoms such as fever, nausea and headaches are the consequences of the inflammatory response in its attempt to control parasite replication and ultimately resolve the infection (Hunt et al., 2006; Schofield and Mueller, 2006; Urban et al., 2005; Stevenson and Riley, 2004). However, uneven ratios between pro- and anti-inflammatory responses to malaria infection resulting from improper regulation could be detrimental to the host and pathology of severe disease has been consistently associated with excessive systemic and local inflammation (Lyke et al., 2004; Dodoo et al., 2002; Day et al., 1999; Kwiatkowski et al., 1990; Grau et al., 1989). Accumulating evidence on the inflammatory mechanisms contributing to the induction of severe disease has been extrapolated from the murine Plasmodium berghei ANKA model and clinical studies in humans (reviewed in Hansen, 2012; Higgins et al., 2011; de Souza and Riley, 2002). Significantly, studies from knockout mice and inhibitory monoclonal antibodies demonstrate that the inflammatory response is central to the development of disease (reviewed in Schofield and Grau, 2005). In addition, elevated circulating levels of TNF-α and other cytokines such as high mobility group box 1 protein (HMGB1) present in the serum of severe patients significantly correlated with CM, were observed (Alleva et al., 2005; Lyke et al., 2004; Kwiatkowski et al., 1990). Observations of brain sections of CM autopsies revealed deposition of TNF-α and interferon γ [IFNγ] (Udomsangpetch et al., 1997). These cytokines are thought to contribute to disease severity by increasing the 50 expression level of cytoadhesion molecules such as ICAM-1, hence enhancing the binding of IEs to endothelial cells (Wassmer et al., 2005; Ho and White, 1999; Ockenhouse et al., 1992). The inflammation model has failed to explain some aspects of CM development; the elevated concentrations of mediators such as TNF-α recorded in patients with uncomplicated vivax infections and also the failure of anti- inflammatory agents to offer beneficial effects on disease severity but rather worsen the clinical course in some situations (Anstey et al., 2009; van Hensbroek et al., 1996). In summary, the data suggest that inflammatory cytokines may be important but not sufficient for CM development. Figure 2.7: A possible mechanism of ICAM-1 and EPCR cooperation in CM pathogenesis. DC8 or DC13-expressing IEs bind to EPCR resulting in stimulation of endothelial cells and release of pro-inflammatory cytokines such as TNF-α which leads to loss of EPCR and thrombomodulin from the surface of endothelial cells with subsequent activation of pathways that promote high expression of ICAM-1 molecules. IEs expressing either DC4 or containing the group A PfEMP1 ICAM-1 motif bind ICAM-1. The initial binding could also be mediated by ICAM-1 binding parasites containing the EPCR CIDR binding domain and that will lead to increased expression of ICAM-1 resulting in increased cytoadhesion, endothelial activation, compromise in barrier activities, brain swelling and death (Adapted from Hviid and Jensen, 2015) 51 2.9.3 Endothelial activation The endothelium consists of a monolayer of endothelial cells lining the vascular system and approximately 1013 endothelial cells can be found in a human adult. Endothelial cells together with pericytes, basal lamina, astrocytes and perivascular endfeet form critical components of the blood–brain barrier (reviewed in Abbott et al., 2006; Medana and Turner, 2006; Galley and Webster, 2004). A healthy endothelium produces nitric oxide which mediates vasodilation, downregulation of endothelial adhesion molecules, anti-inflammatory and antithrombotic processes (Bergmark et al., 2012). In malaria, the endothelium provides a platform where IEs interact with the human host with particular importance in CM where IEs are sequestered in cerebral endothelial microvessels. Endothelial activation appears to be mediated by multiple host and parasite factors (reviewed in Cunnington et al., 2013b; Miller et al., 2013; Grau and Craig, 2012; Kim et al., 2011) which intensify the pathological processes of malaria resulting in damage and apoptosis of ECs, vascular leakage, brain oedema and possibly death (reviewed in Seydel et al., 2015; Shikani et al., 2012). In addition, the repair mechanism of the endothelium is compromised due to significantly low levels of circulating endothelial progenitor cells (Gyan et al., 2009). Endothelial activation appears to be a prominent feature in CM pathogenesis (Conroy et al., 2010; Medana and Turner, 2006; Turner et al., 1998; Turner et al., 1994). It promotes high expression of some of its surface adhesion molecules which in turn are shed into the plasma. For instance, ICAM-1 is known to be significantly expressed on brain endothelial cells and is colocalised with sequestered IEs (Turner et al., 1994). Thus, endothelial activation upsets the vascular integrity by promoting increased IE sequestration through the overexpression of surface adhesion molecules and also by amplifying the inflammatory signals, forming the so-called positive feedback loop 52 (Cunnington et al., 2013b; Kim et al., 2011). Furthermore, activated endothelial cells lead to rapid exocytosis and release of activation markers such as von-Willebrand factor and angiopoietin-2 (Ang-2) from Weibel-Palade bodies into circulation (Matsushita et al., 2005), as a result of reduced nitric oxide bioavailability which usually prevents the release of Ang-2 (Hanson et al., 2015; Yeo et al., 2014). Endothelial activation and the subsequent dysfunction observed in severe falciparum malaria has also been reported in severe P. vivax malaria (Barber et al., 2015; Yeo et al., 2010), suggesting the non-exclusivity of this process. The Angiopoietin (Ang) family of proteins and their Tie-2 receptor are well known for their basic physiological assignment in regulating the endothelial barrier integrity (Conroy et al., 2012). Ang-1-mediated Tie-2 signalling is expressed constitutively and maintains the endothelium in a steady state, thus suppressing inflammation and preventing vascular leakage. Ang-2, however, acts as an antagonist to the protective effects of Ang-1 (Fiedler and Augustin, 2006). During endothelial activation resulting from impaired endothelial nitric oxide production, Ang-2 is rapidly released from endothelial cells and binds competitively to Tie-2 by displacing Ang-1. Consequently, Ang-2 signalling disrupts the stability of the endothelium by facilitating the loss of barrier integrity. Specifically, Ang-2 primes endothelial cells to be activated by other inflammatory cytokines including TNF-α and IL-1, thereby destabilising the microvascular integrity and resulting in increased vascular permeability (Bernabeu and Smith, 2017; Hora et al., 2016; Shikani et al., 2012; Fiedler and Augustin, 2006). A dysfunctional endothelium appears to be an important step in the progression to severe disease including fatal CM and this process is gaining widespread investigation. Notably, previous studies have collectively reported reduced circulating Ang-1 levels and increased Ang-2 levels in severe patients compared to 53 uncomplicated malaria patients (Conroy et al., 2012; Conroy et al., 2009; Lovegrove et al., 2009). Indeed, plasma Ang-1 and Ang-2 levels have been shown to be powerful predictive clinical biomarkers which are able to differentiate paediatric CM and retinopathy-positive patients and also provide information on the degree of retinal damage (Conroy et al., 2012). 2.10 PfEMP1-mediated IgM binding and disease severity Besides mediating adhesion to endothelial receptors, another striking feature of IEs expressing PfEMP1 is their ability to bind to soluble serum factors including non- immune IgM. Binding of non-immune IgM has been strongly connected to virulent P. falciparum phenotypes (Barfod et al., 2011; Creasey et al., 2003; Rowe et al., 2002). Transmission electron microscopy experiments provide initial evidence of fibrillar structures partly comprised of IgM from non-immune individuals linking IEs to uninfected erythrocytes in rosette formation (Scholander et al., 1996). Subsequent experiments have extensively described how IgM-mediated rosetting and more importantly anti-IgM antibodies to some extent can interfere with rosette formation (Clough et al., 1998; Scholander et al., 1996). Further studies demonstrated the occurrence of IgM binding in IEs that adhere to CSA in placental malaria (Creasey et al., 2003). Specifically, IEs expressing VAR2CSA, a PfEMP1 variant is responsible for binding to CSA (Salanti et al., 2004; Salanti et al., 2003). Although these placental IEs did not form rosettes, it appeared that they had dual capacities to bind to CSA and IgM (Barfod et al., 2011; Rasti et al., 2006; Creasey et al., 2003). The Fc portion (Cμ4 domain) of the human pentameric IgM is involved in binding to IEs expressing 54 PfEMP1. The IgM binding site in PfEMP1 has been mapped to specific DBLδ and DBLε domains (Stevenson et al., 2015a; Ghumra et al., 2008; Semblat et al., 2006). Although the molecular basis, as well as the physiological relevance of IgM binding to PfEMP1, is not fully understood (reviewed in Pleass et al., 2016; Czajkowsky et al., 2010), in rosetting for instance, it is thought that IgM together with other serum proteins „cross-bridge‟ and align multiple PfEMP1 molecules thereby tightening and possibly counteracting their relatively weak interactions with carbohydrate receptors on erythrocytes (Stevenson et al., 2015a; Stevenson et al., 2015b). By contrast, IgM binding to VAR2CSA seems to conceal important PfEMP1 epitopes targeted for phagocytic destruction, cleverly eluding immune destruction which is beneficial to parasite survival (Barfod et al., 2011). Some PfEMP1 domain subtypes have been repeatedly documented to bind IgM (Stevenson et al., 2015a; Ghumra et al., 2008; Rasti et al., 2006; Semblat et al., 2006). Given the clinical relevance of IgM binding, Jeppesen et al (2015) set out to investigate the number of IgM binding PfEMP1 in the P. falciparum NF54/3D7 parasite genome using the NF54-G6 clonal parasites, which have their var genes completely erased without affecting the viability or survival of the parasite. Briefly, P. falciparum G6 clonal parasites were transfected with the plasmid pVBH, which bypasses the mechanism that governs mutually exclusive var gene expression but drives the expression of blasticidin S deaminase and essentially allows the reset of PfEMP1 expression by deleting the epigenetic memory of var gene transcription. Thus, beginning with parasite clones with an almost non-existent var background and removal of the blasticidin pressure, transcription is induced which is accompanied by the expression of nearly all var genes in the parasite genome (Fastman et al., 2012; Dzikowski and Deitsch, 2008; Frank et al., 2007). These parasites can then be IgM 55 selected, single sorted based on the ability to bind IgM which can then be further functionally characterised. Taking advantage of this technology, Jeppesen and colleagues (2015) showed that a minimum of five IgM binding PfEMP1 are present in the genome of NF54/3D7 parasite. An important finding from this study was that the functional role of the identified PfEMP1 IgM binders was not limited to mediating rosetting and CSA adhesion, suggesting unknown mechanisms for IgM binding to other PfEMP1 variants. One possible mechanism may be to increase the host receptor binding arsenal involved in sequestration; hence circumvent immune detection and clearance (reviewed in Pleass et al., 2016). It appears that IgM binding may be an important phenotype (Jeppesen et al., 2015) and its numbers in the parasite genome may have been possibly underestimated. It is therefore pertinent to explore the possibility of discovering and characterising new PfEMP1 variants that bind IgM (Chapter seven) since such information may be relevant in guiding the development of interventions and also narrow the gap in the understanding of these adhesive interactions. 2.11 Blood outgrowth endothelial cells (BOECs) In order to elucidate the pathogenesis of SM to facilitate the development of interventions, it is very important to have a model that allows the monitoring of malaria right from parasite infection through the various stages to full blown disease manifestation. Human autopsy evaluations, in vivo animal models and in vitro cells have been the mainstream approaches in studying SM (Milner, Jr. et al., 2015; El- Assaad et al., 2014; Craig et al., 2012; Taylor et al., 2004). Although these studies provide invaluable descriptive data, they have obvious drawbacks. Postmortem 56 analysis evaluates tissues at end point, usually comparing non-fatal cases to fatal cerebral cases. In addition, access to samples is challenging because of ethical reasons, making this approach very unattractive (White, 2011; Medana et al., 2001). In vivo animal models have great advantages as they permit the monitoring of parasite development from inoculation to its transmission and have offered significant insights especially on the host immune factors. Unfortunately, the currently available models such as the rodent P. berghei do not fully replicate the pathophysiological features of CM as P. falciparum is only able to infect humans and not rodents. For instance, the molecular interactions characterising cytoadhesion of IE to the vascular endothelium as observed in CM is not conserved between rodent species and humans. Thus, the contribution from animal models to our current understanding of human malaria is heavily questioned (reviewed in El-Assaad et al., 2014; Craig et al., 2012; White et al., 2010). In the absence of appropriate experimental animal models, in vitro models are relied on for probing IE and endothelial cell interactions. In vitro models include cells derived from non-human origin such as transfected COS or CHO cells (Hasler et al., 1993; Berendt et al., 1989; Oquendo et al., 1989), and human cells including melanoma cells (C32) and primary (human dermal microvascular endothelial cells) or immortalised cells [human brain endothelial cells , HBEC5i] (Wassmer et al., 2006; Swerlick et al., 1992; Dorovini-Zis et al., 1991; Schmidt et al., 1982; Udeinya et al., 1981). Although these routinely used in vitro models for cytoadhesion have provided incredible information on host-parasite interactions, they may not reflect in vivo interactions (Andrews et al., 2005). Recently, Wassmer et al. (2011) isolated endothelial cells from subcutaneous fat tissue with comparable characteristics to brain endothelial cells. Although, this may represent a relevant model to study 57 cytoadhesion, the process of obtaining fat tissues may have ethical issues. The procedure appears to be delicate, time consuming, undesirable and hence cannot be routinely carried out. Clearly, more in vitro models are required to gain an in-depth understanding of disease mechanisms which will open up more possibilities for development of interventions. Blood outgrowth endothelial cells (BOECs), also known as circulating endothelial colony forming cells (ECFCs) represent an excellent model to study parasite binding characteristics since they are an easily accessible source of material with specific endothelial lineage. BOECs are differentiated matured endothelial cells which are a subset of mononuclear cells, present in extremely low quantities (0.010 %) in healthy people (Dauwe et al., 2016; Timmermans et al., 2007; Yoder et al., 2007; Ingram et al., 2005) and the numbers appear to be altered in some disease states demonstrating the extent of endothelial damage. Notably, circulating endothelial cells are markedly elevated in sickle cell disease and the endothelium has been shown to be activated in the crisis state (Sakamoto et al., 2013; Strijbos et al., 2009; Solovey et al., 1997). BOECs can be derived from peripheral blood or cord blood with very high growth capacity when expanded in in vitro cultures and they are believed to be extremely suitable for angiogenesis cell therapy or vascular regeneration (Dauwe et al., 2016; Martin-Ramirez et al., 2012; Timmermans et al., 2007; Hur et al., 2004; Ingram et al., 2004). BOECs are characterised by utilising different culture techniques and or a combination of endothelial cell surface molecules. Specifically, they have a distinct endothelial cobblestone morphology in cultures, express most of the known endothelial cell markers such as CD34, CD146, von Willebrand factor (vWF) and platelet endothelial cell adhesion molecule 1 (PECAM-1; CD31), are negative for the progenitor cell marker CD133 and are capable of taking up acetylated low density 58 lipoproteins (Lin et al., 2000). BOECs provide an invaluable system or tool to study pathological diseases involving endothelial perturbation and investigating differences in the transcription levels of endothelial genes (Chang et al., 2008; Fernandez et al., 2005). Indeed, BOECs have been successfully isolated from patients with several types or isoforms of von Willebrand disease in order to assess the storage as well as secretion of vWF (Wang et al., 2013b). Plasmodium falciparum infection represents a disease where the vascular endothelium is prominently involved, serving as a site for IE sequestration. BOEC provides an alternative in vitro model for studying the endothelial-related pathology of human malaria since it has the capacity to express a potentially diverse range of cytoadhesion receptors. 59 CHAPTER THREE THE ROLE OF AN ICAM-1 BINDING MOTIF IN PFEMP1 IN THE PATHOGENESIS OF CM 3.1 Introduction The remarkable variations in the disease manifestations of individuals with P. falciparum infections even under similar levels of exposure have long been recognised (Miller et al., 2002; Greenwood et al., 1991). Specifically, most individuals resident in malaria endemic populations are either asymptomatic or experience mild disease which can be effectively managed. However, a minor proportion of individuals predominantly children, unfortunately succumb to life-threatening and fatal forms of the disease that usually presents as CM, respiratory distress and SA (WHO, 2016; Storm and Craig, 2014; Miller et al., 2013). SM kills about 400,000 individuals annually with most deaths markedly concentrated in children under five years (WHO, 2016). CM represents the most deadly SM complication with a high fatality rate of about 20 % even after compliance to prescribed treatment regimen. Even more, approximately one-third of survivours have variable levels of neuro-cognitive impediments (reviewed in Birbeck et al., 2010; Idro et al., 2010). During the life cycle of P. falciparum, only early stage ring parasites are seen circulating in the peripheral blood; while mature stage parasites are withdrawn from circulation and preferentially sequester in the microvasculature in order to avoid splenic destruction (reviewed in Miller et al., 2002). Sequestration is believed to be the central precipitating event underlying CM pathogenesis as post-mortem studies provide evidence of extensive accumulation of IEs in brain vessels (Seydel et al., 2015; Milner, Jr. et al., 2014; MacPherson et al., 1985). Variant proteins exported to the surface of IEs are believed to promote sequestration by mediating adhesion to 60 diverse receptors displayed on endothelial cells which result in the blockage of blood flow, inflammation and damage to the endothelial barrier integrity (reviewed in Storm and Craig, 2014; Miller et al., 2002). Majority of the evidence gathered so far implicates the highly polymorphic parasite-derived protein PfEMP1, which are concentrated in knob-like projections on the surface of IEs (Howard et al., 1988; Leech et al., 1984a; Leech et al., 1984b). They are large proteins with variable molecular weights between 200-350 kDa and are encoded by approximately 60 different var genes which are expressed in a mutually exclusive pattern (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995; Howard et al., 1988). Depending on their chromosomal location, upstream sequences and transcriptional orientation, PfEMP1 encoding genes are generally assigned into groups A, B, C or any one of two two hybrid groups of B/A and B/C (reviewed in Hviid and Jensen, 2015; Kraemer and Smith, 2003; Lavstsen et al., 2003). All PfEMP1 have a similar organisational structure comprising variable cytoadhesion domains of Duffy-binding like proteins (DBL) and cysteine rich inter-domain region proteins (CIDR) that are responsible for binding host receptors. Within the DBL and CIDR domains, subgroupings based on sequence identity have been identified and these are believed to be associated with specific receptor interactions (Rask et al., 2010; Smith et al., 2000). Notably, CIDRα2-6 domains of groups B and C frequently adhere to CD36 (Hsieh et al., 2016; Robinson et al., 2003), while CIDRα1 of groups A and B PfEMP1 variants interact with EPCR (Lau et al., 2015; Turner et al., 2013). A fundamental question in malaria pathogenesis has always been whether a specific binding phenotype can be linked to any of different SM phenotypes given the observation that different receptors have been implicated in PfEMP1 binding. The enormous sequence diversity found within PfEMP1 molecules has frustrated efforts in 61 identifying such conserved functional regions. Nevertheless, the discovery that a unique PfEMP1 variant called VAR2CSA specifically interacts with placental CSA during PAM (Salanti et al., 2004; Fried and Duffy, 1996) appears to suggest the existence of some conserved PfEMP1 determinants involved in organ-specific severe disease. This striking example for PAM has spurred the continued search for restricted PfEMP1 variants responsible for other severe disease complications including CM. Indeed, many studies have shown that parasites expressing group A and B/A PfEMP1 that mediate EPCR binding are associated with SM syndromes (Bertin et al., 2013; Turner et al., 2013; Lavstsen et al., 2012). More importantly, the molecular details of the CIDRα subtype within PfEMP1 binding to EPCR were described based on sequence information (Lau et al., 2015). Altogether, these findings have heightened the interest of potentially identifying specific binding phenotypes connected with CM development, the most lethal form of severe disease. Earlier reports have suggested ICAM-1 as a receptor for sequestration of IEs during CM pathogenesis (Chakravorty and Craig, 2005; Kaul et al., 1998; Turner et al., 1994). Immunohistochemical assessment of autopsy specimens of CM provides evidence of increased endothelial ICAM-1 expression together with retained IEs in the brain microvasculature (Taylor et al., 2004; Silamut et al., 1999; Turner et al., 1994). However, the role of ICAM-1 in CM has not always been clear-cut. Indeed, the ability to bind ICAM-1 has been found both in DBLβ domains of groups B and C PfEMP1 associated with UM, and group A DBLβ domains linked to CM (Bengtsson et al., 2013b; Janes et al., 2011; Howell et al., 2008). Additionally, in vitro selection of IEs on human brain endothelial cells expressing PfEMP1 of DC8 and DC13 adhered to EPCR and not ICAM-1 (Turner et al., 2013). Altogether, this indicates that ICAM-1 binding may not be sufficient to initiate CM pathogenesis. 62 In 2013, Bengtsson et al., aimed to understand the molecular underpinnings of group A PfEMP1 associated with ICAM-1 binding in CM. The study searched for orthologous sequences of the group A PFD1235w var gene which is thought to mediate ICAM-1 binding and has been previously associated with the development of SM (Jensen et al., 2004). By using PFD1235w-specific primers and a degenerate group A exon 2–specific primer, pfd1235w-like genes were amplified from parasite isolates obtained from Ghanaian children with falciparum malaria. The PFD1235w- like var gene-encoded form of PfEMP1 obtained from the parasite isolates possessed highly identical three tandem domain (DBLα1.1/1.4-CIDRα1.6-DBLβ3) sequences located N-terminally and was called domain cassette four (DC4) [Figure 3.1]. Figure 3.1: Domain structural organisation of PfEMP1 proteins containing DC4. The PfEMP1 proteins consist of the 3D7 PFD1235w and five PfEMP1 isolated from Ghanaian falciparum parasites. The DC4 protein in defined by three tandem domains of DBLα1.4/DBLα1.1- CIDRα1.6-DBLβ3 and it is enclosed in the red rectangle. A high sequence identity is observed within DC4 among the parasite isolates in comparison with domains outside DC4 (Bengtsson et al., 2013b). 63 The domain involved in ICAM-1 binding was mapped to the DBLβ3 domain four (DBLβ3_D4) and not DBLβ3 domain five (DBLβ3_D5) which is located outside the DC4 region (Figure 3.1). Indeed, up to 80 % sequence similarity was observed among DBLβ3_4 domain of the different parasite isolates. Furthermore, a sequence analysis showed that DBLβ3 of DC4 formed a tight phylogenetic cluster which was different from other ICAM-1 binding and non-binding DBLβ domains (Figure 3.2). IEs expressing DC4 were found to mediate ICAM-1 binding. Importantly, naturally- acquired DC4 DBLβ3 antibodies were cross-reactive and cross-inhibitory to DC4- containing ICAM-1 binding IEs and to a non-DC4, group A DBLβ1 from Dd2VAR32 [DC13] (Bengtsson et al., 2013b). To locate the exact ICAM-1 binding site or region within the DBLβ3_4, hybrid proteins were expressed by domain swapping. Notably, the proteins consisted of possible combinations of the extracellular portions of the ICAM-1 binding DC4 DBLβ3_D4 and a non-ICAM-1-binding DBLβ3_D5 of 3D7 PFD1235w. Hybrid proteins harbouring the C-terminal portions (4C region) of DBLβ3_D4 maintained the ICAM-1 binding property (Figure 3.3) and also DBLβ3 ICAM-1 inhibitory antibodies were able to specifically target this region. The aim of this present study was to dissect further the molecular details characterising ICAM-1 binding by group A PfEMP1 by using sequence information to define a sequence motif that can be used to identify ICAM-1-binding DBLβ domains. Additionally, the relationship between the expression of DBLβ domains involved in ICAM-1 binding and CM development was investigated. This study formed part of a collaborative research with Professor Matthew Higgins‟ laboratory (Oxford University) where the structural biology experiments were done 64 and the data presented in this chapter have been published recently in the Cell Host and Microbe Journal (Lennartz et al., 2017; Appendix E). Figure 3.2: Phylogenetic tree of 16 ICAM-1 binding (red) and 19 non-ICAM-1 binding (blue) DBLβ domains. The round shaded area represents DBLβ domains present in DC4. ´A, B, C‟ represent the var groups in which the DBLβ can be found (Bengtsson et al., 2013b). 65 Figure 3.3: Identification of the ICAM-1 binding region in PFD1235w DBLβ3_D4. A model of the PFD1235w domains of DBLβ3_D4 and DBLβ3_D5 showing the extracellular portions (green, magenta, pink). Schematic diagrams of PFD1235w domains DBLβ3_D4 (gray), DBLβ3_D5 (white) and hybrids (H1–H6) of the extracellular portions (A-C). T1 represents a DBLβ3_D4 shortened at the C-terminal side. ICAM-1 binding was localised to the 4C region of DBLβ3_D4 (Bengtsson et al., 2013b). 3.2 Hypothesis and specific objectives Plasmodium falciparum IEs associated with CM express a particular subset of group A PfEMP1 that bind to ICAM-1 and EPCR. Such PfEMP1 contain a specific amino acid sequence (motif) in the DBLβ domain that mediates the ICAM-1 binding. Specific objectives 1. To evaluate the binding interactions between ICAM-1 and recombinant group A PfEMP1 DBLβ domains 66 2. To measure motif-specific antibody responses in plasma samples from individuals living in malaria endemic regions 3. To compare the transcription levels of motif-encoding var genes and EPCR binding CIDRα domains in defined categories of malaria patients 4. To determine the binding of IE expressing PfEMP1 containing the motif to ICAM-1 and EPCR under flow conditions 3.3 Methods 3.3.1 Study sites, ethics and sample collection The study cohorts were recruited from Hohoe Municipal Hospital in the Volta Region of Ghana (Kweku et al., 2008), from Korogwe District Hospital in the Korogwe District of Northeastern Tanzania (Lavstsen et al., 2012), and from the Centre National Hospitalier Universitaire Hubert Koutoucou Mega in Cotonou, Benin (Moussiliou et al., 2015). The study sites are characterised by marked differences in the intensity of P. falciparum malaria transmission and these were selected to increase the possibility of acquiring enough parasite isolates. The ethics and study protocols as well as permit to conduct the study were reviewed and approved by the Institutional Review Board of the Noguchi Memorial Institute for Medical Research, Ghana (Study number 026/13-14), the Ethical Review Committee of the Ghana Health Service (MOH; file GHS-ERC 08/05/14; Appendix A), the Ethical Review Board of the National Institute for Medical Research, Tanzania (National Institute of Medical Research/HQ/R.8a/Vol.IX/559) and by the Ethics Committee of the Research Institute of Applied Biomedical Sciences, Cotonou, Benin (N°21/CER/ISBA/13). Patients were only enrolled if their parents or guardians voluntarily consented to partake after the 67 goals of the study were explained and also if they met the study requirements. Study participants could exit from the study voluntarily without any consequences. Following informed consent, children younger than 12 years of age with differential SM clinical syndromes were enrolled in the study and underwent clinical investigations. Thick and thin blood smears were prepared on the same microscope slide from the blood collected. An aliquot of blood (7 μL) was placed at one end of a microscope slide and evenly spread out in a circle of approximately 1-2 cm diameter using one corner of a clean microscope slide to make the thick smear. Approximately 2 μL of blood was placed on the same microscope slide with the thick smear (almost in the mid portion). A second microscope slide was used to spread the blood across the slide quickly but gently to produce a thin film. The blood smear was air dried, fixed for 10 s in 100 % methanol (VWR, USA) and stained for 15 min with 20 % Giemsa (Merck, Germany) diluted in Giemsa buffer pH 7.2. The slide was gently rinsed with water, air dried and then examined under oil with the 100 X objective of a light microscope. Parasitaemia was estimated as parasites per 200 white blood cells. Patients who consented were grouped according to the clinical definitions outlined by the WHO on clinical and laboratory criteria (WHO, 2000). Patients with CM were defined as being microscopy positive for P. falciparum infection with a Blantyre coma score (BCS) ≤ 2, attributed to falciparum infections only. SA patients were defined as having haemoglobin (Hb) < 5g/dL and BCS >2 in addition to infection with falciparum. Other malaria patients were defined as having BCS >2 and Hb levels >5 g/dL with detectable peripheral falciparum parasites. Plasma samples from malaria-exposed Tanzanian individuals (1-17 years) involved in a previous study investigating risk factors for anaemia and febrile malaria (Lusingu et 68 al., 2004) and also nine plasma samples from immune Liberian adults from another previous study (Theisen et al., 2000) were additional samples used in this study to assess the acquisition of antibody responses to identified motif in individuals from different malaria transmission settings 3.3.2 Sample processing Processing of blood samples was carried out under aseptic conditions and all work was done in a microbiological biosafety II cabinet which was regularly cleaned with detergent and 70 % ethanol. Blood samples were centrifuged (800 x g; 5 min) to separate plasma samples. The pellet which contained IEs was washed in RPMI 1640 with 25 mM Hepes modified (Sigma-Aldrich, Germany), supplemented with 50 µg/mL gentamicin sulfate (Sigma-Aldrich, Germany), [referred to as incomplete RPMI] and centrifuged (500 x g; 5 min). The wash process was repeated once. The washed pelleted erythrocytes containing parasites was resuspended in Glycerolyte 57 freezing medium (Fenwal, USA) in a ratio of five volumes of freezing medium to three volumes of cell pellet in two steps. Firstly, one volume of freezing medium was added in dropwise with gentle swirling and the mix was allowed to stand for 5 min before slow addition of the remaining four volumes. The resulting suspension was gently mixed, aliquoted into cryovials (1.0 mL per cryovial) and placed in Mr. Frosty freeze containers to allow slow freezing at -80 ⁰C overnight before being transferred to liquid nitrogen for long-term storage. A portion of the washed pelleted IEs was stored in TRIzol reagent (Thermofisher Scientific, USA) for purification of RNA to assess var gene transcript levels. Briefly, TRIzol reagent (Thermofisher Scientific, USA) was allowed to equilibrate to room temperature (room temp) and about 50-200 µL of the pelleted erythrocytes was completely dissolved in nine volumes of TRIzol 69 reagent by pipetting several times to uniformly mix the pellet. The resulting mix was finally vortexed to obtain a dark brown suspension with no traces of clumps and was stored at -80 ⁰C freezer until use. 3.3.3 Recombinant proteins Recombinant DBLβ domains containing the ICAM-1 motif or non-motif DBLβ proteins and a head structure of PFD1235w (CIDRα1.6- DBLβ3) were produced in E. coli SHuffle C3030 cells [New England Biolabs, UK] (Bengtsson et al., 2013b). Briefly, these proteins were produced from genomic DNA amplified by PCR using published primers (Appendix B).The PCR products were sub-cloned into a modified pET15b vector and expressed as N-terminal, hexahistidine-tagged (his-tagged) proteins in E. coli SHuffle C3030 cells (New England Biolabs, UK). A hybrid of PFD1235w DBLβ3_D4 (amino acid 739-1,221) and PFD1235w DBLβ3_D5 (amino acid 1,239-1,689) termed D5_motif were cloned by overlapping PCR amplification and expressed in E. coli SHuffle C3030 cells. The expressed proteins were purified by immobilised metal ion affinity chromatography using HisTrapTM HP 1 mL column (GE Healthcare, Denmark). In addition to these proteins, previously produced DBLβ proteins (Table 3.1) were also used (Bengtsson et al., 2013b). The motif peptide (LYAKARIVASNGGPGYYNTEVQKKDRSVYDFLYELHLQNGGKKGPPPATHP YKSVNTRDKRDATDDTTP) derived from PFD1235w DBLβ_D4 was produced by Schaefer-N, Denmark. Human ICAM-1 combined with the Fc region of human IgG1 (ICAM-1-Fc) was cloned, expressed as His-tagged protein in HEK293-F cells (Invitrogen, USA) according to the manufacturer‟s instructions and described in (Bengtsson et al., 2013a) was previously produced. The recombinant protein was purified from the supernatant on a HiTrap Protein G High Performance column (GE 70 Healthcare, Denmark), eluted using 0.2 M glycine/HCl buffer (pH 2.5), neutralised in suspension using Tris/ HCl buffer (1 M, pH 9) and finally buffer-exchanged into phosphate buffered saline (PBS) (Bengtsson et al., 2013b). 3.3.4 Rat immunisations All procedures concerning animal experiments and immunisations complied with the Danish national regulations. The animal procedures described in this study were carried out according to the guidelines elaborated in act numbers LBK 1306 (23/11/2007) and BEK 1273 (12/12/2005). Permission to carry out experiments was granted by the Danish Animal Procedures Committee ("Dyreforsøgstilsynet") and detailed in permit number 2008/561-1498. Before immunisation, sera was collected from each animal and used in flow cytometry and binding assays as negative controls. Recombinant PfEMP1 DBLβ constructs (3D7 PFD1235w_D4 [new ID: PF3D7_0425800_D4]; and IT4VAR13 (new ID: ABM88750) and synthetic peptide, (LYAKARIVASNGGPGYYNTEVQKKDRSVYDFLYELHLQNGGKKGPPPATHP YKSVNTRDKRDATDDTTP) [Schaefer, Denmark] from 3D7 PFD1235w_motif were used to immunise rats (Wistar; 8-12 weeks of age; three animals per immunisation group) to generate rat antisera. Procedures to generate rat antisera preparations have been previously described (Bengtsson et al., 2013b). Briefly, rats were subcutaneously injected with 25 µg of protein in Freund's incomplete adjuvant followed by two booster vaccinations of 15 µg in incomplete Freund's adjuvant. Post- immunisation sera were taken two weeks following the last immunisation. 71 3.3.5 Antibody purification Rat IgG specific for DBLβ proteins and the motif (anti-PFD1235w_D4; anti- PFD1235w_motif; anti-IT4VAR13) were purified from rat antisera using previous procedures (Joergensen et al., 2010). Briefly, 1 mg of protein in each 1 mL was dialysed overnight against coupling buffer and further coupled to Hi-Trap NHS- activated High Performance columns following the manufacturer‟s protocol (GE Healthcare, Denmark). Antibodies were diluted (1:1 in PBS), affinity purified, eluted in glycine buffer (0.1 M, pH 2.75) and neutralised in 1M Tris/HCl (pH 9.0). Pre- immunisation serum or rat IgG (Sigma-Aldrich, Germany) was used as negative controls in binding assays. Pooled plasma from nine immune Liberian adults (Theisen et al., 2000) was used for affinity purification of DBLβ and motif specific human IgG (anti-PFD1235w_D4; anti- PFD1235w_motif; anti-IT4VAR13). Human IgG (Sigma- Aldrich, Germany) was used as negative control in binding assays. 3.3.6 ELISA-based binding inhibition assay ICAM-1 binding to different recombinant DBLβ proteins was measured by ELISA as previously described before (Bengtsson et al., 2013b). Maxisorp plates (Nunc, Thermofisher Scientific, Denmark) were coated with different recombinant DBLβ domains (50 µL; 2-10 g/mL; 0.1 M glycine/HCl buffer pH 2.75) overnight at 4⁰C and blocked with blocking buffer (PBS, 0.5 M NaCl, 1 % Triton-X-100, 1 % BSA, 0.03 mM phenol red, pH 7.2; 1 h; room temp). The plates were emptied and washed four times in PBS-Triton-X-100 (PBS, 0.5 M NaCl, 1 % Triton X-100, pH 7.4). Recombinant ICAM-1–Fc chimera (0.5-16 µg/mL; 50 µL) was added to duplicate wells and incubated (1 h; room temp). Following washing, bound chimera was detected with rabbit anti-human IgG-HRP (Dako, Denmark; 1:3,000) targeting the Fc- 72 portion of the ICAM-1-Fc. A total of 26 recombinant DBLβ proteins were screened for ICAM-1 binding. Similar to the protocol described above motif-specific IgG reactivity was assayed in plasma samples collected from immune adults. Plates were coated with motif containing DBLβ (50 µL; 2 or 4 g/mL; 0.1 M glycine/HCl buffer pH 2.75; overnight at 4 °C) and blocked with blocking buffer (1h; room temp). Diluted plasma (1:100) were incubated (1 h; room temp) and bound human IgG was detected using rabbit anti-human IgG (Dako, Denmark; 1:3000; in blocking buffer; 50 μL). Inhibition of ICAM-1 binding to DBLβ domains by rat antisera was performed using a modified version of the ELISA protocol. Briefly, plates were coated with ICAM-1 (50 µL; 2 or 4 g/mL ICAM-1; 0.1 M glycine/HCl buffer pH 2.75; overnight at 4 °C) and blocked with blocking buffer (1h; room temp). His-tagged recombinant DBLβ domains (0.5-16 µg/mL) were mixed with affinity purified antisera (1:5) or purified human IgG (0.25-32 µg/mL) and subsequently incubated (1 h; room temp) with pre- coated ICAM-1 plates. Following washing, bound DBLβ was detected with Penta-His antibody conjugated to HRP (Qiagen, UK; 1:3,000). All antisera were initially assessed for His-tag specific antibodies by ELISA and only sera that were negative for His-tag reactivity were included in the assay. 3.3.7 RNA purification and DNase treatment This work was carried out in the fume hood due to toxic fumes from chloroform and TRIzol. RNA is very sensitive to ubiquitous RNases, thus tubes and tips that have not been in contact with skin were used and “RNase Away” spray was used to clean pipettes. Gloves were won throughout the whole purification period and were changed frequently to prevent contamination. RNA was extracted from both field isolates and 73 selected ring stage laboratory parasite isolates (see section 3.3.13). Frozen TRIzol samples were thawed to room temp, 500 µL of thawed sample was pipetted into a 1.5 mL microcentrifuge tube and 500 µL of TRIzol (Thermofisher Scientific, USA) added. Two hundred microlitres of chloroform (Sigma-Aldrich, Germany) was added, the suspension was shaken vigorously for about 15 s, allowed to sit at room temp for 2-3 min and centrifuged (12,000 x g; 15 min; 4⁰C). The aqueous phase was collected, and 500 µL of isopropanol (Sigma-Aldrich, Germany) and 5 µL glycogen (Thermofisher Scientific, USA) were added. The suspension was mixed a few times, allowed to stand for 10 min at room temp and then centrifuged at 12,000 g for 10 min at 4 ⁰C to pellet RNA. The supernatant was gently decanted, the pellet washed in 1 mL of 75 % ethanol in 0.1 % diethylpyrocarbonate in deionised water (DEPC-H20), prior to centrifugation (7500 x g; 5 min; 4 ⁰C). The supernatant was gently aspirated without disturbing the pellet and dried (7-10 min; room temp). Dried samples were resuspended in 12 µL of DEPC-H20, heated at 65 ⁰C for 5 min and quickly placed on ice. Extracted RNA was immediately treated to remove genomic DNA contamination. DNase treatment was done according to the manufacturer by adding 2 µL DNase1 (Sigma-Aldrich, Germany), 1.5 µL of 10X DNase buffer (Sigma-Aldrich, Germany) to 11.5 µL of extracted RNA. Following incubation (20 min; 37 ⁰C), 2 µL of stop solution (Sigma-Aldrich, Germany) was added and then incubated again (70 ⁰C; 15 min) to inactivate the enzyme. The treated RNA was stored at -80 ⁰C freezer or processed for confirmation of the absence of DNA in RNA samples by quantitative real-time polymerase chain reaction (qPCR) with seryl-tRNA synthetase primers (p90) (Salanti et al., 2003). Briefly, 0.5 µL of RNA was added to 0.2 mL PCR tube containing 10 µL of SYBR Green PCR mastermix (Qiagen, UK), 8 µL of DEPC-H20 74 and 2 µM of p90. qPCR was carried out using a Rotorgene thermal cycler system (Corbett Research, UK) with the following cycling conditions: 95 C for 15 min, followed by 40 cycles of 95 ⁰C for 30 s, 50 ⁰C for 40 s, and 65 ⁰C for 50 s, with final elongation at 68 ⁰C for 40 s. 3.3.8 cDNA synthesis and qPCR Samples free of genomic DNA contamination were reverse transcribed to obtain cDNA. For the synthesis of first strand cDNA, 15 µL of RNA was placed in 0.2 µL PCR tube and 0.02 µg/µL of random primers (Thermofisher Scientific, USA), as well as 1.74 µM Deoxynucleotide Triphosphates mix (Invitrogen, USA) were added. The reaction mix was vortexed, briefly span, incubated for 10 min at 65 ⁰C and placed on ice immediately. To the reaction tube, 6 µL of 10x first strand buffer (Thermofisher Scientific, USA), 3 µL of dithiothreitol (Thermofisher Scientific, USA), 1 µL of RNaseOUT recombinant enzyme (Thermofisher Scientific, USA) and 1 µL of Superscript II enzyme (Thermofisher Scientific, USA) were added. The reaction mixture was vortexed and briefly centrifuged. cDNA synthesis was carried out in a PCR machine (MWG-Biotech, Germany) and synthesis settings used were incubation at 25 ⁰C for 10 min, 42 ⁰C for 50 min, 70 ⁰C for 15 min and 4 ⁰C for a minimum of 4 min. cDNA was stored at -20 ⁰C until use for var transcript analysis. cDNA was tested for absence of contaminating genomic DNA by qPCR with p90 primers (Salanti et al., 2003) as described above for the absence of contaminating DNA in purified RNA samples. Primers specific for var gene sub-types encoding group A DBLβ domains predicted to bind to ICAM-1 (motif primers: Q183/Q186) and/or var gene sub-types encoding CIDRα1 domains involved in EPCR-binding (“DBLα2/1.1/2/4/7”) (Mkumbaye et al., 75 2017) (Appendix B) were used to amplify these var gene groups from cDNA. For laboratory isolates, var gene specific primers for each of the var gene present in 3D7 (Dahlback et al., 2007; Salanti et al., 2003), HB3 (Soerli et al., 2009) and IT4 (Wang et al., 2012) were used. qPCR was performed in 20-μL reaction mix using QuantiTect SYBR Green PCR master mix (Qiagen, UK) on a Rotorgene thermal cycler system (Corbett Research, UK). The amplification cocktail included 0.2 μL of cDNA, 10 μL of Quantitect SYBR Green PCR Master Mix (Qiagen, UK) and 2 µM primers. qPCR cycling conditions were 95 ⁰C for 15 min, followed by 40 cycles of 95 ⁰C for 30 s, 50 ⁰C for 40 s, and 65 ⁰C for 50 s, with final elongation at 68⁰C for 40 s. Data were acquired at the end of elongation of each cycle and the specificity of amplifications was assessed by melting-curve analysis. The cycle threshold (Ct) was set at 0.025. The transcript abundance was determined relative to the average transcript abundance of the housekeeping gene seryl-tRNA synthetase using the formular: ΔCtvar_primer = Ctvar_primer − Ctaverage_control primers as previously described (Dahlback et al., 2007). ΔCt var_primer values were converted into Transcript Units (Tu = 2 (5-ΔCt)) as described previously (Lavstsen et al., 2012). Briefly, var transcript levels with a Ct value of 5 or higher than the average of the endogenous housekeeping gene Ct value was given a value of 5 and the Tu calculated as 2(5- ΔCtvar_gene). Notably, Tu value of 1 was given to ΔCtvar_gene values ≥5. ΔCtvar_primer = 4 is equivalent to Tu = 2, ΔCt = 3 ≈ Tu = 4, ΔCt = 2 ≈ Tu = 8, ΔCt = 1 ≈ Tu = 16, ΔCt = 0 ≈ Tu = 32 which is equivalent to the average value of the transcription of the endogenous housekeeping gene. 3.3.9 Parasite cultures Culturing of parasites was carried out under aseptic conditions and all work was done in a microbiological biosafety II cabinet which was routinely cleaned with detergent 76 and 70 % ethanol. The well-established laboratory P. falciparum clonal lines 3D7, HB3 and IT4 were used in this study. The HB3 isolate was originally obtained from Honduras (Bhasin and Trager, 1984), 3D7, a clone from NF54 which may possibly be of African origin (Walliker et al., 1987) and IT4 which was isolated from Brazil (Udeinya et al., 1983). These isolates (3D7, HB3 and IT4) were maintained for long- term in vitro and selected for IE surface expression of specific PfEMP1 (Bengtsson et al., 2013b; Joergensen et al., 2010). Plasmodium falciparum parasites were grown in sterile flasks of 25 cm2 or 75 cm2 (Thermofisher Scientific, Denmark) and cultured using a well-described method (Cranmer et al., 1997) with slight modification. Briefly, parasites were maintained in RPMI 1640 with 25 mM Hepes modified (Sigma-Aldrich, Germany), 50 µg/mL gentamicin sulphate (Sigma-Aldrich, Germany), 4 mM Glutamine (Sigma-Aldrich, Germany), 0.5 % Albumax II (Invitrogen, USA), [now referred to as culture medium] and human erythrocytes (O+). Parasites in flasks were cultured in a gas mixture of 2.0 % O2, 5.5 % CO2, and 92.5 % N 2 (Strand Møllen, Denmark) and incubated at 37 ⁰C. The culture medium was changed every other day with pre-warmed medium (37 ⁰C) and Giemsa-stained smears were also made from 3 µL of parasite culture to estimate parasitaemia. Cultures were routinely maintained at 1 % parasitaemia by adding washed human erythrocytes, culture medium and gassed. Human erythrocytes (O+) used for culturing were obtained from the Copenhagen Hospital Biobank (Denmark), washed twice in incomplete RPMI and centrifuged (500 x g; 8 min) to remove plasma and leucocytes. Washed erythrocytes were resuspended at 50 % haematocrit in incomplete RPMI and stored at 4 ⁰C for about two weeks. Mycoplasma infection was checked regularly using the MycoAlert Mycoplasma Detection Kit (Lonza, USA) according to the manufacturer‟s instructions. The genotype of parasites was verified by amplifying the 77 polymorphic proteins, merozoite surface 2 (MSP 2) and glutamate rich protein (GLURP) using standard methods (Snounou et al., 1999). 3.3.10 Thawing of frozen parasites All solutions used for thawing were pre-warmed to 37 ⁰C. The parasite stabilate was allowed to thaw rapidly at 37 ⁰C after careful removal from the liquid nitrogen. Following thawing, the content was transferred to a 50 mL falcon tube and 200 μL 12 % NaCl (VWR, USA) was added to 1 mL of packed cell volume in dropwise with gentle swirling. After 5 min of incubation at room temperature (room temp), 10 mL of 1.6 % NaCl (VWR, USA) was added in drops, followed by another 10 mL of 0.9 % NaCl (VWR, USA) containing 0.2 % dextrose (Baxter, USA). The mixture was centrifuged (500 x g; 5 min), washed once in incomplete RPMI and resuspended in culture medium at 2 % haematocrit. The cell suspension was transferred to a 25 cm2 culture flask, gassed for 30 s and incubated at 37 ⁰C. The next day, parasitaemia was assessed and medium replaced with fresh culture medium. 3.3.11 Gelatin purification This procedure separates mature stage trophozoite-IEs from the young and immature ring stages and uninfected erythrocytes, thereby synchronising the parasite culture. Briefly, cultures with predominant late stages (3-5 %) were centrifuged (500 x g; 5 min) and the supernatant carefully decanted. Pre-warmed gelatin (Sigma-Aldrich, Germany) [3 mL of 0.75 % in H2O] was added to resuspend the parasite pellet and incubated (37 °C; 20 min) to enrich for late stages. Mature IEs in the top layer of the parasite enriched gelatin suspension were carefully transferred to a round bottom tube, washed twice in incomplete RPMI (10 mL) and centrifuged (500 x g; 5 min). The 78 purified mature IEs were used for antibody selection (laboratory parasites) of specific PfEMP1 expression. 3.3.12 Magnetic activated cell sorting (MACS) purification Late trophozoites and schizonts (at least 2 %) were purified from uninfected erythrocytes and ring-stage IEs using MACS. This purification takes advantage of the accumulation of haemozoin, a degradative product of haemoglobin making late stage IEs susceptible to being captured by a strong magnet. The MACS column (Miltenyi Biotec, Germany) was fitted onto a magnetic stand; a 20 G needle and a 3-way stop were fitted onto the attached column. The MACS column was washed with 20 mL PBS supplemented with 2 % foetal bovine serum (Sigma-Aldrich, Germany) [2 % FBS] and to remove trapped air bubbles in the column. About 10 mL of 2 % FBS was allowed to run through the tap fitted onto column at a slow rate (about 2 drops per s). The parasite suspension (in 2 % FBS) was added to the column and allowed to flow through and the column was washed with 20 mL 2 % FBS until a clear run through was observed. A plunger was inserted on top of the column and a syringe filled with 15 mL of 2 % FBS was fitted onto the plunger. The column with the fitted syringe was removed from the magnet and the 2 % FBS in the syringe pushed forcibly through the column into a 50 mL falcon tube to elute the MACS-purified IEs. The syringe was filled again with 10 mL of 2 % FBS to elute any residual IEs still present in the column. The concentration of the cell suspension was enumerated on a Neubaeur haemocytometer and cells were used for IE surface reactivity flow cytometry assay or antibody specific selection. 79 3.3.13 Parasite selection for surface reactivity Late stage P. falciparum clonal lines (3D7, HB3, IT4) were selected for IE surface expression of specific PfEMP1 (Bengtsson et al., 2013b; Joergensen et al., 2010). Briefly, 3D7 IEs were selected with the human monoclonal antibody AB01 (15 µg), which targets 3D7 PFD1235w DBLγ13_D6 (PF3D7_0425800_D6) (Barfod et al., 2011) or with a polyclonal depleted rat anti-serum against PF11_0008 CIDRβ4_D6 (1:20 in incomplete RPMI). Plasmodium falciparum IT4 IEs were selected using sterile filtered depleted rat anti-serum (1:20 incomplete RPMI) against IT4VAR13 DBLβ5_D4 (ABM88750_D4), while the selection of HB3 IEs was done using sterile filtered depleted rat anti-serum (1:20 in incomplete RPMI) against HB3VAR03 DBLβ3_D4 (new ID: KOB63865_D4). For 3D7 selection, protein A coupled Dynabeads (25 µL; Invitrogen, USA) were aliquoted into 12 mL centrifuge tube and 8 mL of incomplete RPMI was added to wash the beads. The tube containing the beads was placed on a magnetic stand; the tube inverted a few times and then allowed to stand for about 1 min. The medium was aspirated and the wash process repeated. Sterile filtered AB01 (15 µg diluted in 1 mL incomplete RPMI) was added to the beads and incubated (30 min; room temp) on a rocking table. The tube containing the mixture was placed on a magnetic stand and the wash process repeated thrice to remove unbound antibodies. Gelatin purified 3D7 IEs were resuspended in 2 mL of incomplete RPMI and added to the AB01 coated beads and incubated (30 min; room temp) on a rocking table. For the 3D7 PF11_0008, HB3 or IT4 selection, gelatin-purified IEs resuspended in 2 mL of incomplete RPMI were added to sterile depleted rat antisera, incubated (30 min; room temp) and the unbound parasites removed by washing three times with 10 mL of incomplete RPMI, followed by centrifugation (500 x g, 5 min). Simultaneously, Dynabeads M-280 biotinylated 80 (50 µL; Invitrogen, USA) was aliquoted into 12 mL round bottom centrifuge tube and 8 mL of incomplete RPMI was added to wash the beads. The tube containing the beads was placed on a magnetic stand and the beads washed thrice as described before. Anti-rat IgG biotinylated (15 µL in 1 mL incomplete RPMI) was added to the beads and incubated (30 min; room temp) gently on a rocking table. The tube containing the mixture was placed on a magnet and the wash process repeated thrice to remove unbound antibodies. The bound PF11_0008, HB3 or IT4 parasites were added to the antibody-coated beads and incubated (30 min; 37 °C). All selected parasites were subsequently washed thrice as described before, resuspended in culture medium (7 mL) and 200 µL of human erythrocytes was added. The selected parasite suspensions were transferred to 25cm2 culture flasks, gassed and maintained in an incubator (37 °C). The beads were removed next day when parasites were at the ring stage. Briefly, the culture medium was aspirated and the parasites resuspended in fresh complete medium (10 mL). The parasite suspension was transferred to round bottom tubes, placed in a magnetic stand, inverted gently a few times and allowed to stand for 1 min. The medium containing the parasites were transferred to culture flasks, gassed and maintained at 37 °C. Giemsa-stained smears were prepared to assess parasitaemia and confirm parasite stage. The parasites are known to frequently alter their PfEMP1 expression and so the procedure was regularly performed every 2-3 weeks to maintain the predominantly expressed phenotype. The success of IE selection was routinely verified in transcription analysis using ring stage parasites and also flow cytometry assays by IE surface staining using the specific antisera or monoclonal antibody. Cultures with more than 60 % antibody-labeled IEs were used in the experiments. 81 3.3.14 IEs Surface reactivity to specific antibodies by flow cytometry The phenotypic identity of the P. falciparum clonal isolates (3D7, HB3, IT4) was assessed using flow cytometry as described before (Joergensen et al., 2010). Briefly, late stage IEs were MACS purified and 100 μL (2 × 106 cells/mL) of IEs was added to wells containing specific rat antisera (1:40) for the various isolates (anti - PFD1235w_D4; anti-HB3VAR03_D4; anti-PF11_0008_D6; anti-IT4VAR13). The samples were incubated (30 min; 4 ⁰C), washed twice by adding 200 μL of 2 % FBS and centrifuged (4 min; 500 x g; 4⁰C). Thereafter, IEs were incubated (30 min; 4 ⁰C) with FITC-conjugated goat anti-rat IgG (Vector Laboratories, USA) [1:150 in 2 % FBS; 100 μL/ well] and ethidium bromide (2 μg/mL; Sigma-Aldrich, Germany). The reaction mix was subsequently washed twice in 2 % FBS and the cells resuspended in 100 μL of 2 % FBS for acquisition of data on an FC500 MPL flow cytometer (Beckman Coulter, Denmark). Data were analysed using WinList version 6.0 (Verity Software House Inc.) and IEs were gated based on ethidium bromide positive cells. 3.3.15 Inhibition of IEs in flow-based adhesion assays Flow adhesion and inhibition assays were performed on laboratory-adapted parasites (3D7, IT4, HB3) selected to express specific PfEMP1 using previously described methods (Lennartz et al., 2015). The assays were performed by Dr. Yvonne Adams (University of Copenhagen). Notably, 3D7 parasites were selected to express PFD1235w or PF11_0008, IT4-selected IEs expressed IT4VAR13 and selected HB3 IEs expressed HB3VAR03 (see 4.3.6). Channels of microslides (VI0.1; Ibidi, Germany) were coated with recombinant ICAM-1-Fc [50 µg/mL], (Bengtsson et al., 2013a), EPCR-his [10 μg/mL] (Nunes-Silva et al., 2015) or with recombinant CD36 (rCD36; 20 μg/mL, R&D Systems, USA) and incubated in a humidified petri dish at 82 4°C overnight. Adhesion assays using human brain microvascular endothelial cells (HBMEC; Sciencell, USA) between passages two and five were seeded onto either fibronectin (2 μg/cm2; Millipore, Germany) pre-coated venaEC (Cellix, Ireland) [5x105 cells/mL] or microslides (VI0.4; Ibidi, Germany; 3x105 cells/mL) for 72–96 h. Stimulation of HBMEC was done by incubating with TNF-α (10 ng/mL; Thermofisher Scientific, USA) for 18 h. Microslides or venaEC (Cellix, Ireland) were blocked (1 h; 37 oC) with 1 % bovine serum albumin (BSA; Saveen Werner AB, Sweden) in PBS, mounted onto a Leica inverted phase contrast microscope and varying shear stresses (0.5 – 2 dyn/cm2) were generated by connecting to an NE- 1002X microfluidic pump (World Precision Instruments, UK). Cultured late-stage IEs adjusted to 3 % parasitaemia in RPM1 1640 with 2 % normal human serum (Copenhagen Hospital Biobank, Denmark) was subjected to a shear stress by passing or flowing over the microslides or venaEC for 5 min followed by another 5 min to wash off unbound IEs. The number of bound IEs per mm2 for five separate fields was counted at 20x magnification. The flow was stopped and adherent IEs were removed after each run and the experiments repeated for the next shear stress. All experiments were performed in triplicates and repeated at least three independent times. Flow channels were pre-incubated with 40 μg/mL anti-ICAM-1 antibody (clone 15.2, AbD Serotec, USA), 10 μg/mL anti-EPCR (polyclonal, R&D Systems, USA) or with 20 μg/mL anti-CD36 (monoclonal, Beckman Coulter, Germany) to test the specificity of IE adhesion to ICAM-1-Fc, EPCR-his, rCD36 and HBMEC. Inhibition of IE adhesion was carried out using the above protocol. Briefly, mature IEs were pre-incubated with affinity-purified IgG or human plasma (purified on D4 domain of 3D7 PFD1235w [PF3D7_0425800]) or motif antisera (targeting motif in 3D7 PFD1235w [PF3D7_0425800]), before being flowed on the microslides. 83 Negative controls included in the assay were affinity-purified non-immune rat or human IgG (Sigma-Aldrich, Germany). 3.3.16 Immunofluorescence assay Equivalent molarity (4 mM) of ICAM-1-Fc and goat-F(ab)2-anti-human IgG-Fc Dylight 488 (Abcam, UK) or EPCR-His with mouse-anti-PENTA-His (Qiagen, UK) and anti-mouse IgG-Alexa 568 (Abcam, UK) were incubated (4 °C; 4 h), aliquoted and thereafter frozen ( -20 °C) until ready for use. IEs (5 % parasitaemia) expressing PFD1235w, HB3VAR03, or IT4VAR13 were pelleted by centrifugation, washed with PBS and subsequently incubated with 2 % normal human serum (37 °C; 10 min). IEs were then washed twice with 1× PBS (Sigma-Aldrich, Germany) and incubated with thawed aliquots of the antibody: protein complex diluted 10x in 2 % Ig-free BSA (Sigma-Aldrich, Germany) diluted in PBS on a rocking table (37 °C; 10 min). IEs were washed twice with 2 % Ig-free BSA in PBS, air dried and mounted with ProLong Gold Antifade DAPI mounting medium (Life Technologies, USA) and examined using a Zeiss AxioImager Z1 equipped with a 63× oil inversion lens. Images were analysed with Zen Blue (Zeiss) and Fiji-ImageJ open source biological imaging software (Schindelin et al., 2012). Immunofluorescence assays were performed by Dr. Yvonne Adams (University of Copenhagen). 3.3.17 Bioinformatics analysis Multiple alignments of DBL domains with binding affinity for ICAM-1 were generated using MUSCLE v. 3.7 software (Edgar, 2004) and the MEGA software 84 (Tamura et al., 2007) was used to create sequence distance trees. Bioinformatics analysis was done by Professor Anja Jensen (University of Copenhagen). 3.3.18 Data analysis Clinical data of study participants were summarised as median, 25th and 75th percentile of values. Levels of var gene transcript among clinical groups were compared using the Wilcoxon-Mann-Whitney Rank Sum Test. To analyse whether study site influenced the association between ICAM-1 transcript levels and clinical outcome, a comparison was done using a logistic regression model with CM as outcome variable and study site (Benin, Ghana and Tanzania) as well as ICAM-1 transcript level as explanatory variables. Inhibition of ICAM-1 binding by antibodies was compared using the Wilcoxon-Mann-Whitney Rank Sum Test using SigmaPlot 13.0 (Systat Software Inc., United Kingdom). Adhesion inhibition experiments involving IEs were analysed by one-way ANOVA in GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, USA) and IE binding to recombinant receptors or HBMEC was analysed by two-way ANOVA with Tukey‟s Multiple Comparison. 3.4 Results 3.4.1 Binding to ICAM-1 by DBLβ hybrid protein containing ICAM-1 binding motif Bengtsson et al., (2013) showed that the C-terminal portion (4C region) of PFD1235w DBLβ3_D4 was responsible for ICAM-1 binding. In this study, the 4C region of DBLβ3_D4 was transferred to a related non-ICAM-1 binding DBLβ to generate a hybrid protein. Both the non-ICAM-1 binding DBLβ domain (DBLβ3_D5) and the 85 hybrid protein containing the minimal region involved in ICAM-1 binding (D5_motif) were tested using ELISA. The hybrid D5_motif protein bound to ICAM-1, whereas DBLβ3_D5 non-motif did not mediate binding to ICAM-1 (Figure 3.4A). Thus, the ICAM-1 binding property was gained by the non-ICAM-1 binding DBLβ protein. 3.4.2 Adhesion of IEs expressing motif-containing ICAM-1 binding DBLβ to ICAM-1 Further tests were carried out to determine whether antibodies raised against the ICAM-1 binding region would be capable of functionally inhibiting ICAM-1 binding to IEs. Briefly, rats were immunised with either the motif peptide or the DBLβ3_4 (D4) region mediating ICAM-1 binding. The resulting rat antisera were tested for their capacity to inhibit ICAM-1 binding of IEs expressing 3D7 PFD1235w, a group A PfEMP1 which is known to mediate ICAM-1 binding, a phenotype associated with cerebral IE sequestration (Bengtsson et al., 2013b; Turner et al., 1994). Purified anti- motif peptide IgG or DBLβ3_4 domain affinity purified on a 3D7 PFD1235w motif peptide were each incubated with selected mature IEs expressing PFD1235w, and thereafter allowed to adhere to recombinant ICAM-1 receptor under flow conditions. Controls in the assay included anti-ICAM-1 antibody, affinity purified non-immune rat IgG and rat antisera raised against IT4VAR13 which is a non DBLβ3_4 domain- containing ICAM-1 binder. Purified antibodies raised against DBLβ3_4 (α-D4) or the peptide (α-motif) significantly inhibited the binding of 3D7 PFD1235w IEs to ICAM- 1 (p = 0.004, one-way ANOVA), whereas ICAM-1 binding by 3D7 PFD1235w IEs was not blocked by IT4VAR13 antibodies (Figure 3.4B). To test the functional significance of this inhibition, similar experiments were repeated with immune plasma affinity purified on both recombinant D4 and the motif peptide. Similar to the rat 86 antibodies, human IgG against the motif peptide and D4 showed a significant inhibition of the binding of IEs to ICAM-1 (p = 0.004; one way ANOVA; 3.4C). Thus, the data suggest that the motif sequence is sufficient to facilitate binding to ICAM-1. 3.4.3 Binding of ICAM-1 to recombinant DBLβ containing ICAM-1 binding motif predicted from sequence analysis In order to determine the residues critical for ICAM-1 binding, structural analysis was done using a crystal structure of a group A PfEMP1 DBLβ domain in complex with ICAM-1 (performed by Professor Matthew Higgins‟ laboratory, Oxford University). Based on the data generated from the structural analysis together with the sequences in Bengtsson et al., 2013, a highly conserved sequence motif (I[V/L]x3N[E]GG[P/A] xYx27GPPx3H) harbouring the determinants of ICAM-1 binding by group A PfEMP1 was elucidated (Lennartz et al., 2017). The motif was used to search sequence databases resulting in the retrieval of 145 DBLβ domains with majority from group A PfEMP1 and a few B/A containing the motif and as such were predicted to mediate ICAM-1 binding (Lennartz et al., 2017). Based on these data, 26 randomly selected DBLβ domains were produced as recombinant proteins. ELISA was used to investigate ICAM-1 binding of 16 motif-containing DBLβ and 10 non-motif group A DBLβ domains (Appendix B). Among the 26 DBLβ proteins, eight were previously identified to bind ICAM-1 (Bengtsson et al., 2013b), but were identified as harbouring the motif in the sequence analysis (Lennartz et al., 2017). Ten proteins harbouring the motif and eight non-motif DBLβ proteins with unknown ICAM-1 binding properties were picked by sequence analysis of DBLβ sequences available from GenBank. The ELISA results showed that all of the 16 motif-containing DBLβ proteins bound 87 ICAM-1, although variability was observed in the degree of binding, whereas the 10 non-motif DBLβ were unable to bind (Figure 3.4D). Figure 3.4: ICAM-1 binding of predicted DBLβ-containing ICAM-1 binding motif. (A). ICAM-1 binding by ELISA (OD 490 nm; ± SD) of recombinant 3D7 PFD1235w DBLβ3_D4 (motif-containing), PFD1235w DBLβ3_D5 (non-motif), and a hybrid of DBLβ3_D5 containing the ICAM-1 binding motif region of DBLβ3_D4 (D5_motif). (B and C). Inhibition of 3D7 PFD1235W IEs binding to ICAM-1 under flow conditions using anti-ICAM-1 antibodies, affinity- purified anti-PFD1235w DBLβ3_D4 (anti-D4) IgG, anti-PFD1235w_motif IgG, anti-ITVAR13 and control IgG. IgG antibodies were affinity purified from rat anti-serum (B) and human serum (C). Results are shown as ±SD of a minimum of three independent experiments done in triplicate and data analysis was done using one-way ANOVA. * indicate significance (p = 0.0004). (D). ICAM-1 binding by ELISA (OD 490 nm; ± SD three replicates) of 26 recombinant group A DBLβ domains (4 DBLβ1, 14 DBLβ3, 2 DBLβ6, 2 DBLβ7, 2 DBLβ11, 1 DBLβ12 and 1 DBLβ unknown sub-class) from the structural analysis (Lennartz et al., 2017). All recombinant DBLβ-containing motifs bound ICAM-1, confirming the prediction of binding domains. „„DC‟‟ stands for the domain cassette harbouring the domain. „„ND‟‟ stands for unknown DC; the DC type is unknown as only the DBLβ sequence is available. „„No‟‟ stands for a domain that is not part of a documented DC. Presence of DC13 prior to DBLβ is indicated. Source: Lennartz et al., 2017 88 3.4.4 Phylogenetic analysis of ICAM-1 and non-ICAM-1 binding DBLβ domains Phylogenetic analysis of DBLβ domains known to interact with ICAM-1 and DBLβ S3 sequences obtained from whole genome-wide studies revealed that group A and B/A PfEMP1 containing the ICAM-1 binding motif formed one phylogenetic group which differed from the other non-motif DBLβ in PfEMP1 of group A non-ICAM-1 binders, groups B and C ICAM-1 binders. Further analysis revealed that the PfEMP1- containing motif also contained an adjacent EPCR CIDRα domain. Indeed, they formed a separate phylogenetic cluster from non-motif containing PfEMP1 with EPCR binding domain and the motif shown to be present in about 14 % of all var genes encoding an EPCR binding CIDRα domain. Conversely, groups B and C PfEMP1 that mediate binding to ICAM-1 that are also linked to CD36 binding via CIDRα subdomain were found to be spread across the phylogenetic tree (Figure 3.5). Taken together, the data indicated the existence of at least two evolutionary divergent groups of ICAM-1 binding PfEMP1. Group A PfEMP1 with binding affinity for ICAM-1 formed a subgroup which was uniquely different from groups B and C PfEMP1 mediating ICAM-1 binding. 89 Figure 3.5: Phylogenetic tree of ICAM-1 binding and non-ICAM-1 binding DBLβ. The phylogenetic tree was constructed using 1,823 DBLβ S3 sequence data from the seven published genomes and 226 annotated whole genome sequencing data. Shaded and open circles stand for DBLβ experimentally demonstrated as ICAM-1 and non ICAM-1 binders respectively. Colors represent the CIDR domain located N-terminal to DBLβ: red stands for EPCR binders containing ICAM-1-binding motif; orange stands for EPCR binders without ICAM-1-binding motif; green stands for non-EPCR binders (group A); blue stands for CD36 binders; magenta stands for VAR1. Source: Lennartz et al., 2017. 3.4.5 Cross-reactive adhesion inhibition by antibodies raised against motif- containing DBLβ motif-peptides The sequence analysis data showed that the motif sequences were strikingly conserved among ICAM-1 binding group A DBLβ domains and that suggested that the motif might elicit serologically cross-reactive responses that may correlate with protection following natural falciparum infections. To test this, immune plasma samples from Liberian resident adults were pooled and purified separately on three different DBLβ domains containing the motif (Dd2VAR32, KM364031, and PFD1235w [PlasmoDB: PF3D7_0425800] DBLβ_D4) by affinity chromatography. Inhibition of ICAM-1 90 binding to different recombinant DBLβ proteins using the purified plasma IgG pool was assessed by ELISA. The pooled plasma IgG purified on the different motif- containing domains demonstrated the capacity to block ICAM-1 binding to a diverse selection of motif-containing DBLβ domains of group A PfEMP1 except for KF984156 (Figure 3.6A). For instance, the IgG purified on the Dd2VAR32 motif domain achieved greater than 74 % ICAM-1 inhibition in 10/13 motif-containing DBLβ domains; the IgG did not inhibit ICAM-1 binding of a group B DBLβ domain (Figure 3.6A). As a control, similar inhibitory experiments were carried out using the plasma pool affinity purified on a related non-ICAM-1 binding DBLβ domain without the motif (PFD1235w DBLβ _D5). The resulting IgG showed very little or no inhibition of ICAM-1 binding of the different recombinant DBLβ domains screened (Figure 3.6A). To provide complementary data to support this cross-reactivity functionality, the ability of malaria-exposed children to acquire antibodies that could recognise the specific ICAM-1 binding site (motif peptide) present in the motif-harbouring DBLβ proteins and the extent to which these antibodies could interfere with ICAM-1 binding was determined. Briefly, a synthetic motif was used to screen plasma samples obtained from falciparum-exposed Tanzanian children. Majority of plasma samples (69/76; > 90 %) contained antibodies that had reactivity against the motif peptide. Further, plasma samples were affinity purified on the motif peptide and subsequently tested in ICAM-1 binding inhibition assays using DBLβ (D4 of PFD1235w). Notably, inhibition of ICAM-1 binding was markedly higher in plasma samples that demonstrated high motif peptide recognition (ELISA OD >1) relative to those with reduced reactivity towards the motif peptide (ELISA OD < 1; p=0.001) (Figure 3.6B). The data presented thus far showed that group A motif-containing ICAM-1 binding 91 PfEMP1 elicit cross-reactive IgG that prevents ICAM-1 adhesion to motif-containing DBLβ proteins. Figure 3.6: Binding inhibition of ICAM-1 to motif-containing PfEMP1 by plasma antibodies. (A). ICAM-1 binding inhibition by plasma pooled from adults. Purified IgG from plasma pool of immune adults inhibited ICAM-1binding of different motif-containing DBLβ domain variants (DBLβ_D4 domain from Dd2var32, KM364031, and PFD1235w) and no inhibition on a closely related, but non-ICAM-1-binding DBLβ_D5 domain from PFD1235w. ICAM-1 inhibitory capacity: >74% (black), 51%–74% (dark gray), 21%–50% (light gray), and 0%–20% (white). (B). ICAM-1 binding inhibition by plasma antibodies from children. Plasma with low (ELISA OD < 1) and high (ELISA OD > 1) motif reactive DBLβ IgG in malaria-exposed Tanzanian children (1-17 years; Lusingu et al., 2004) inhibited ICAM-1binding to motif peptide. Results are shown as boxplots with median. Whiskers = 5% and 95% percentiles. Source: Lennartz et al., 2017 92 3.4.6 Motif-encoding var transcript levels in SM patients The transcription level of var genes encoding group A PfEMP1 was analysed to determine if there was a link to the different clinical malaria syndromes. In short, a specific primer set targeting the motif and in addition to primer sets specific for DNA sequences encoding EPCR binding CIDRα domains (highly transcribed in SM, Turner et al., 2013) were designed. Ring stage parasites obtained directly from 115 children with either CM or non-cerebral SM (Table 3.1) were purified and used to synthesize cDNA. qPCR was performed with the designed primer sets using the synthesized cDNA as templates. The relative transcription level of the var genes was determined by normalising to an endogenous housekeeping gene (seryl-tRNA synthetase) and further converted to transcript units, Tu (Lavstsen et al., 2012). High median transcript levels (Tu = 7.5) were recorded for motif-encoding DBLβ genes in CM in comparison with SA (Tu = 3.0) and other SM (Tu = 2.4). The median motif transcript values did not significantly differ between SA and other malaria; both groups were combined as one entity and subsequently compared with the CM group (Table 3.2). Median levels of transcripts encoding motif-containing DBLβ in CM group markedly differed from the combined non-cerebral SM group (p = 0.020). Analysis of var transcripts encoding CIDRα EPCR binders recorded a median level of Tu = 82.7 for CM, Tu = 46.4 for SA and median level of Tu = 77 was recorded for other malaria (Table 3.2). Thus, in comparison with motif transcripts, high transcripts of CIDRα EPCR binders were found among all the SM groups, consistent with previous findings that show high transcription of EPCR binding CIDRα domains in SM (Bertin et al., 2013; Turner et al., 2013; Lavstsen et al., 2012). A similar analysis of CIDRα encoding transcripts was done by combining the non-cerebral groups as one group and comparing with the CM group. The results showed no significant difference between 93 CM and the combined non-cerebral SM group (p=0.751). Taken together, the data suggest a connection between the expression of motif-encoding var genes and development of CM. Table 3.1: Data on the study participants used for P. falciparum var gene expression experiments Children admitted to the hospital with malaria were classified according to the WHO guidelines as CM: cerebral malaria: Blantyre coma score (BCS) < 2 and no other identifiable course for the coma; SA: severe anaemia: haemoglobin <5g/dL and BCS >2; OM: other malaria, children admitted to hospital a with BCS >2 and haemoglobin >5g/L. Median (25 %; 75 % percentiles). Source: Lennartz et al., 2017. Table 3.2: Var subtypes transcript levels in children admitted with malaria Transcription levels of var genes measured using motif-specific primers, EPCR binding CIDRα primers and cDNA from ring stage parasites isolated from study participants. The relative transcription level was determined using an endogenous housekeeping gene (seryl t-RNA synthetase) and then converted to Tu (arbitrary units). Data are presented as median with 25 % and 75 % percentiles. Source: Lennartz et al., 2017 94 3.4.7 Selection of parasites expressing PfEMP1 domains predicted to bind both EPCR and ICAM-1 Results from the bioinformatics and sequence analysis indicated that all PfEMP1 containing the motif additionally harboured an adjacent CIDRα domain known to mediate EPCR binding (Lennartz et al., 2017). This suggested that IEs expressing a combination of these domains to bind both ICAM-1 and EPCR. The study further hypothesized that PfEMP1 harbouring the motif and being able to bind EPCR are likely to be involved in CM pathogenesis. To test this, HB3 and 3D7 IEs were antibody-selected (see section 3.3.13) to obtain IE expressing HB3VAR03 and PFD1235w, two PfEMP1 proteins known to mediate binding to ICAM-1 and EPCR. Additionally, IT4 IEs were antibody selected to enrich for IE expressing IT4VAR13, a dual CD36 and ICAM-1 binding PfEMP1. Finally, 3D7 IEs were selected to express PF11_0008 PfEMP1 (PlasmoDB: PF3D7_1100200), which is known to bind PECAM-1 (Joergensen et al., 2010). Surface expression of HB3VAR03, 3D7 PFD1235w, PF11_0008 and IT4VAR13 were confirmed by var transcription and flow cytometry and the data are shown in figure 3.7. Var transcription using gene-specific primer sets for the different parasite isolates showed the prominent transcription of PFD1235w in 3D7 IEs (Figure 3.7B), VAR03 in HB3 IEs (Figure 3.7D), and VAR13 in IT4 IEs (Figure 3.7F). In 3D7 PF11_0008 PF11 IEs (Figure 3.7H), in addition to the dominant expression of the selected var gene, minimal transcripts of other var genes were also observed. Results from the flow cytometry experiments in figure 3.7 (3.7A, 3.7C, 3.7E, 3.7G) showed that the different IE populations were homogenously labeled by the specific antisera and this was reflected by a shift in the fluorescence intensity of the positive population in comparison with unstained IEs. 95 Figure 3.7: var/PfEMP1 expression of HB3, IT4 and 3D7 IEs. Specific primers for the different IEs were used in qPCR on cDNA isolated from ring stage- selected IEs. Transcription profile shows the dominant var transcripts following antibody selection. Flow cytometry data of different late stage-selected IEs stained with ethidium bromide and specific antisera. (A). 3D7 PFD1235w ethidium bromide stained IEs with (grey histogram) and without (black histogram) rat PFD1235w DBLβ3_D4 antiserum. (B). Var gene transcript levels present in 3D7 PFD1235W IE normalised with a housekeeping gene (seryl-tRNA synthetase). The domain organisation of the prominently expressed PfEMP1, PFD1235w is indicated. (C). HB3VAR03 ethidium bromide stained IEs with (grey histogram) and without (black histogram) rat VAR03_D4 antiserum. (D). Var gene transcript levels present in HB3VAR03 IE normalised with a house keeping gene (seryl-tRNA synthetase). The domain organisation of the prominently expressed PfEMP1, VAR03 is indicated. (E). IT4VAR13 ethidium bromide stained IEs with (grey histogram) and without (black histogram) rat VAR13_D4 antiserum. (F). Var gene transcript levels present in IT4VAR13 IE normalised with a housekeeping gene (seryl-tRNA synthetase). The domain organisation of the prominently expressed PfEMP1, VAR13 is indicated. (G). 3D7PF11_0008 ethidium bromide stained IEs with (grey histogram) and without (black histogram) rat PF11_0008_D6 antiserum.(H) Var gene transcript levels present in 3D7 PF11_0008 IE normalised with a house keeping gene (seryl-tRNA synthetase). The domain organisation of the prominently expressed PfEMP1, PF11_0008 is indicated. Source: Lennartz et al., 2017 96 3.4.8 Adhesion of selected IEs to immobilised recombinant ICAM-1 and EPCR receptors The capacity of selected IEs (3D7 PFD1235w, HB3VAR03, IT4VAR13) to adhere to recombinant receptors were analysed in a flow-based system using physiologically relevant shear stresses (0.5-5 dyn/cm2). Recombinant human receptors ICAM-1, EPCR, CD36, or combinations of ICAM-1/EPCR or ICAM-1/CD36 were coated on channels of microslides and the selected IEs were passed on the pre-coated microslides for them to adhere. The binding assays were performed in triplicates, repeated at least three independent times and the average number of IEs/mm2 per surface area estimated. Data are shown in figure 3.8. HB3VAR03 and PFD1235w IEs were observed to bind ICAM-1 and EPCR independently from 0.5-1.0 dyn/cm2 with higher numbers bound at 1.0 dyn/cm2 (Figures 3.8A; 3.8B). Binding to individual receptors markedly decreased at 2.0 dyn/cm2 with nearly no detectable binding in HB3VAR03 IEs (Figure 3.8A). When both receptors were available at the same time, a high number of IEs were bound at 1.0 dyn/cm2 compared with 0.5 dyn/cm2 similar to results obtained for binding to the individual receptors. However, IEs were still bound at 2.0 dyn/cm2, which is comparable to numbers observed at 1 dyn/cm2 (Figure 3.8A; 3.8B). This observation at 2.0 dyn/cm2 sharply contrasts the data generated from binding to individual receptors alone. The dual ICAM-1 CD36 binding IEs (IT4VAR13) showed increased binding from 0.5-1.0 dyn/cm2 to ICAM-1 receptor alone or when both receptors were simultaneously available (Figure 3.8C). Higher number of IT4VAR13 IEs adhered when both receptors were present in comparison with adhesion to CD36 receptor only. Binding to CD36 however, decreased after 0.5 dyn/cm2 to almost no-detectable levels at 1.0 dyn/cm2. At a high shear stress of 2.0 dyn/cm2, binding to individual or 97 combined receptors sharply decreased (Figure 3.8C). Thus, the observed result was noticeably different from the binding observed for the EPCR and ICAM-1 dual binding parasites and it suggested that the former parasites (HB3VAR03 and PFD1235w IEs) are able to withstand binding at high shear stress. This data were further supported by immunofluorescence assays which showed binding of ICAM-1 and EPCR on the surfaces of both HB3VAR03 IEs and 3D7 PFD1235w IEs, whereas ICAM-1 was only localised on the surface of IT4VAR13 IEs (Figure 3.8E-3.8G). Control experiments were conducted with IEs selected to express PF11_0008 PfEMP1 (PlasmoDB: PF3D7_1100200) which is known to bind to PECAM-1 (Figure 3.8D). No binding was observed to any of the receptors investigated. 98 Figure 3.8: Binding of P. falciparum laboratory isolates to recombinant protein receptors. (A-D). Channels of microslides were pre-coated with rICAM-1, rEPCR, rICAM-1 and rEPCR; rCD36; or rICAM-1 and rCD36. Binding of (A) HB3VAR03 IEs (predicted ICAM-1 and EPCR binder), (B) 3D7PFD1235w IEs (predicted ICAM-1 and EPCR binder), (C) IT4VAR13 IEs (ICAM-1 and CD36 binder) and (D) 3D7PF11_0008 IE (PECAM-1 binder) to the immobilised 2 receptors was performed in parallel at variable shear stresses (0.5 to 5.0 dyn/cm ). Results are triplicates 2 of at least three independent experiments and presented as bound IEs per mm ± SEM (y-axis) plotted against shear stress (x-axis). Data were analysed by two-way ANOVA with Tukey‟s Multiple Comparison. (E-G). Images from immunofluorescence assays showing IEs of (E) HB3VAR03, (F) 3D7PFD1235w, and (G) IT4VAR13. Complexes of ICAM-1or EPCR were incubated with the different IEs to assess binding by immunofluorescence. Results show overlays of ICAM-1 (green), EPCR (red), and nuclear (blue) staining. Inserts show single channels. Scale bar, 2 µm. Source: Lennartz et al., 2017 99 The physiological consequence of the dual binding property of motif-containing PfEMP1 was investigated using HBMEC which is known to constitutively express ICAM-1 and EPCR on their surface. Binding was done using both resting and TNF-α stimulated HBMEC cells (passages 3-5). Blocking antibodies to each receptor were used to evaluate the influence of either ICAM-1 or EPCR binding alone or the effect of the combined receptors in binding. Shear stresses (0.5-2.0 dyn/cm2) similar to the experiments done with recombinant proteins were used. The data are presented in figure 3.9. It was observed that HB3VAR03 IEs were bound to HBMEC that had not been pre-incubated with any receptor antibody between 0.5-2.0 dyn/cm2 shear stresses (Figure 3.9A). By contrast, pretreatment with antibodies against either EPCR or ICAM-1 receptors slightly reduced the binding of HB3VAR03 IEs to HBMEC from 0.5-0.75 dyn/cm2. Furthermore, a significant blockage in the binding of HB3VAR03 IEs to HBMEC was already observed at a low shear stress of 0.5 dyn/cm2 in the presence of combined antibodies against EPCR and ICAM-1 (80 %, p = 0.0361). At increasing shear stress, a similar binding reduction pattern of HB3VAR03 IEs to HBMEC was further observed in the presence of either single receptor or combined receptor blockage. At a shear stress of 1.0 dyn/cm2, a significant reduction in adhesion of HB3VAR03 IEs to HBMEC treated with individual receptor antibodies was recorded (Figure 3.9A). Inhibition of 3D7 PFD1235w IEs binding to HBMEC blocked with either single receptor antibody or combined anti-EPCR and anti-ICAM-1 antibodies were observed at all the tested shear stresses for the various conditions (Figure 3.9B). This observation differed in comparison with the pattern of inhibition observed in HB3VAR03 IEs. Equivalent experiments were carried out with the dual ICAM-1 CD36 binding isolate IT4VAR13 IEs. HBMEC treated with anti-EPCR antibodies did 100 not affect the binding of IT4VAR13 IEs to ICAM-1 as expected, whereas HBMEC blocked with either ICAM-1 antibodies or the combined antibodies were inhibited at all shear stresses (Figure 3.9C). Similar adhesion inhibition assays were done with HBMEC stimulated overnight with TNF-α using all the parasite lines. In the absence of any antibody treatment, increased binding to stimulated HBMEC by all IEs was observed (Figure 3.9D-3.9F) in comparison with binding on resting HBMEC at shear stresses between 0.5-2.0 dyn/cm2. When stimulated and blocked with anti-EPCR antibodies, the number of HB3VAR03 IEs and 3D7 PFD1235w IEs bound were comparable to the numbers observed in stimulated cells only (Figures 3.9D, 3.9E). This may be due to the decreased EPCR expression following TNF-α treatment (Fukudome and Esmon, 1994). Thus, in the absence of EPCR, the parasites depended solely on ICAM-1 to attach to HBMEC. Treatment of HBMEC with either anti-ICAM-1 antibodies or combination of ICAM-1 and EPCR antibodies at all shear stresses blocked or prevented the binding of all IEs to stimulated HBMEC (Figure 3.8D-3.8F). 101 Figure 3.9: Binding of P. falciparum laboratory isolates to HBMEC under flow conditions. Binding of (A and D) HB3VAR03 IEs (predicted ICAM-1 and EPCR binder), (B and E) 3D7PFD1235w IEs (predicted ICAM-1 and EPCR binder), and (C and F) IT4VAR13 (ICAM-1 and CD36 binder) to resting (A-C) human brain microvascular endothelial cells (HBMEC) or TNF-α stimulated (D-F) HBMEC on pre-coated chips was performed in parallel at variable shear stresses (0.5 to 5.0 dyn/cm2). To assess specific adhesion, channels coated with HBMEC were pre-incubated with anti-ICAM-1 (40 µg/mL), anti-EPCR (10 µg/mL), or anti-ICAM-1 and EPCR combined (40 and 10 µg/mL, respectively). Results are triplicates of at least three independent experiments and presented as 2 bound IEs per mm ± SEM (y-axis) plotted against shear stress (x-axis). Data were analysed by two- way ANOVA with Tukey‟s Multiple Comparison. Source: Lennartz et al., 2017 102 3.5 Discussion The virulent parasite protein PfEMP1 is well recognised for its notorious sequence variability within and between the genome of different isolates. A mechanism adopted by the parasite to subvert the host immune system (reviewed in Chan et al., 2014; Miller et al., 2002). This has constituted a major bottleneck in considering PfEMP1 as an attractive vaccine immunogen. This significant impediment is highlighted in a study that used crystal structures of CIDRα1 bound to EPCR in an effort to identify conserved features that could be targeted for therapeutics against SM (Lau et al., 2015). They showed that the overall organisational structure of the CIDRα1 domain of PfEMP1 is preserved to mediate interaction with its receptor, however, the residues that directly interact with EPCR are significantly variable (Lau et al., 2015). Thus, remarkable sequence variability is observed even in PfEMP1 with the same binding phenotype and that significantly hampers the possibility of generating extensive cross- reactive natural responses against these CDIRα1 residues that directly interact with EPCR. Notwithstanding, this study used sequence information and structural analysis of a group A PfEMP1 protein bound to ICAM-1 to identify a highly conserved sequence motif. This motif was used to identify an array of DBLβ domains of group A PfEMP1 predicted to mediate ICAM-1 binding. Indeed, the recombinant DBLβ proteins containing an ICAM-1 binding motif predicted from the in silico analysis all successfully bound to ICAM-1 in contrast to the group A DBLβ lacking the motif. Furthermore, antibodies raised against the motif effectively blocked the interaction of IEs mediating ICAM-1 binding. Altogether, the data showed that the motif mediates ICAM-1 binding (Figure 3.4). Previous studies have demonstrated that group A PfEMP1 are prominently expressed by parasites isolated from children with SM complications including CM and also 103 antibodies directed against group A PfEMP1 are acquired relatively faster and are believed to be cross-reactive (Bengtsson et al., 2013b; Cham et al., 2009; Joergensen et al., 2010; Jensen et al., 2004). This study provides evidence to support this notion and also demonstrates further that functionally important conserved epitopes indeed occur in group A PfEMP1 and are capable of eliciting cross-reactive responses. Such responses would be of great importance in combating CM development. qPCR data revealed markedly high transcription of motif-containing DBLβ in parasites obtained from CM in comparison with parasites from non-cerebral severe patients, showing a direct connection between the expression of motif-containing ICAM-1 DBLβ and development of CM (Table 3.2). Even more, data gathered from this study showed that individuals in malaria-endemic regions are naturally exposed to motif-containing DBLβ early in life; such individuals accumulated broadly cross-reactive functional IgG capable of inhibiting the interaction between ICAM-1 and motif-containing DBLβ domains of different falciparum genomes (Figure 3.6). The identification of high levels of transcripts encoding motif-containing ICAM-1- binding DBLβ in CM provoked the question of why there have been inconsistent reports on the importance of ICAM-1 in CM development. While doing data mining of all currently available PfEMP1 sequences, it was discovered that all PfEMP1 that contained the motif additionally harboured an immediate upstream CIDRα domain and that potentially explained to some extent the discrepancy surrounding the connection between ICAM-1 and CM (Figure 3.5). Furthermore, it suggested why ICAM-1 could not have been the absolute receptor driving CM pathogenesis and also pointed to a likelihood of ICAM-1 and EPCR cooperation in CM. Previous studies have shown that IEs expressing PfEMP1 are capable of mediating adhesion to multiple receptors (Avril et al., 2016; Adams et al., 2014; Oleinikov et al., 2009; 104 McCormick et al., 1997). Recently, Avril et al (2016) showed that CIDRα mediating EPCR-binding and ICAM-1-binding DBLβ domains exist in a single PfEMP1. However, there were no documented reports on the ability of these PfEMP1 to engage both EPCR and ICAM-1 concurrently. Additionally, the benefits this dual adhesion offers the parasites were unknown. In this study, IEs expressing a single PfEMP1 capable of mediating adhesion to EPCR and ICAM-1 were selected (Figure 3.7) and assessed for binding under conditions that mimic in vivo blood flow, to understand the physiological impact of dual binding. IEs increased their ability to adhere at higher shear stress only when both EPCR and ICAM-1 were available for concurrent binding (Figures 3.8, 3.9). Upon TNF-α stimulation of cells, the IEs expressing the dual PfEMP1 domains markedly depended on ICAM-1 for binding, owing possibly to a decreased expression of EPCR (Fukudome and Esmon, 1994). Altogether, the results suggested that dual binding to EPCR and ICAM-1 equipped the parasite with flexible options that permitted enhanced cytoadhesion at physiologically higher shear stresses and also enabled the parasite to remain adherent during alterations in receptor expression resulting from cell stimulation. Thus, the process of increased cytoadhesion found in dual binding IEs facilitates the development of CM. Stronger adhesion of IEs to both ICAM-1 and EPCR in the brain may drive disease pathogenesis through common pathways that involve both receptors and thus promote CM development. Indeed, adhesion of IEs to EPCR has been shown to disrupt the activation of protein C and further block downstream signaling pathways that regulate the vascular barrier properties that maintain the endothelium in an anticoagulant and anti-inflammatory state (Petersen et al., 2015; Turner et al., 2013). Even more, IE adhesion to EPCR results in a significant depletion of the normally low levels of EPCR on endothelial cells and an increase in adhesion molecules such as ICAM-1 105 (Moxon et al., 2013) that may promote further sequestration of IEs and increased pathology in the brain (Moxon et al., 2013). Thus, it is conceivable that the parasite is equipped with dual receptor binding capabilities to resist endothelial changes such as loss of EPCR functionality which occurs during IE binding or adhesion and still be retained in the brain by depending on an overexpressed ICAM-1 receptor for cytoadhesion. Sequestration of IEs undoubtedly plays an important role in CM pathology (Ponsford et al., 2012; Silamut et al., 1999) and it is strongly associated with inflammation and more importantly contributes greatly to increased total parasite biomass, a critical determinant for development of CM (Storm and Craig, 2014; Cunnington et al., 2013a). The combined activities of sequestration and a perturbed inflammatory response give rise to endothelial dysfunction which may lead to alterations in the blood-brain barrier permeability, cerebral edema and coma (Sahu et al., 2015; Seydel et al., 2015; Storm and Craig, 2014; Idro et al., 2010). Brain swelling and increased intracranial pressure are notable features recorded in children with CM and it is a high risk factor for death (Seydel et al., 2015). Consistent with that, Seydel et al (2015) in a recent study reported severe brain swelling in 84 % of children who died from CM related complications in comparison with 27 % of survivours. Although the molecular details and sequence of CM pathogenesis remain partially elucidated, it is well recognised to be a complicated disease involving multiple factors including sequestration as well as other parasite and host factors that possibly converge to drive disease pathogenesis. Considering parasite proteins mediating sequestration, PfEMP1 appears not to be the only candidate involved. Emerging data show the possible involvement of other variant parasite proteins such as RIFIN and STEVOR proteins 106 (Goel et al., 2015; Niang et al., 2014). Thus, it is important for future studies to investigate the possible influence of all these aspects on the onset of CM pathogenesis. In summary, this study provides evidence for the importance of an ICAM-1-binding motif in group A PfEMP1 with dual binding affinity for host proteins EPCR and ICAM-1. Such binding property allows for increased cytoadhesion and that constitutes an important risk factor for CM. The global burden associated with CM particularly in children remains unacceptably high even in those who recover. Thus, vaccines that will inhibit parasite sequestration in the brain and other supportive therapies that will aid full recovery from neurological deficits remain vital. Of interest, the residues characterising the motif are extremely conserved, thus, eliciting extensive cross- reactive responses and making them attractive candidates to be included in a vaccine cocktail that seeks to prevent CM complications. 107 CHAPTER FOUR ANTIBODY RESPONSES TO PFEMP1 LINKED WITH THE DEVELOPMENT OF PAEDIATRIC CM 4.1 Introduction SM syndromes in children, which include SA, respiratory distress and CM are thought to be driven by specific cytoadhesion properties of the parasite and an imbalanced host immune response (reviewed in Storm and Craig, 2014). Although massive disease intervention campaigns have been implemented for several decades, the burden of malaria remains unacceptably high particularly in children below the age of five in sub Saharan Africa (WHO, 2016). An effective vaccine to complement existing strategies remains a number one global health priority. A striking immuno-epidemiological observation is that children from malaria endemic regions who survive few clinical episodes of malaria in the early stages of life become refractory to severe disease (Griffin et al., 2015; Goncalves et al., 2014; Reyburn et al., 2005; Gupta et al., 1999). Acquired immunity to febrile illness however, is remarkably slow and requires several years of persistent exposure to the malaria parasites. Moreover, lifelong exposure does not appear to offer sterile protection as adults in endemic regions are known to frequently harbour low levels of parasites, even though they are asymptomatic (reviewed in Fowkes et al., 2016; Bejon et al., 2014). Although the detailed mechanism of this natural acquisition is only partially understood, earlier studies have demonstrated that passive transfer of sera from immune adults to acutely ill children with malaria resulted in a profound reduction in disease severity (Sabchareon et al., 1991; McGregor et al., 1963; Cohen et al., 1961), providing strong evidence that antibodies targeting the asexual parasite stages are important. This antibody mediated clinical protection appears to be directed towards 108 the parasite variant antigens displayed on the surface of IEs particularly the PfEMP1 antigens (Chan et al., 2012; Ofori et al., 2002; Dodoo et al., 2001; Bull et al., 1998). PfEMP1 are encoded by the approximate 60 different members of the var multigene family and through a highly coordinated process only one or few PfEMP1 are expressed and exposed on the membrane of IEs (Hviid and Jensen, 2015). Periodically, the parasite alters the surface expression of these PfEMP1 variants to escape immune destruction (Scherf et al., 2008) and this likely translates into the reported slow rate of acquired clinical immunity. In chapter three, a group A PfEMP1, harbouring an ICAM-1 binding motif, was identified via sequence analysis of several parasite genomes and parasites expressing this particular motif bind both ICAM-1 and EPCR concomitantly (Chapter three; Lennartz et al., 2017). This dual binding phenotype was associated with a high risk of developing CM and the PfEMP1 containing this motif among others included members of DC4 and DC13 (lennartz et al 2017). These findings suggest that antibodies directed against these motif-containing DCs could be central to acquisition of clinical immunity to CM. Therefore, this study aimed at investigating whether antibodies to the motif associated with CM are naturally-acquired in malaria-exposed populations and the possibility of experimentally inducing such motif-specific antibodies by immunisation. 4.2 Hypothesis and specific objectives Motif-containing ICAM-1 DBLβ proteins induce cross–reactive antibodies that inhibit binding of IEs associated with CM to ICAM-1. 109 Specific objectives 1. To measure antibodies against motif-containing ICAM-1 binding and non- motif non-ICAM-1 binding DBLβ domains in plasma of malaria patients. 2. To generate motif-specific rat antibodies by immunisations and evaluate the reactivity and cross-adhesion inhibitory activities of these antibodies. 4.3 Methods 4.3.1 Study demographics The study was carried out in the Hohoe Municipality (7º99N, 0°289E) which is located in the central zone of Ghana, approximately 220 km north east of Accra, with a population of 165,000 inhabitants. The study site was the Municipal Hospital located in Hohoe, with a population of 63,000 people (Kweku et al., 2015), has the responsibility of attending to referred cases from neighbouring towns. The climate in Hohoe Municipality is tropical (22 ⁰C – 37 ⁰C) and the vegetation is a semi-deciduous forest. Two rainy seasons are observed in a year and the major rainy season occurs from April to July each year. An average rainfall of approximately 1592 mm is recorded annually with more than 80 % of the rains occurring during the major rainy season. Malaria transmission is intense and all year round, with seasonal peaks observed after the major rains in June. The reported entomological inoculation rate is 65 infective bites per person per year (Kweku et al., 2015; Kweku et al., 2008). Malaria remains the principal cause of outpatient clinic visits (46.2 %) and constitutes about 30 % of total admissions. Among the top 10 causes of mortality, malaria and anaemia make up 16 % and 12 % respectively (Kweku et al., 2015). Plasmodium 110 falciparum, the dominant malaria parasite species in Hohoe remains sensitive to Artemisinin-based combination therapy (Quashie et al., 2013; Koram et al., 2005). 4.3.2 Patient samples and ethics Children (< 12 years of age) admitted to the Hohoe Municipal Hospital with a microscopy diagnosis of P. falciparum malaria during the 2014 malaria transmission season were recruited after informed consent was obtained. Briefly, plasma samples (n=79) and P. falciparum isolates (n=13) were obtained from enrolled patients. Additional P. falciparum isolates (n=19) were obtained from a previous study (Lavstsen et al., 2012) on Tanzanian children diagnosed with malaria. Enrolles were categorised into SM or UM according to the WHO clinical and laboratory criteria (WHO, 2000). The criteria for SM included SA (Hb < 5g/dL) and hyperparasitaemia (> 250,000 parasites/µL). CM patients (BCS < 2) were not identified in this study. UM patients were identified by having parasitaemia less than 250,000 parasites/µL and Hb > 5 g/dL. Plasma samples from 20 healthy Danes with no previous exposure to malaria were also included in this study. Both study sites received clearance from their respective ethics committee and a signed written informed consent from parents or legal guardians was obtained for each enrolled child (see chapter three section 3.3.1). 4.3.3 Recombinant proteins Recombinant DBLβ domains containing the ICAM-1 motif or non-motif DBLβ proteins and a head structure of PFD1235w (CIDRα1.6- DBLβ3) were produced in E. coli SHuffle 3030 cells [New England Biolabs, UK] (Bengtsson et al., 2013b). Briefly, these proteins (Appendix C) were produced from genomic DNA amplified by 111 PCR using published primers (Bengtsson et al., 2013b), a new primer set (IT4VAR16 DBLβ_D4 primers, Appendix C) or produced as synthetic genes (DBLβ_D4 of CDO62031, CDO61797, and CDO63496) by Eurofins (Germany). The PCR products were subcloned into a modified pET15b vector and expressed as N-terminal, hexahistidine-tagged (His-tagged) proteins in E. coli SHuffle 3030 cells (New England Biolabs, UK). The expressed proteins were purified by immobilised metal ion affinity chromatography using HisTrapTM HP 1 mL column (GE Healthcare, Denmark). The motif peptide derived from PFD1235w DBLβ_D4 (LYAKARIVASNGGPGYYNTEVQKKDRSVYDFLYELHLQNGGKKGPPPATHP YKSVNTRDKRDATDDTTP) was produced by Schaefer-N, Denmark. Recombinant GLURP-R2 (amino acids 705-1178) was produced in E. coli cells described previously (Theisen et al., 1995) and was kindly donated by Professor Michael Theisen. Human ICAM-1 combined with the Fc region of human IgG1 (ICAM-1-Fc) was cloned and expressed as a His-tagged protein in HEK293-F cells (Invitrogen, USA) as described previously (Bengtsson et al., 2013a). Recombinant ICAM-1 protein was purified from the supernatant on a HiTrap Protein G High- Performance column (GE Healthcare, Denmark), eluted using 0.2 M glycine/HCl buffer (pH 2.5), neutralized in suspension using Tris/ HCl buffer (1 M, pH 9) and finally buffer-exchanged into PBS (Bengtsson et al., 2013a). 4.3.4 DBLβ-specific antibodies and purification of motif-specific antibodies All procedures concerning animal experiments and immunisations complied with the Danish national regulations. The animal procedures described in this study were carried out according to the guidelines elaborated in act numbers LBK 1306 (23/11/2007) and BEK 1273 (12/12/2005) and permission to carry out experiments 112 was granted by the Danish Animal Procedures Committee ("Dyreforsøgstilsynet") detailed in permit number 2008/561-1498. Before immunisation, sera were collected from each animal and used in flow cytometry and binding assays as negative controls. Recombinant PfEMP1 DBLβ constructs (3D7 PFD1235w_D4 [new ID: PF3D7_0425800_D4]; Dd2VAR32 [new ID: KOB85388] and KM364031_D4) and synthetic peptides were used to immunise rats to generate rat antisera. Procedures to those preparations have been previously described (Bengtsson et al., 2013b). Briefly, rats (Wistar; 8-12 weeks of age; three animals per immunisation group) were subcutaneously injected with 25 µg protein in complete Freund's adjuvant followed by two booster vaccinations of 15 µg in incomplete Freund's adjuvant. Post- immunisation sera were taken two weeks following the last immunisation. Rat IgG specific for DBLβ proteins and motif (anti-PFD1235w_D4; anti- PFD1235w_motif; anti-IT4VAR13), were purified from rat antisera using previous procedures (Joergensen et al., 2010). Briefly, 1 mg of protein in each 1 mL was dialysed overnight against coupling buffer and further coupled to Hi-Trap NHS- activated High-Performance columns following the manufacturer‟s protocol (GE Healthcare, Denmark). Antibodies were diluted (1:1 in PBS), affinity purified, eluted in glycine buffer (0.1 M, pH 2.75) and neutralized in 1M Tris/HCl (pH 9.0). 4.3.5 ELISA The anti-DBLβ reactivity was measured in plasma samples collected from the malaria patients. Maxisorp plates (Nunc, Thermofisher Scientific, Denmark) were coated with different recombinant DBLβ domains (50 µL; 2 µg/mL; 0.1 M glycine/HCl buffer pH 2.75) overnight at 4 ⁰C. The plates were emptied and washed once with PBS-Triton- X-100 (PBS, 0.5 M NaCl, 1 % Triton X-100, pH 7.4) and blocked with blocking 113 buffer (PBS, 0.5 M NaCl, 1 % Triton-X-100, 1 % BSA, 0.03 mM phenol red, pH 7.2) for 30 – 60 min at room temp. The plasma samples were diluted 1:100 in blocking buffer, and 50 μL of prepared sample was added to each duplicate well and incubated (1 h; room temp). The plates were washed four times and bound antibodies in plasma were detected with an anti-human IgG-HRP (Dako, Denmark; 1:3000; in blocking buffer; 50 μL). Following 1 h of incubation and subsequent washing, plates were developed using O-phenylenediamine dihydrochloride tablets (Dako, Denmark) according to the manufacturer‟s instructions. The colour reactions were stopped by the addition of 50 μL of 0.5 M H2SO4, and the optical density (OD) values were read at 490 nm on a VERSAmax microplate reader (Molecular Devices, USA). Antibody reactivity was expressed as arbitrary units calculated as (ODsample − ODbackground)/ (ODpositive control sample – ODbackground) × 100 % (Jensen et al., 2003). In all 26 recombinant proteins (25 DBLβ domains and 1 GLURP-R2) were used to screen 79 plasma samples and on each plate a positive hyperimmune African plasma pool and a negative non-exposed Danish pool were included. Following the ELISA protocol above, antibodies raised in rats against motif- containing DBLβ domains of 3D7 PFD1235w (PF3D7_0425800), KM364031 and Dd2VAR32 (KOB85388) were tested for reactivity against the recombinant DBLβ constructs. Inhibition of ICAM-1 binding to DBLβ domains by these rat antisera was performed using a modified version of the ELISA protocol. Briefly, plates were coated with ICAM-1 (50 µL; 2 or 4 µg/mL ICAM-1; 0.1 M glycine/HCl buffer pH 2.75; overnight at 4 °C) and blocked with blocking buffer (1 h; room temp). His- tagged recombinant DBLβ domains (0.5-16 µg/mL) were mixed with antisera (1:5) or purified human IgG (10 µg/mL) and subsequently incubated (1 h; room temp) with pre-coated ICAM-1 plates. Following washing, bound DBLβ was detected with a 114 Penta-His antibody conjugated to HRP (Qiagen, UK; 1:3,000). All antisera were initially assessed for His-tag specific antibodies by ELISA and only sera negative for His-tag reactivity were included in the assay. 4.3.6 Plasmodium falciparum parasite culture The P. falciparum clonal isolates (3D7, HB3, IT4) were cultured as described before (see chapter three section 3.3.9) and selected for IE surface expression of specific PfEMP1 (Bengtsson et al., 2013b; Joergensen et al., 2010). Briefly, 3D7 IEs were selected with the human monoclonal antibody AB01, which targets 3D7 PFD1235w DBLγ13_D6 [PF3D7_0425800_D6] (Barfod et al., 2011). Plasmodium falciparum IT4 IEs were selected using a rat antiserum against IT4VAR13 DBLβ5_D4 (ABM88750_D4), while the selection of HB3 IEs was done using a rat antiserum against HB3VAR03 DBLβ3_D4 (new ID: KOB63865_D4). The procedure was routinely done, the success of IE selection was verified in flow cytometry assay (see chapter three section 3.3.14) and only cultures with more than 60 % antibody-labelled IEs were used in the experiments. A total of 33 P. falciparum parasite isolates obtained from Ghanaian and Tanzanian children were maintained in in vitro cultures for 1-28 days as described before (see chapter three section 3.3.9). Parasites were maintained in RPMI 1640 with 25 mM Hepes modified (Sigma-Aldrich, Germany), 50 µg/mL gentamicin sulfate (Sigma- Aldrich, Germany), 4 mM Glutamine (Sigma-Aldrich, Germany), 0.5 % Albumax (Invitrogen, USA), with 10 % pooled normal human serum (labelled as complete medium) and human erythrocytes (O+). The cultures were regularly checked for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza, USA) according to the manufacturer‟s protocol. The degree of parasite clonality was 115 determined by MSP 2 and GLURP genotyping using previously established methods (Snounou et al., 1999). 4.3.7 ICAM-1 Adhesion-Inhibition of IE under physiological flow conditions Flow adhesion and inhibition assays were performed on patients‟ isolates and laboratory adapted parasites using previously described methods (Lennartz et al., 2015) and was performed by Dr. Yvonne Adams (University of Copenhagen). Microslides (VI0.1; Ibidi, Germany) were coated with recombinant ICAM-1-Fc (50 µg/mL, Bengtsson et al., 2013a), at 4 oC overnight. Microslides were blocked (1 h; 37 oC) with PBS + 1 % BSA, mounted onto a Leica inverted phase contrast microscope and a shear stress (1 dyn/cm2) was generated by connecting microslides to an NE- 1002X microfluidic pump (World Precision Instruments, UK). Cultured late stage IEs adjusted to 3 % parasitaemia were subjected to a shear stress by flowing over the microslides for 5 min followed by another 5 min to wash off unbound IEs. The number of bound IEs per mm2 for five separate fields was counted at 20x magnification and the experiment was performed in triplicates and repeated at least three more times independently. Inhibition of ICAM-1 binding was carried out using P. falciparum isolates (3D7, HB3, IT4) following the above described protocol. Briefly, mature IEs were incubated with affinity-purified IgG (purified on D4 domains of 3D7 PFD1235w [PF3D7_0425800]) or motif antisera (targeting motif in 3D7 PFD1235w [PF3D7_0425800], Dd2VAR32 [KOB85388] or KM364031) before being allowed to flow on the microslides. Controls included in the assay were affinity-purified non- immune rat IgG and antisera against IT4VAR13 (new ID: ABM88750). ICAM-1 specific binding to IEs was tested by pre-incubating microslide channels with the anti- 116 ICAM-1 antibody (40 µg/mL, clone 15.2, AbD Serotec, USA). Inhibition assay on P. falciparum field isolates was tested on isolates that recorded ≥ 10 IE/mm2 bound ICAM-1 in the adhesion assay. The assay was done using a pool of rat antisera (1:100 dilution) raised against motif derived peptides in 3D7 PFD1235w [PF3D7_0425800] and Dd2VAR32 [KOB85388]. 4.3.8 Immunofluorescence assay Immunofluorescence assays with mature IEs expressing PFD1235w, HB3VAR03, or IT4VAR13 were performed following previously described procedures (Ghumra et al., 2008) and was done by Dr. Yvonne Adams (University of Copenhagen). IEs (50 µL, 5 % parasitaemia) were centrifuged, washed twice with PBS with 1 % Ig-free BSA (Sigma-Aldrich, Germany) and incubated with sera (1:50; 1 h) on ice. Cells were washed thrice (PBS with 1 % Ig-free BSA) and incubated with anti-rat-FITC secondary antibody (Vector laboratories, USA) on ice for 1 h in the dark. Following washing, thin slides were prepared, parasite nuclei were stained with 5 µL ProLong Gold anti-fade DAPI (Thermofisher Scientific, USA) and the thin slides covered with slips. Images were obtained with a Nikon Eclipse TE2000 microscope and x63 objective. 4.3.9 Data analysis. Patient data were presented as median and interquartile range (25th and 75th quartiles) except for male frequency which was summarised as percentages. Plasma antibody reactivity was expressed as ELISA units (EU, percentage) and a score was assigned for each plasma sample/protein combination in relation to the calculated EU. Briefly, EU between 0-25 % of the combination was assigned a score of 0; 26-50 % were 117 assigned a score of 1; 51-75 % were scored as 2; 76-100 % were scored as 3; above 100 % were scored as 4. The sum of all scores was calculated for each DBLβ recombinant protein and each plasma sample. Data were analysed by Student‟s unpaired t-test using the SigmaPlot 13.0 software (Systat Software Inc., United Kingdom). Statistical significance was set at p ≤ 0.01. Experiments involving ICAM-1 adhesion-inhibition of laboratory-adapted IEs and IEs from patients were analysed by One-way ANOVA in GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, USA). Multiple alignments of DBL domains with binding affinity for ICAM-1 were generated using MUSCLE v. 3.7 software (Edgar, 2004) and the MEGA software (Tamura et al., 2007) was used to create sequence distance trees. 4.4 Results 4.4.1 Characteristics of patients for the antibody reactivity profile This hospital based study enrolled a total of 79 Ghanaian patients (<12 years of age). The characteristics of the qualified participants are shown in Table 4.1. SM was more frequently recorded in males whilst there was no gender bias in the occurrence of UM. Overall, the median age between SM patients (3.8+0.3) significantly differed from those of UM (5.6+0.4) (Student‟s unpaired t-test, p = 0.0020). 118 Table 4.1: General characteristics of patients related to figure 4.1 Data are shown as median (1st and 3rd interquartile values) except male gender which is shown as percentages. Patients were grouped into SM (35) and UM (44). Within each disease severity group, patients were distributed into age categories (1-4 years, >4 years). Overall, SM was most frequent in males. Also, the median age was noticeably lower in patients with SM compared to patients with uncomplicated malaria. * Student‟s unpaired t-test between SM and UM, p<0.01. 119 4.4.2 Plasma antibody responses to DBLβ domains of group A PfEMP1 In chapter three, DBLβ domains of group A PfEMP1 with a conserved motif that bind ICAM-1 were found to be connected with the risk of CM development. Hence, this study sought to profile antibody responses against a large set of recombinant DBLβ constructs. A total of 25 different recombinant DBLβ domains which included 12 with motif-binding ICAM-1 and 13 non-motif non-ICAM-1 binding proteins derived from different P. falciparum parasite genomes were used to examine the antibody reactivity in plasma samples of 79 children by ELISA. The assay also included the C-terminal repeat region (R2) of GLURP protein, an asexual stage non PfEMP1 protein considered a vaccine candidate (Dodoo et al., 2000). Each ELISA plate contained a positive hyper-immune African control to normalise data. Plasma antibody reactivity was expressed as ELISA units (EU) and a score was assigned for each plasma sample/protein combination in relation to the calculated EU (see section 4.3.9). The mean sum of all scores was further calculated for each DBLβ recombinant protein and each plasma sample and was used to compare responses between motif-containing and non-motif DBLβ. Figure 4.1A shows reactivity profiles categorised into two domain subsets namely motif-containing (left panel) and non-motif DBLβ with both plotted against the age of the patient donors. In general, the pattern of antibody recognition to the different DBLβ domains greatly varied among the children (Figure 4.1A). Notably, low antibody levels were recorded against all screened DBLβ proteins for some donors (e.g. donors 2, 14, 16; Figure 4.1A), conversely a broad spectrum of DBLβ specific antibody responses with high titres were measured in plasma samples from other children (e.g. donors 50, 67, 70; Figure 4.1A). Comparative plasma antibody responses between the two domain subsets revealed that significantly lower mean score of antibody titres against motif-containing ICAM-1 binding DBLβ were 120 recorded (Figure 4.1B left panel) relative to antibody titres against the non-motif DBLβ proteins (Figure 4.1B right panel) (Student‟s unpaired t-test, p < 0.001). Thus, plasma samples from most children were markedly more reactive towards non-motif DBLβ than towards motif-containing ICAM-1 binding DBLβ proteins (Student‟s unpaired t-test, p < 0.001). 4.4.3 Antibody responses determined by age and disease severity Antibody responses within the two DBLβ subsets (with or without the ICAM-1 motif) were stratified by age (1-4 years, > 4 years) and disease severity (UM, SM) and shown in figure 4.1C. Antibody reactivity compared among the age groups indicated an age- dependent accumulation in both motif containing (Figure 4.1C, left panel) and non- motif DBLβ groups (Figure 4.1C, right panel). Notably, children in the 1-4 years group recorded strikingly lower titres in comparison to children more than 4 years old (Student‟s unpaired t-test, p<0.001). Within the younger age group (1-4 years), motif- containing specific antibody response outcomes remained markedly lower in comparison with responses against non-motif DBLβ proteins (Student‟s unpaired t- test, p<0.001). By contrast, there was no significant difference in antibody reativity to both motif and non-motif proteins in the comparatively older age group (> 4years old). Further, comparable mean sum of scores for antibody responses were recorded for both SM and UM in motif-containing ICAM-1 binding DBLβ within the same age groups. An equivalent pattern was observed between disease severity groups of the non-motif DBLβ binders. 121 Figure 4.1: Plasma antibody reactivity to P. falciparum DBLβ domains. Plasma samples obtained from 79 pediatric patients with either severe or uncomplicated P. falciparum infections were tested for their reactivity against a broad selection of DBLβ domains and the responses were expressed as ELISA units (EU). (A). Antibody responses categorised into DBLβ domains with motif (left column) or non-motif (centre column); a non-DBLβ domain (negative control), R2 (right column). The antibody responses are plotted against age (left edge). The numbers along the right edge indicate the total number of plasma samples analysed (1-79). Each square stands for a specific DBLβ/plasma combination and colours indicate reactivity (%) relative to a positive control pool as indicated in the figure key and detailed in 4.3.5. (B-C). Both figures similar to (A) show plasma antibody reactivity to each DBLβ domain; the responses were scored on a scale of 0 to 4 relative to the IgG reactivity to that particular domain. (B). The overall antibody reactivity to each DBLβ domain was calculated as the mean sum of the score (bar charts) with 95 % confidence intervals (C.I.; error bars) from all 79 plasma samples and shown for motif-containing (left panel) and non-motif (right panel) DBLβ domains. The white bars in each panel represent the percentage of children with a positive antibody response. (C). Antibody responses relative to age and disease severity are shown for motif- containing ICAM-1 binding DBLβ domains (left panel) and non-motif DBLβ domains (right panel). Calculation of the overall antibody recognition for all DBLβ domains was similar to those performed in (B). (D). A plasma pool was generated from the donors and used to determine the ability to inhibit ICAM-1 binding by DBLβ_D4 of Dd2VAR32 [KOB85388]. Gray bars represent percentage binding by DBLβ_D4 of Dd2VAR32 to ICAM-1 in the absence (left) or presence of (right) pooled plasma antibody. ***Student‟s t-test, p ≤ 0.001. 122 4.4.4 ICAM-1 binding-inhibition by antibodies against motif-containing DBLβ domains Plasma samples randomly obtained from ten children based on reactivity to motif DBLβ protein were pooled and used to test for the ability to inhibit ICAM-1 binding in an ELISA. The samples were pooled to increase the signal in the inhibition of binding assay. Briefly, the pooled plasma was mixed with a recombinant motif- containing DBLβ protein and subsequently incubated with recombinant ICAM-1 on pre-coated plates. The data presented in figure 4.1D showed that the pooled plasma contained antibodies that could markedly (p < 0.001) inhibit (> 80 %) ICAM-1 binding of a heterologous motif-containing DBLβ (Dd2VAR32_D4 [KOB85388_D4]) protein. This result suggested indirectly that the motif-containing DBLβ could induce cross-reactive antibodies that inhibit ICAM-1 binding. In order to investigate this hypothesis, antisera were generated from rats immunised with four recombinant motif-containing DBLβ constructs (DBLβ_D4 domains of 3D7 PFD1235w [PF3D7_0425800], PF11_0521 [PFD3D7_1150400], KM364031 or Dd2VAR32 [KOB85388]) identified as domains involved in ICAM-1 binding in chapter three. Similar inhibitory ELISA experiments as described above were carried out using rat antisera against these motif DBLβ domains (14 motif-binding ICAM-1 DBLβ and two non-motif ICAM-1 binding DBLβ). The results showed that the individual antisera had the capacity to interfere with ICAM-1 binding of homologous and several heterologous motif-containing DBLβ domains (Figure 4.2A). Conversely, these rat antisera showed minimal binding–inhibition of non-motif proteins of DBLβ_D4 domains of IT4VAR13 [ABM88750] (~ 30 %) and ITVAR16 [new ID AAS89259] (~ 40 %) [Figure 4.2A]. Experiments using pooled antisera against motif-containing 123 DBLβ resulted in pronounced inhibition (> 75 %) of ICAM-1 binding of all motif domains (Figure 4.2A). The cross-inhibitory function of the specific IgG against the motif (the ICAM-1 interaction site) present in the DBLβ domain was determined. Briefly, serum IgG collected from rats immunised with any of the three motif-containing DBLβ domains (D4 of 3D7 PFD1235w; KM364031; or Dd2VAR32) were affinity purified on a peptide of the 3D7 PFD1235w motif and was tested in similar ELISA experiments as described before for the antisera against the motif DBLβ domains. Purified IgG against the motif exhibited the capacity to block ICAM-1 binding of nearly all except two DBLβ motif proteins (KM364031; KF984156). Conversely, purified motif IgG had no effect on the level of binding of the non-motif constructs DBLβ_D4 of IT4VAR13 and of IT4VAR16 (Figure 4.2B). 124 Figure 4.2: Inhibition of ICAM-1 binding by antibodies raised against motif- containing DBLβ domains. (A). Individual or pooled antisera generated from rat immunisations with 3D7 PFD1235w_D4 [new ID PF3D7_0425800], PF11_0521_D4 [new ID PFD3D7_1150400], KM364031_D4, and Dd2VAR32_D4 [new ID KOB85388] were used to test for the inhibition of DBLβ: ICAM-1 binding in ELISA. A phylogenetic tree indicating the relatedness of the different domains is shown to the extreme left. (B). Similar inhibitory experiments carried out with 3D7 motif- specific IgG affinity purified from rats immunised with DBLβ_D4 of PFD1235w, KM364031 or Dd2var32. IT4VAR16 and IT4VAR13 indicate non-motif containing DBLβ. Level of inhibition is shown as: black (>75%), dark gray (50-75%), gray (20-50%), and white (<20%). As described in chapter three, an important finding was that IEs selected to express motif-containing DBLβ domain (HB3VAR03, 3D7 PFD1235w) on their surface had dual binding affinity for human receptors EPCR and ICAM-1. The ability of motif- specific IgG to broadly inhibit the adhesion of ICAM-1 using laboratory adapted isolates (HB3, IT4 and 3D7) was therefore evaluated in binding assays under physiological flow conditions. Briefly, antisera raised against the motif-harbouring 125 proteins [3D7 (PFD1235w) and Dd2VAR32 (KM364031)] and IgG purified on the PFD1235w motif peptide were utilised. Both affinity purified motif IgG antibodies were individually incubated with selected mature IEs expressing PFD1235w, HB3VAR03 or IT4VAR13 and thereafter allowed to bind recombinant ICAM-1 receptor under a physiologically relevant shear stress of 1.0 dyn/cm2. Affinity purified non-immune rat IgG and antisera against IT4VAR13 were included as controls. The results showed that both IgG purified on the motif peptide significantly inhibited the adhesion of HB3VAR03 (p < 0.0001) and 3D7 PFD1235w IE (p = 0.0001) to recombinant ICAM-1 (Figure 4.3). Specific IgG targeting the Dd2VAR32 motif were also observed to interfere with the binding of all three parasite isolates. Notably, antibodies targeting IT4VAR13 or non-immune control IgG did not interfere with ICAM-1 binding of HB3VAR03 nor 3D7 PFD1235w IE (Figure 4.3). Taken together, the data presented thus far showed that motif-containing DBLβ elicit cross-reactive inhibitory antibodies that block the interaction between IEs expressing surface specific native PfEMP1 and ICAM-1. 126 Figure 4.3: Inhibition of ICAM-1 binding to P. falciparum lab isolates by antibodies raised against motif DBLβ domains. Adhesion inhibition of ICAM-1 binding of HB3VAR03 IE (left), 3D7 PFD1235w IE (centre), and IT4VAR13 IE (right) to ICAM-1 under 2 physiological flow (1 dyn/cm ) in the presence of anti-ICAM-1 antibodies, rat affinity purified anti- PFD1235w_D4 IgG, anti-PFD1235w_motif IgG, anti-KM364031_motif IgG, anti-Dd2VAR32_motif IgG, anti-IT4VAR13 and control IgG. At least three independent experiments performed in triplicates are reported. Data are represented as mean± S.D. *** indicate significance, p <0.01. 4.4.5 ICAM-1 adhesion-inhibition of recombinant motif domains and P. falciparum isolates by motif-specific peptide antibodies Supplemental experiments aimed to test the binding-inhibitory functionality of antibodies against the motif peptide (minimum ICAM-1 binding region) were performed. Briefly, rats were immunised with either the PFD1235w motif peptide alone or combined with a Dd2VAR32 motif peptide. The resulting antisera from either the single peptide or the double peptide immunisations were tested for their ability to inhibit ICAM-1 binding in ELISA as described before. The data showed that the motif peptide antisera from each immunised rat interfered with the ICAM-1 binding of a broad selection of motif-containing DBLβ domains to recombinant ICAM-1. This inhibition was increased when antisera from rats immunised with both 127 peptides was used. Conversely, the peptide antibodies did not inhibit ICAM-1 binding of non-motif containing IT4VAR13 and IT4VAR16 DBLβ domains (Figure 4.4). Figure 4.4: Inhibition of ICAM-1 binding by motif peptide-specific antibodies to motif DBLβ proteins. Raised antisera from rats immunised with one (PFD1235w) or two (pool: 3D7 PFD1235w and Dd2VAR32) motif peptides tested in ELISA inhibited ICAM-1 binding of several motif-containing DBLβ but were not effective at inhibiting non-motif DBLβ binding (IT4VAR16, IT4VAR13) to ICAM-1. The capacity of peptide antisera to disrupt IEs capable of adhering to ICAM-1 and EPCR concomitantly was assessed. IEs selected to express PFD1235w or HB3VAR03 were incubated with either single peptide or double peptide antisera (pool) and subsequently allowed to bind recombinant ICAM-1 receptor coated on a microslide under flow conditions. Similar experiments were done with IT4VAR13 IE. Antibodies raised against single peptide markedly interfered with the dual binding parasite isolates of both 3D7 PFD1235w IE (homologous) and HB3VAR03 IEs (heterologous) to ICAM-1 and more evidently, a greater binding inhibition was recorded when the motif peptide pool antisera was used. Contrastingly, both the single or pool peptide 128 antisera were not capable of disrupting the ICAM-1 binding of IT4VAR13 parasite isolate, a CD36 and ICAM-1 dual binder (Figure 4.5A). In addition, peptide antisera were incubated with HB3VAR03, 3D7 PFD1235w or IT4VAR13 selected IEs to determine whether they reacted with the PfEMP1 expressed on the surface of these IEs. Data from immunofluorescence assays showed that the peptide antisera only recognised the surface of motif-expressing HB3VAR03 and 3D7 PFD1235w IE and not the non-motif IT4VAR13 control (Figure 4.5B). Similar flow adhesion-inhibitory experiments were carried out using P. falciparum isolates obtained from Ghanaian (n=14) and Tanzanian patients (n=19). A total of 22 parasites isolates (3UM, 14 SM and 5CM) showed adhesion to ICAM-1. Motif- specific peptide antisera inhibited a considerable number (11/22) of ICAM-1 binding parasite isolates, notably, one from a child with UM, six with SM, and four with CM at variable degrees. Adhesion blocking activity was around 25-40 % for three of the parasite isolates, while 45-68 % was recorded for the other eight isolates. Taken together, the data showed that the children were infected with IEs that harboured the motif (Figure 4.5C) and more importantly, vaccination with motif peptides induced cross-reactive antibodies that inhibited the binding of four out of five P. falciparum isolates obtained from paediatric CM patients. 129 Figure 4.5: Adhesion inhibition of ICAM-1 binding of P. falciparum parasites by motif peptide-specific antibodies. (A). Adhesion inhibition of ICAM-1 binding of HB3VAR03 2 IE (top), 3D7 PFD1235w IE (centre), and IT4VAR13 IE (bottom) under flow conditions (1dyn/cm ) in the presence of anti-ICAM-1 antibodies, rat plasma anti-PFD1235w_motif and rat plasma anti- PFD1235w/ Dd2VAR32_motif (pool). At least three independent experiments performed in triplicates are reported. Data are represented as mean ± S.D. * indicates significance (B). Antibodies raised against the motif peptides were incubated with laboratory isolates namely HB3VAR03 IE (1-3), 3D7 PFD1235w IE (4-6) and IT4VAR13 IE (7-9). Peptide antibodies recognised the surface expression of PfEMP1 only in HB3VAR03 IE (1-3), 3D7 PFD1235w IE (4-6) using immunofluorescence assay (representative image). Antibodies used were anti-PFD1235w DBLβ_D4, anti-IT4VAR13 DBLβ_D4 and anti- PFD1235w/ Dd2VAR32_motif (pool). (C). Adhesion inhibition of ICAM-1 binding of P. falciparum field isolates (n=20) by motif specific antibody pool. Inhibition assay was done only if 2 parasite isolate bound to ICAM-1 was ≥ 10 IE/mm in the adhesion assay. Dotted line represents 25 % inhibition cut-off. A phylogenetic tree indicating the relatedness of the different domains is shown to the extreme left on figure A. Blue shading indicates non-motif containing DBLβ. Level of inhibition is shown as: black (>75%), dark gray (50-75%), gray (20-50%), and white (<20%). 130 4.5 Discussion The protective role of naturally-acquired antibodies against P. falciparum infection has long been recognised (Sabchareon et al., 1991; Cohen et al., 1961). Earlier studies have demonstrated that the surface exposed PfEMP1 antigen is the principal target of protective immunity since antibodies directed against this antigen have been linked with a decreased risk of developing clinical malaria (Chan et al., 2012; Nielsen et al., 2002; Ofori et al., 2002). Additionally, available evidence indicates that increased exposure to P. falciparum leads to the production of a broadly effective PfEMP1 antibody response, resulting in cross-reactive protection (Cham et al., 2009; Mackintosh et al., 2008; Krause et al., 2007). Thus, on one hand, the data from these studies provide a strong rationale for the development of an anti-PfEMP1 based vaccine. On the other hand, the enormous diversity of PfEMP1 found within and between different parasite populations makes them unattractive candidates. Despite this widespread diversity, there is increasing evidence suggesting that there may be structurally conserved epitopes that may be considered potential vaccine targets or constituents (reviewed in Bull and Abdi, 2016). Consistent with these observations, in chapter three, it was demonstrated that CM is precipitated by group A PfEMP1 expressing IEs with dual and simultaneous binding affinity for EPCR and ICAM-1. These IEs were found to contain a structurally conserved ICAM-1 binding DBLβ motif capable of eliciting cross-reactive antibodies. Given its association with CM development, this study investigated in detail the antibody responses against an array of group A PfEMP1 with the motif-containing ICAM-1 or non-motif DBLβ recombinant proteins. The study further explored the potential utility of motif containing DBLβ domains and peptides to mount cross- reactive antibody responses interfering with the ICAM-1 binding of several 131 recombinant DBLβ domain constructs as well as IEs expressing motif-containing PfEMP1. Antibody reactivity to group A PfEMP1 recombinant DBLβ proteins with an ICAM-1 binding motif or non-motif was measured in 79 plasma samples obtained from children with SM or UM. Overall, there was a striking degree of variability in antibody responses to the DBLβ recombinant proteins in the different plasma donors , with higher titres and broader antibody recognition observed in some donors (Figure 4.1). Most studies have previously demonstrated that host age is a major determinant during the acquisition of naturally protective partial immunity in endemic populations, possibly resulting from frequent exposures to falciparum parasites (Joergensen et al., 2007; Magistrado et al., 2007; Ofori et al., 2002; Bull et al., 2000). This is further supported by the fact that the heaviest toll of malaria infections are carried by children younger than 5 years, particularly in regions of ongoing transmission (Gupta and Day, 1994; Greenwood et al., 1991). In keeping with this observation, children were grouped according to age to compare antibody responses between the groups. An overall trend of an age-related build-up of antibody repertoire to both motif and non- motif DBLβ proteins was evident and this trend resonated with what had been previously reported for other PfEMP1-based studies (Cham et al., 2010; Cham et al., 2009; Magistrado et al., 2007; Ofori et al., 2002). The mean anti-DBLβ response increased significantly from the four year old or younger age group to the more than four years old group for both types of DBLβ domains. Also, most plasma samples from children recorded markedly higher responses to non-motif DBLβ proteins compared with responses towards motif proteins only in the younger age group (Figure 4.1). A possible explanation for this observation may be that the children had 132 limited exposure to IEs expressing motif-containing DBLβ domains. There was however, no difference in antibody responses to both motif and non-motif proteins in the older age groups, suggesting that natural immunity to motif proteins is acquired later in comparison with non-motif proteins. When patients were stratified according to disease severity, no appreciable difference in antibody reactivity was detected between SM and UM in both DBLβ protein groups within the same age group. However, some earlier studies have observed higher anti- PfEMP1 responses in UM patients and thus suggested that may have prevented the development of severe forms of malaria (Duffy et al., 2016; Rovira-Vallbona et al., 2012; Mayor et al., 2009; Tebo et al., 2002). The data presented in chapter three clearly showed that IEs that transcribed group A motif-containing ICAM-1 binding PfEMP1 were exclusively involved in CM development. This study however, was not able to obtain plasma from CM patients and that may possibly account for the observed results. In keeping with the finding of chapter three, it would have thus been predicted that significantly reduced motif antibody levels would be quantified in CM individuals in comparison with non-cerebral SM or UM. Earlier studies have demonstrated that immune plasma from individuals in malaria- endemic areas contained IgG inhibitory of ICAM-1 binding (Lennartz et al., 2017; Bengtsson et al., 2013b). Consistent with that, a plasma pool generated from the study donors and affinity purified on motif domain was able to inhibit ICAM-1 binding of a heterologous motif DBLβ domain. The results presented here likely fits into the proposed mechanism where anti-PfEMP1 antibodies offer clinical protection by blocking tissue-specific sequestration, resulting in non-adherent parasites effectively destroyed by the spleen and resulting in a considerable reduction in parasite load (Dodoo et al., 2001). Thus, it may be possible to develop a vaccine based on this 133 ICAM-1 binding motif to prevent CM by circumventing parasite sequestration in the brain. A vaccine based on the motif involved in ICAM-1 binding must be able to evoke a strong anti-adhesive immune response. Consistent with this notion, the potential of using individual DBLβ domains or motif peptides representing the group A ICAM-1 binding site to trigger cross-reactive and anti-adhesive IgG was investigated. Rat immunisations were done with either motif peptides (ICAM-1 binding epitope only) or motif-containing DBLβ domains. Significantly, rat antisera raised against motif peptides effectively blocked a broad selection of DBLβ domains involved in ICAM-1 binding (Figure 4.4). It is essential that a vaccine based on the motif is capable of inhibiting IE adhesion regardless of the origin of the parasite strain. Notably, antisera generated from only two motif-specific peptides were effective at blocking significantly adhesion of ICAM-1 binding of several malaria parasites including two laboratory falciparum isolates expressing motif-specific PfEMP1 (Figure 4.5A) and falciparum parasites isolated from four out of five paediatric CM patients (Figure 4.5C). Some limitations were identified in this study. The number of plasma samples used for screening the antibody response was inadequate and thus did not provide enough statistical power to detect profound differences between SM and UM. The study could also not evaluate motif reactivity in plasma from CM pateints because none of the plasma donors had CM complications. However, these limitations to some extent were counteracted by the inclusion of additional experiments with the parasite isolates from CM patients which validated the cross-inhibitory functionality of antibodies targeting DBLβ motif and thus demonstrated the importance of the motif in CM. Further 134 investigations will have to be carried out on a large number of samples particularly from CM patients, though it is recognised that it has become increasingly difficult to access samples from CM patients. It is widely accepted that the clinical epidemiology of malaria including transmission seasons, endemicity and intensity play decisive roles in shaping the development of immunity (Cham et al., 2009; Mayor et al., 2009; Giha et al., 2000; Rogier et al., 1999; Snow et al., 1994). Thus, future studies would have to evaluate the antibody responses against this motif using samples from different geographic regions while evaluating these influential factors to determine if replicative responses can be generated and that would be in line with its consideration as a potential global vaccine candidate. In addition, future longitudinal studies would be crucial for establishing the involvement of these antibodies in the development of immunity. Notwithstanding, this study showed that antibodies that are cross-adhesion inhibitory of a large set of motif DBLβ domains and IEs expressing native PfEMP1 associated with CM can be experimentally induced, in addition to being acquired following natural falciparum infections. A motif-specific peptide (representing the ICAM-1 binding epitope) known to be involved with paediatric CM elicits cross-reactive IgG that can functionally protect against CM, making it a promising vaccine candidate that should further be investigated. 135 CHAPTER FIVE MARKERS OF INFLAMMATION AND ENDOTHELIAL ACTIVATION IN GHANAIAN CHILDREN WITH MALARIA 5.1 Introduction The aetiology of SM remains difficult to explain, notably why a small fraction of infected children succumb to severely fatal complications. The host immune system is believed to be one of the major contributors to severe disease development (reviewed in Clark et al., 2006; van der Heyde et al., 2006). During the erythrocytic cycle, the presence of the malaria parasites, accompanied by the release of parasite toxins into the blood circulation, directly stimulates immune cells to secrete both pro- and anti-inflammatory cytokines which are important for combating the infection and hence offering protection from malaria (reviewed in Schofield and Grau, 2005; Clark and Schofield, 2000). However, overproduction of pro-inflammatory cytokines could have detrimental consequences for the host. Indeed, circulating levels of TNF-α, IL-1 and high mobility group box 1 (HMGB1) protein present in the serum of malaria patients have been reported to correlate significantly with malaria severity (Alleva et al., 2005; Kwiatkowski et al., 1990; Grau et al., 1989). An imbalanced inflammatory response is thought to contribute to disease severity by increasing the expression level of endothelial surface adhesion molecules such as ICAM-1 (Turner et al., 1994; Ockenhouse et al., 1992), thereby increasing IE sequestration to endothelial cells in microvascular beds resulting in flood flow impediment (reviewed in Miller et al., 2002). The increased sequestration of IEs together with a dysregulated host inflammatory response, are believed to activate the endothelium and contributes to the increased expression of adhesion molecules and microvascular leakage, exacerbating the 136 pathological processes of malaria (reviewed in Liles and Kain, 2014; Cunnington et al., 2013b; Kim et al., 2011). Indeed, immunohistochemical evaluations provide evidence of endothelial cell activation during malaria (Silamut et al., 1999; Turner et al., 1998; Turner et al., 1994). During endothelial activation, some of these endothelial receptors including EPCR and ICAM-1 are shed as soluble forms into circulation possibly through the action of metalloproteinases (Qu et al., 2007; Fiore et al., 2002; Xu et al., 2000) and these may serve as biomarkers of inflammation and endothelial activation. Indeed, studies have reported increased levels of these soluble molecules in acute malaria suggesting that they form a critical component or markers of the onset of disease pathology (Moussiliou et al., 2015; Adukpo et al., 2013; Cserti- Gazdewich et al., 2010). Furthermore, the activated endothelial cells can fuse with Weibel Palade bodies and facilitate the rapid release of their contents, including, von- Willebrand factor and Ang-2 directly into circulation (Fiedler and Augustin, 2006). High plasma concentrations of Ang-2 were found to correlate with death in individuals infected with falciparum malaria (Prapansilp et al., 2013; Yeo et al., 2008). The present study quantified plasma levels of HMGB1, Ang-2, sEPCR and sICAM-1 in paediatric Ghanaian with malaria on the day of clinical presentation (admission samples, week 0) and two weeks following treatment (convalescent samples, week 2). This was to examine whether there are alterations in the levels of these soluble markers during the acute phase and after resolution of the infection. This panel of biomarkers were investigated based on their role in disease pathogenesis and no study has assessed their kinetics concurrently in Ghanaian children with malaria. 137 5.2 Hypothesis and specific objectives High levels of endothelial markers of activation are associated with disease severity Specific objective 1. To evaluate the levels of biomarkers of activation and inflammation in the plasma of acute and convalescent children with UM and SM. 5.3 Methods 5.3.1 Study outline This study was carried out within the frame of a broader study which aimed to build malaria vaccine capacity in Ghana. All ethics-related protocols and permit to conduct the study were granted by the institutional review board of Noguchi Memorial Institute for Medical Research and the Ethical Review Committee of the Ghana Health Service (file GHS-ERC 08/05/14). Participation in the study was voluntary and the patients were enrolled after parents or guardians signed the written informed consent. Patients were treated by adhering to the standard guidelines of the Ministry of Health in Ghana. Children (<12 years of age) reporting with malaria, or non-malarial febrile illness to the Hohoe Municipal Hospital, Ghana were enrolled into the study during the 2015 and 2016 malaria transmission seasons. Malaria transmission in this township occurs throughout the year with marked seasonal peaks after the major rains in June. On average, inhabitants are exposed to approximately 65 infectious bites per person per year (Kweku et al., 2015; Kweku et al., 2008). A detailed description of the study site can be found in chapter four section 4.3.1. The enrolled children immediately underwent clinical investigations and blood samples were taken for disease diagnosis (parasite evaluation and haematology) and 138 research purposes. Malaria was diagnosed by a rapid diagnostic kit and by light microscopy from Giemsa-stained thick and thin peripheral blood smears. The children were then placed into categories following the WHO criteria for malaria diagnosis (WHO, 2000) as described before (see section 4.3.2). Febrile children (FC) (> 37.5 °C) reporting to the hospital who tested negative via a rapid diagnostic test and microscopic investigation were enrolled. Age-matched healthy community school children (CC) who tested negative for malaria via a rapid diagnostic test and microscopic investigation with no previous malaria treatment in the past two weeks before enrollment were also included in the study. 5.3.2 Sample collection and processing Blood was collected into EDTA (6 mL) and Heparin (4 mL) vacutainers (Beckon Dickson, Germany) from consenting children on the day of admission (week 0) and on week two following initial presentation (convalescent patients). For healthy community controls, blood samples were collected at only one time point. Blood was immediately centrifuged to separate plasma from pelleted erythrocytes. Plasma samples were aliquoted into cryovials and stored immediately in a -80 °C freezer. The samples were then shipped on dry ice to Copenhagen and further kept in a freezer at - 80 °C until they were used for quantifying inflammation markers by ELISA. 5.3.3 ELISA for markers of Inflammation/endothelial activation All reagents and samples were allowed to equilibrate to room temperature prior to analysis. Plasma samples, controls and standards were assayed in duplicates. Measurements for the markers of inflammation or endothelial activation were carried out on plasma samples collected at admission (week 0), week two following initial 139 presentation (week 2) and recovery, as well as community controls. All biomarkers were quantified using commercial kits employing a sandwich ELISA and carried out at room temperature. Concentrations were determined by interpolation of a four- parameter-fit standard curve generated for the various markers. 5.3.3.1 Quantitative determination of sICAM-1 in human plasma samples The plasma concentration of soluble ICAM-1 (sICAM-1) was quantified using a commercial ELISA kit (DCIM00; R&D Systems, USA) according to the manufacturer‟s instructions. Precoated microplate strips with human ICAM-1 non- allele-specific monoclonal antibody were incubated with standards (0.625 ng/mL-40 ng/mL) diluted in calibrator diluent RD5P (1:5); 100 µL/well) and samples (1:20 in calibrator diluent RD5P (1:5); 100 µL/well) for 1 h on a horizontal orbital microplate shaker at 500 rpm ± 50 rpm. The wells were aspirated, washed four times (400 µL wash buffer/per well) and padded dry on clean paper towels. The plates were incubated with human ICAM-1 non-allele-specific conjugate (200 µL/well) for 1 h on a shaker which was followed by washing (400 µL/well; four times). An aliquot of 200 µL of substrate solution was added to each well and incubated for 30 min in the dark. Stop solution (2N H2SO4; 50 µL) was added to all wells to halt the colour reaction and the plates were read at 450 nm and 540 nm using a VERSAmax reader (Molecular Devices, USA). 5.3.3.2 Quantitative determination of sEPCR in human plasma samples The assay was carried out using a commercial kit (dy2245; R&D Systems, USA) and following the manufacturer‟s protocols. Plates were coated with the capture antibody (100 µL; 800 ng/mL in PBS) and incubated overnight at room temperature. Plates 140 were washed three times with 400 µL per well with wash buffer (0.05 % Tween in PBS) and blocked with reagent diluent (300 µL; 1 % BSA in PBS) for 1 h. Diluted plasma samples (1:20, in reagent diluent; 100 µL) and standards (0.313 ng/mL-20 ng/mL in reagent diluent; 100 µL) were added to wells and incubated for 2 h. The plates were aspirated, washed three times and incubated with detection antibody (1:200; 100 µL) for 2 h. Following washing, plates were incubated with streptavidin conjugated to horseradish peroxidase (1:200; 100 µL) for 20 min in the dark. Washed plates were subsequently incubated with substrate solution (100 µL) in the dark for another 20 min and reaction curtailed by the addition of 50 µL of stop solution (2N H2SO4) to each well. Plates were read using a VERSAmax plate reader (Molecular Devices, USA) at 450 nm and 540 nm to account for background correction. 5.3.3.3 Measurement of Ang-2 concentration in human plasma samples The plasma concentration of Ang-2 was quantified using a commercial ELISA kit (DANG20 duo set; R&D Systems, USA) according to the manufacturer‟s instructions. Precoated microplate strips with a monoclonal antibody specific for human Ang-2 were allowed to equilibrate to room temperature and the assay diluent, RD1-76 reagent (100 µL/well), was added to the plates. Plates were incubated with standards (46.9 pg/mL-3000 pg/mL in reagent diluent; 50 µL) and samples (1:5 in reagent diluent; 50 µL) for 2 h on a horizontal orbital microplate shaker at 500 rpm ± 50 rpm. The wells were aspirated, washed four times with 400 µL of wash buffer per well and blotted on clean paper towels. Human Ang-2 conjugate (200 µL/well) was added to each well and incubated for 2 h on a plate shaker. Washed plates were subsequently incubated with 200 µL of substrate solution per well for 30 min in the dark. The reaction was stopped with 50 µL/well of stop solution (2 N H2SO4) and the OD was 141 determined at 450 nm and 540 nm using a VERSAmax reader (Molecular Devices, USA). 5.3.3.4 Measurement of HMGB1 concentration in human plasma samples Plasma HMGB1 was measured according to the manufacturer‟s protocol (ST51011; IBL International, Germany). Reagent diluent (100 µL) was added to each well of precoated microtitre plates. Plates were incubated with 10 µL of standards (2.5 ng/mL-80 ng/mL in reagent diluent) and plasma samples (10 µL) for 22-24 h at 37 ⁰C. Plates were washed five times with 300 µL per well with wash buffer (< 0.5% Tween in PBS). In each well, 100 µL of enzyme conjugate was added and plates incubated for 2 h at 25 ⁰C. Washed plates were further incubated with 100 µL per well with colour reagents (ready to use TMB and 0.005 M H2O2) for 30 min in the dark. The reaction was stopped by the addition of 50 µL/well of stop solution (0.35 M H2SO4) and the plates read at 450 nm using a VERSAmax plate reader (Molecular Devices, USA). 5.3.4 Data analysis Statistical analyses were performed using the SigmaPlot13 (Systat Software Inc, United Kingdom). Clinical information and soluble marker data on study groups were summarised as median and inter quartile range (25 %; 75 % quartiles). Continuous variables were analysed using the Kruskal-Wallis Rank Sum Test with Dunn‟s multiple comparison test. Comparison between admission and convalescent samples were analysed using the Mann-Whitney U Test. Statistical significance was set at p < 0.05. 142 5.4 Results 5.4.1 Baseline information on study groups The demography and clinical information of enrolled children are summarised in Table 5.1. A total of 191 children were enrolled in this study. They included 41 children with SM (4 CM, 3 SA, 1 CM/SA, 33 hyperparasitaemia), 69 children with UM, 13 children with FC and 68 CC. In the SM group, the majority were males whereas, in the UM group, gender was uniformly spread. The median ages were comparable among the study participants. Also, median parasitaemia was not statistically different between malaria groups. Expectedly, higher median Hb levels were detected in healthy CC than in SM (p < 0.001) and UM (p = 0.045), with higher levels in UM compared to SM (p < 0.001; Kruskal-Wallis, Dunn‟s Test). There was also no significant difference between either FC and CC or FC and UM (Kruskal- Wallis, Dunn‟s Test). Median temperature recordings were markedly lower in CC than in SM (p < 0.001), UM (p < 0.001) and FC (p < 0.001; Kruskal-Wallis, Dunn‟s Test). 143 Table 5.1: Baseline clinical profile and the levels of soluble markers of inflammation and endothelial activation. Variable SM(n=41) UM(n=69) FC(n=13) CC(n=68) Age(years) 4.45(2.45-5.58) 3.85(2.70-6.75) 2.90(2.90-2.90) 5.00(3.00-6.00) Male (%) 28(68.3 %) 34(49.3 %) 11(84.6 %) 29(42.6 %) α Haemoglobin(g/dL) 7.80(7.10-9.10) 10.00(9.25-11.25) 11.20(9.65-11.95) 11.40(9.80-12.20) o β Temperature ( C) 38.50(37.50-39.60) 39.00(37.80-39.50) 38.60(38.35-39.30) 36.35(36.00-36.70) Parasites*1000/µL 52.51(17.56-130.67) 38.66(18.80-78.18) NA NA sICAM-1 (ng/mL) 876.36(770.47-982.28) 790.97(685.78-958.86) 623.76(499.70-818.35) 339.24(288.97-399.03) sEPCR(ng/mL) 16.27(11.52-22.51) 16.81(11.48-23.76) 11.74(8.36-22.99) 4.39(2.60-7.56) Ang-2 (pg/mL) 5926.30(2941.35-7895.76) 3873.22(2786.54-6008.00) 2379.39(1727.87-3641.16) 1474.67(1123.91-2095.18) HMGB1(ng/mL) 9.66(6.48-19.30) 10.80(5.65-18.87) 4.84(3.03-12.63) 4.96(3.03-7.94) Data are presented as median (interquartile range) except male gender (%). Groups were compared using Kruskal-Wallis Rank Sum Test followed by Dunn‟s Post Hoc Test for multiple comparisons. Mann-Whitney U Test was used to compare parasite density between UM and CM. α-indicates that the median Hb levels were significantly higher in CC than in SM (p < 0.001) and UM (p = 0.045). β-indicates that the median temperature was significantly lower in CC than in SM, UM and FC (p < 0.001). UM = uncomplicated malaria, SM = severe malaria (includes cerebral malaria (CM; n = 4), severe malarial anaemia (SA; n = 3), CM/SA (n = 1), hyperparasitaemia (n = 33)), FC = febrile control (n=13) and CC = community control (n=68), NA=not applicable. 144 5.4.2 Soluble ICAM-1 (sICAM-1) levels Data on sICAM-1 were evaluated in admission samples (week 0) and in follow-up convalescent samples (week two) of SM, UM, FC and CC study participants and the data are summarised in figure 5.1. In week 0, significantly elevated levels were recorded for malaria groups (SM=876.36 ng/mL; UM=790.97 ng/mL) or FC group (623.76 ng/mL) in comparison with CC group (339.24 ng/mL; p < 0.001, Kruskal- Wallis, Dunn´s Test). Although the median sICAM-1 concentration was higher in the SM group compared with UM, the difference was not statistically significanct (Figure 5.1A). Levels of sICAM-1 decreased in convalescent samples (week two) when compared with week 0 levels in the malaria groups (UM week 0 versus UM week two; p < 0.001; SM week 0 versus SM week two; p < 0.001; Mann-Whitney U Test) [Figures 5.1B and 5.1C]. There was no difference in sICAM-1 levels among the different age categories in both SM and UM (p > 0.05) [Appendix D; Dunn‟s Test]. Figure 5.1: Median sICAM-1 levels among study groups. (A) sICAM-1 levels were assessed by ELISA in children with SM (41), UM (69), non-malarial febrile illness (FC; 13) and healthy community controls (CC; 68) on admission day (week 0). Data are presented as box plots with median and with 25th and 75th percentile distribution of data. Whiskers represent minimum and maximum values with outliers. (B-C) sICAM-1 levels on week 0 compared with week two samples in both SM (B) and UM (C). Each horizontal line represents the data points for each individual measurement. Multiple variables were analysed using the Kruskal-Wallis Rank Sum Test with Dunn‟s Multiple Comparison Test. Comparisons between two variables were done using Mann-Whitney U Test. Wk 0 = week 0; wk2 = week two 145 5.4.3 Soluble EPCR (sEPCR) levels Quantification of the sEPCR levels in the study groups in admission samples (week 0) showed that malaria patients and children in the FC group generally had higher levels of sEPCR (SM = 16.27 ng/mL; UM = 16.81; ng/mL; FC = 11.74 ng/mL, p < 0.001) compared with the healthy CC group (4.39 ng/mL; Kruskal-Wallis, Dunn´s Test). There was no significant difference between UM and SM patients (Figure 5.2A). Following two weeks after initial presentation, sEPCR levels decreased significantly in comparison with week 0 levels in the SM group (p < 0.001; Mann-Whitney U Test; Figure 5.2B). A similar trend was recorded for the UM group when week 0 was compared to week two (p < 0.001, Mann-Whitney U Test) [Figure 5.2C]. There was no significant difference in sEPCR levels among the different age groups in both SM and UM (p > 0.05; Dunn‟s Test) [Appendix D] Figure 5.2: Median sEPCR levels among study groups. (A) sEPCR levels were assessed by ELISA in children with SM (41), UM (69), non-malarial febrile illness (FC; 13) and healthy community controls (CC; 68) on admission day (week 0). Data are presented as box plots with median and with 25th and 75th percentile distribution of data. Whiskers represent minimum and maximum values with outliers. (B-C) sEPCR levels on week 0 compared with week two samples in both SM (B) and UM (C). Each horizontal line represents the data points for each individual measurement. Multiple variables were analysed using the Kruskal-Wallis Rank Sum Test with Dunn‟s Multiple Comparison Test. Comparisons between two variables were done using Mann-Whitney U Test. Wk 0 = week 0; wk2 = week two 146 5.4.4 Plasma Ang-2 levels Plasma Ang-2 levels were evaluated in week 0 and in week two plasma samples of children in the SM, UM, FC and CC study groups and data are summarised in figure 5.3. In week 0 samples, children with malaria (SM, UM) recorded significantly higher Ang-2 median levels (SM = 5926.23 pg/mL; UM = 3873.22 pg/mL) when compared with the CC group (1471.67 pg/mL, p < 0.001; Dunn‟s Test; Figure 5.3A). The median Ang-2 levels were markedly lower in the FC group (2379.39 pg/mL) when compared with the SM group (p = 0.039; Dunn´s Test). Median Ang-2 concentration did not differ significantly between SM and UM (p > 0.05, Dunn‟s Test; Figure 5.3A). When Ang-2 levels in the malaria group were compared between week 0 samples and week two samples, the median levels were significantly reduced in the latter (p < 0.001, Mann-Whitney U Test; Figure 5.3B-5.3C). Comparable median levels of plasma Ang-2 were observed among the different age categories in both malaria groups (p > 0.05; Kruskal-Wallis, Dunn´s Test; Appendix D). Figure 5.3: Median plasma Ang-2 levels among study groups. (A) Plasma Ang-2 levels were assessed by ELISA in children with SM (41), UM (69), non-malarial febrile illness (FC; 13) and healthy community controls (CC; 68) on admission day (week 0). Data are presented as box plots with median and with 25th and 75th percentile distribution of data. Whiskers represent minimum and maximum values with outliers. (B-C) Ang-2 levels on week 0 compared with week two convalescent samples in both SM (B) and UM (C). Each horizontal line represents the data points for each individual measurement. Multiple variables were analysed using the Kruskal-Wallis Rank Sum Test with Dunn‟s Multiple Comparison Test. Comparisons between two variables were done using Mann-Whitney U Test. Wk 0 = week 0; wk2 = week two 147 5.4.5 Plasma HMGB1 levels Plasma concentrations of HMGB1 were determined in admission samples and in convalescent samples (week two) and the data are summarised in figure 5.4. Median levels of plasma HMGB1 were significantly increased in SM and UM when compared to the healthy CC group (SM = 9.66 ng/mL; UM = 10.80 ng/mL; CC = 4.96 ng/mL; p < 0.001; Kruskal-Wallis, Dunn´s Test; Figure 5.4A) on admission day (week 0; Figure 5.4A). The levels were however comparable between SM and UM in admission samples (p > 0.05 Dunn‟s test; Figure 5.4A). In week two convalescent samples, plasma HMGB1 levels remain high in both malaria groups similar to levels reported for week 0 (p > 0.05; Mann-Whitney U Test) [Figures 5.4B and 5.4C]. There was no difference in plasma HMGB1 levels among the different age groups in both malaria groups (Appendix D; Dunn‟s Test). Figure 5.4: Median plasma HMGB1 levels among study groups. (A) Plasma HMGB1 levels were assessed by ELISA in children with SM (41), UM (69), non-malarial febrile illness (FC; 13) and healthy community controls (CC; 68) on admission day (week 0). Data are presented as box plots and with median with 25th and 75th percentile distribution of data. Whiskers represent minimum and maximum values with outliers. (B-C) HMGB1 levels on week 0 compared with week two convalescent samples in both SM (B) and UM (C). Each horizontal line represents the data points for each individual measurement. Multiple variables were analysed using the Kruskal-Wallis Rank Sum Test with Dunn‟s Multiple Comparison Test. Comparisons between two variables were done using Mann-Whitney U Test. Wk 0 = week 0; wk2 = week two 148 5.5 Discussion This study quantified a panel of markers including sICAM-1, sEPCR, Ang-2 and HMGB1 in plasma samples; comparing the levels between children with peripheral smear-positive P. falciparum infections, aged control children with febrile non- malaria infections and healthy community control children. The finding that levels of all inflammatory and endothelial activation mediators measured were markedly elevated in children presenting with acute malaria in comparison with healthy community controls are in agreement with the notion that acute malaria is partly a disease of systemic inflammation (reviewed in Storm and Craig, 2014; Miller et al., 2013; Clark et al., 2006). The adhesion molecule ICAM-1 is a cytokine-inducible glycoprotein expressed at low levels on diverse cells including resting endothelial cells (Gearing and Newman, 1993). The physiological role of ICAM-1 is to mediate leucocyte adhesion and trafficking to inflammatory sites by binding to the leucocyte function-associated antigen and Mac 1, which are expressed on leucocytes (Diamond et al., 1990; Simmons et al., 1988). Expression of ICAM-1 is elevated following endothelial cell activation by various stimuli including cytokines (Gearing et al., 1992; Becker et al., 1991). ICAM-1 is a well-recognised major receptor for the virulent malaria parasite protein PfEMP1, sequestering IEs in vital organs including the brain (reviewed in Chakravorty and Craig, 2005). Corroborating evidence comes from histological studies that show extensive accumulation of IEs localised with raised levels of ICAM- 1 in the brain tissues (Silamut et al., 1999; Turner et al., 1994). Membrane-bound ICAM-1 can be cleaved from the cell surface leading to the detection of increased concentration of its soluble forms in plasma during infection and that appears to 149 correlate with disease severity (Adukpo et al., 2013; Cserti-Gazdewich et al., 2010; Tchinda et al., 2007; Elhassan et al., 1994; Jakobsen et al., 1994; Hviid et al., 1993). In this study, sICAM-1 levels were found to be significantly higher in Ghanaian children with falciparum malaria than in comparable healthy community control children on admission days (Figure 5.1A), supporting and extending previous findings (Park et al., 2012; Turner et al., 1998; Hviid et al., 1993). Soluble ICAM-1 levels did not differentiate children with SM from those with UM although a trend of higher median sICAM-1 levels was detected in SM (Figure 5.1A). Although this observation is consistent with previous findings (Park et al., 2012; McGuire et al., 1996; Deloron et al., 1994), there are other studies that have reported higher levels of sICAM-1 in individuals with SM in comparison with UM or controls (Cserti-Gazdewich et al., 2010; Tchinda et al., 2007; Turner et al., 1998). The site of endothelial inflammatory reactions may be an important determinant for disease presentation. In keeping with that, children with CM may experience inflammatory reactions in brain endothelial cells whereas children with UM with increased levels of sICAM-1 may have inflammatory reactions in endothelial beds in other parts of the body. It is also possible that children diagnosed with UM with high levels of sICAM-1 reported here could have advanced to severe disease if they had not sought treatment. The raised levels of sICAM-1 in acute malaria may indicate an increased activation of endothelial cells likely resulting in increased receptor expression and subsequent shedding from the vascular endothelium and activated lymphocytes (Turner et al., 1998; McGuire et al., 1996). Like ICAM-1, EPCR serves as a receptor for specific PfEMP1 variants and has been implicated in SM complications (Lau et al., 2015; Turner et al., 2013). EPCR is a 64 kDa type 1 transmembrane glycoprotein expressed on a wide variety of cells, with 150 particularly low expression on brain microvascular cells (Laszik et al., 1997). It is involved in the regulation of inflammation, coagulation as well as in generalised cytoprotective processes on endothelial cells by converting protein C to activated protein C [APC] (Rao et al., 2014; Griffin et al., 2012; Stearns-Kurosawa et al., 1996; Fukudome and Esmon, 1994). There is considerable overlap in the PfEMP1 and APC binding interfaces on EPCR and PfEMP1 shows a greater binding affinity for EPCR (Lau et al., 2015). A soluble form of EPCR (sEPCR) is found circulating in plasma of healthy individuals and exhibits comparable binding affinity for APC (Fukudome et al., 1996; Regan et al., 1996). The plasma levels of sEPCR in a steady state are thought to be influenced by variations in the genetic sequence of EPCR (Uitte de et al., 2004; Xu et al., 2000). Many studies have determined the levels of sEPCR in an attempt to link these levels with severity of many infectious diseases (Moussiliou et al., 2015; Medina et al., 2004; Uitte de et al., 2004). In malaria, although sEPCR was shown to mediate binding to IEs and block their adhesion to human brain microvasculature endothelial cells (Petersen et al., 2015), the reports on sEPCR and disease associations have been conflicting. One study showed that elevated levels positively correlated with severe disease (Moussiliou et al., 2015) whereas other studies found reduced levels in SM (Shabani et al., 2016; Hansson et al., 2015). The present study showed that sEPCR levels were increased in malaria patients in comparison with healthy controls (Figure 5.2A), consistent with observations in other diseases characterised by inflammation. Notably, in sepsis, a condition with similar symptomatology to malaria, the consensus is that raised levels are detected in individuals with sepsis in comparison with healthy individuals (Kendirli et al., 2009; Liaw et al., 2004; Kurosawa et al., 1998). The findings reported here are partially consistent with a previous study on Beninese children where raised sEPCR levels 151 were recorded in SM (CM) with lower levels in convalescent children (Moussiliou et al., 2015). However, no significant differences were detected between children with SM and UM in this study which is at variance with the Beninese study where sEPCR clearly discriminated the two disease syndromes (Moussiliou et al., 2015). In other studies, reduced levels of sEPCR were detected in SM, a marked departure from this study (Shabani et al., 2016; Hansson et al., 2015; Moxon et al., 2013). The observed differences could be due to the fact that the present study did not investigate the role of EPCR genetic variants. Indeed, earlier studies have demonstrated a connection between the prevalence of the rs867186-GG genotype and SM, although this is not always consistent (Shabani et al., 2016; Hansson et al., 2015; Naka et al., 2014; Schuldt et al., 2014). Thus, this study cannot possibly rule out the influence of genetic differences on the steady-state levels and whether those levels predisposed some individuals to be more or less susceptible to severe disease, although a study in Ghana showed that genetic variations did not affect susceptibility to SM (Schuldt et al., 2014). The Angiopoietin family of proteins and their Tie-2 receptors are well-recognised regulators of the endothelial barrier integrity. On one hand, Ang-1-mediated Tie-2 signalling maintains the endothelium in a steady state by suppressing inflammation. Ang-2 on the other hand competitively opposes the protective effects of Ang-1 by priming endothelial cells to become responsive to inflammatory cytokines, which destabilises the microvascular integrity resulting in increased vascular permeability (reviewed in de Jong et al., 2016; Fiedler and Augustin, 2006). The importance of an activated and dysfunctional endothelium in the pathogenesis of SM cannot be overemphasized as previous studies have unequivocally documented significantly 152 decreased levels of circulating Ang-1 and increased Ang-2 levels in severe patients (Conroy et al., 2010; Conroy et al., 2009; Lovegrove et al., 2009). This study documented elevated plasma levels of Ang-2 in admission samples of SM and UM in comparison with healthy community control children (Figure 5.3). The data generated are compatible with prior studies that demonstrate altered Ang-2 levels in severe disease in comparison with healthy controls (Jain et al., 2011; Yeo et al., 2008). Yeo et al., (2008) showed that Ang-2 is a superior marker of SM-related deaths compared to lactate in Indonesian adults. Furthermore, Ang-2 levels alone have been proven to be a clinically robust marker of disease severity or mortality in children (Conroy et al., 2012). In the present study, plasma Ang-2 levels could not discriminate children with SM from UM, although increased Ang-2 levels were detected in the former. Thus, the data point to the potential function of Ang-2 dependent endothelial activation in the early onset of malaria. The HMGB1 protein is an abundantly expressed nuclear non-histone protein believed to be an important late-onset cytokine mediator of systemic inflammation (Wang et al., 1999a). It is actively secreted from stimulated immune cells or passively secreted from necrotic cells (Scaffidi et al., 2002; Andersson et al., 2000; Wang et al., 1999b). Once liberated from cells, HMGB1 triggers inflammatory responses via interaction with its numerous binding receptors which result in downstream pathways to activate immune cells and endothelial cells to secrete more cytokines including TNF-α, which in turn can induce more HMGB1 production (Clark et al., 2006; Park et al., 2006; Fiuza et al., 2003; Andersson et al., 2000). Thus, secretion of HMGB1 into the circulation forms a positive feedback loop, a process that sustains a long-term inflammatory state (Andersson and Tracey, 2011; Clark et al., 2006; Alleva et al., 2005). Evidence of the importance of HMGB1 in promoting inflammation comes 153 from sepsis, a condition with a similar clinical resemblance to malaria. These studies reported elevated levels of plasma HMGB1 that correlated with the degree of organ dysfunction and poor disease outcome (Sunden-Cullberg et al., 2005; Wang et al., 2001). The suggestive role of HMGB1 in modulating the outcome of malaria infection has also been demonstrated. Indeed, studies have reported significantly increased levels of plasma HMGB1 in fatally ill African children relative to healthy controls or those who survived from severe disease (Angeletti et al., 2013; Higgins et al., 2013; Alleva et al., 2005). However, none of these studies have examined the kinetics of HMGB1. Thus, information pertaining to HMGB1 levels post recovery is not available. In this study, markedly increased levels of plasma HMGB1 were detected in malaria patients and febrile children compared with age-matched healthy community control children in admission samples [week 0] (Figure 5.4A). This confirms findings from earlier malaria studies (Angeletti et al., 2013; Alleva et al., 2005) as well as reports from bacterial sepsis and other acute systemic inflammation-related conditions (Ito et al., 2011; Sunden-Cullberg et al., 2005). The levels of plasma HMGB1 were found to be comparable among the three clinical groups suggesting that HMGB1 may be a marker of general inflammation. In a previous study carried out on Ugandan children with falciparum malaria, higher HMGB1 levels were used to discriminate between SM and UM in admission samples and the levels were subsequently used to predict a fatal outcome (Higgins et al., 2013), which is at variance with this study. Notwithstanding, the data presented here demonstrate that plasma levels of HMGB1 may not always correlate with disease severity or fatal outcome which is in accordance with previous reports (Angeletti et al., 2013; Angus et al., 2007; Gibot et al., 2007; Sunden-Cullberg et al., 2005). 154 Soluble EPCR and sICAM-1 levels were raised in both malaria and febrile control groups, which is in line with previous studies that implicate endothelial activation in many infections (Moxon et al., 2014; Wang et al., 2013a; Saposnik et al., 2012). The levels of all the soluble markers assessed in this study except HMGB1 markedly declined following treatment in convalescent patients when two weeks post-treatment samples were compared with admission samples (Figures 5.1B-C, 5.2B-C, 5.3B-C). This suggests that the alterations in the levels of the various soluble markers screened at disease presentation were likely influenced by the infection rather than host genetic variations. The plasma levels of HMGB1 however, were found to persist in both malaria groups in convalescent samples at two weeks post treatment (Figure 5.4B-C), a finding reported for the first time in malaria studies. A similar observation has been documented in previous sepsis studies where they showed that levels remain increased even one week after treatment (Angus et al., 2007; Sunden-Cullberg et al., 2005). The relevance of this prolonged elevation is not entirely known but it may suggest a central role of HMGB1 in both febrile conditions and in disease progression by possibly protracting the symptoms of the disease. This study cannot rule out the potential influence of genetic variations on the observed data and future studies involving more time points may provide more details on when levels decline in malaria patients. A notable observation in this study was that all the markers of inflammation and endothelial activation assessed could not discriminate between SM and UM as documented in some studies. A possible explanation for this difference may be related to the study population and sample size. In most previous studies the study cohort included well-categorised CM cases (Adukpo et al., 2013; Conroy et al., 2012; Cserti- Gazdewich et al., 2010). In the present study, however, a majority of the children in 155 the SM group consisted of children with hyperparasitaemia (n=33) [Table 5.1]. Only four CM cases were detected during the sampling season, supporting the observation that SM incidences are markedly declining in endemic regions (WHO, 2016). Thus, our sample size possibly affected our ability to detect any statistical difference between the malaria groups. It is thus predicted that significantly elevated levels of the various markers would have been detected with enough representation of more fatal cases, since a pattern of higher soluble marker levels was observed in severe cases than those with uncomplicated cases. Some limitations apart from the effect of sample size were identified. Previously, it had been shown that markers of endothelial activation remain elevated in malaria patients compared with healthy controls up to a month after resolution of infection, possibly resulting from accumulated vascular injury encountered after multiple malaria infections (Moxon et al., 2014). In this study, the healthy control group was only sampled at one time point. Hence the levels of soluble markers in convalescent samples could not be compared to the healthy control group and as such, this study cannot totally rule out if the declined levels observed in convalescent samples are significant. Furthermore, the levels of soluble markers were not evaluated in asymptomatic non-febrile children. Contributions from parasite density alone to endothelial activation remains to be determined and such data may have important consequences given that most individuals in endemic populations are asymptomatic. It is well accepted that SM pathology is complex, comprising both parasite cytoadhesion and host inflammatory processes. Although it is entirely unclear which of the factors initiates the pathological process, it is highly plausible that both processes occur simultaneously and jointly reinforce each other. In chapter three, it was shown that IEs expressing a specific PfEMP1 motif can concomitantly bind 156 EPCR and ICAM-1 and that correlates with a high risk of developing CM. Thus, the additive actions of cytoadhesion of IE to both receptors and a hyperactive immune response may trigger pathways that would promote high expression of ICAM-1 molecules, loss of EPCR expression, increased release of pro-inflammatory cytokines such as TNF-α and endothelial activation (reviewed in Hviid and Jensen, 2015). Increased production of TNF-α mediates the release of other cytokines including HMGB1 that further causes the release of more TNF-α leading to a vicious cycle resulting in the upregulation of ICAM-1 and the further amplification of IE sequestration and associated shedding of soluble forms. Endothelial activation may trigger the exocytosis of Ang-2 from Weibel Palade bodies resulting in increased blood-brain barrier leakage and elevation of the soluble forms of EPCR and ICAM-1 in circulation. Indeed, microvascular obstruction, inflammation, cerebral oedema, blood-brain-barrier dysfunction and breakdown are notable features in paediatric CM (reviewed in Storm and Craig, 2014; Miller et al., 2013; Clark et al., 2006). Thus, targeted host response studies combined with analysis of specific PfEMP1 motifs could unravel new mechanisms of protection from cerebral IE sequestration including the development of therapeutic interventions. Overall, this study demonstrates that endothelial activation characterised by high levels of soluble adhesion molecules and inflammatory mediators are observed in Ghanaian children with acute malaria. 157 CHAPTER SIX CHARACTERISATION OF BLOOD OUTGROWTH ENDOTHELIAL CELL (BOEC) IN VITRO SYSTEM AND ITS POTENTIAL USE TO STUDY THE ADHESION PROPERTIES OF PLASMODIUM FALCIPARUM-INFECTED ERYTHROCYTES 6.1 Introduction The endothelium is made up of a monolayer of endothelial cells lining the vascular system and is capable of interacting with its environment. In its steady state, the vascular endothelium regulates blood homeostasis, suppresses coagulation, and acts as a barrier to molecules within the circulation. By contrast, activation of the endothelium can disrupt all these properties resulting in coagulation and vascular leakage (reviewed in Aird, 2012; Pate et al., 2010; Galley and Webster, 2004; Cines et al., 1998). In P. falciparum infection, the vascular endothelium is an active participant. Significantly, IEs interact with adhesion receptors enriched on the surface of endothelial cells, stimulating the endothelial cells to upregulate the expression of these receptors on their surface in response to the infection. Indeed, cytoadhesion of IEs, mediated by the parasite protein PfEMP1, to host endothelial receptors including ICAM-1 and EPCR is a prominent pathological feature in SM. This adhesion process results in a build-up of sequestered IEs in the microvascular beds of vital organs (Hviid and Jensen, 2015; Storm and Craig, 2014). IE sequestration has been linked to a significant risk of developing CM as previous post-mortem studies reveal high numbers of sequestered IEs in brain vessels of patients who died from CM (Milner, Jr. et al., 2014; Milner, Jr. et al., 2014); (Taylor et al., 2004; Silamut et al., 1999; MacPherson et al., 1985). Sequestration, accompanied by the release of toxic parasite molecules and a distorted host inflammatory cytokine response, lead to marked 158 increase in expression of adhesion proteins, endothelial dysfunction and a collapse in the vascular barrier integrity (Seydel et al., 2015; Cunnington et al., 2013b; Miller et al., 2013; Dorovini-Zis et al., 2011). Research attempting to unlock the processes involved in parasite cytoadhesion and endothelial dysfunction related to malaria pathology has been largely based on evaluations of autopsy samples and use of animal models (Milner, Jr. et al., 2014; Craig et al., 2012; Franke-Fayard et al., 2010; White et al., 2010; Taylor et al., 2004). Although these models have offered insightful snapshots into disease mechanisms, they have some challenges. The currently available animal models only partially replicate the pathological features of SM. Notably, sequestration of erythrocytes infected with P. falciparum in brain tissues has not been observed. Hence their relevance to disease understanding in humans has been intensely challenged (El- Assaad et al., 2014; Franke-Fayard et al., 2010; White et al., 2010). As a result, the study of parasite sequestration in the brain has been confined to the evaluation of autopsy samples which are carried out at end point. The challenge associated with this particular assessment is that it is impossible to investigate the early phase of clinical symptoms. Additionally, there are ethical and cultural restrictions or barriers in obtaining cerebral tissues for investigations (El-Assaad et al., 2014; White, 2011; Taylor et al., 2004; Medana et al., 2001). In vitro sequestration systems remain essential for studying the adhesive interactions between IEs and endothelial cells in the absence of suitable animal models. Most models used have been derived from non-human sources such as transfected Chinese hamster ovary cells as well as human sources, which include melanoma cells and primary cells (Rogerson et al., 1995; Hasler et al., 1993; Swerlick et al., 1992; Schmidt et al., 1982). Although important knowledge has been gained from these 159 routinely used in vitro cells, they have some limitations (Andrews et al., 2005). Significantly, primary cell sources are usually pooled samples obtained from different individuals and thus may not ideally reflect in vivo situations in malaria patients while others are known to exhibit a limited life span as well as phenotypic drift. Even more, the processes involved in obtaining some of the physiologically relevant endothelial cells are invasive and time consuming and as such cannot be routinely applied (Wassmer et al., 2011). Given the clinical importance of parasite adhesive interactions with the host endothelium, there is a recognised need to develop more tools since that could result in opportunities to guide the development of medicines and adjunctive therapies. Blood outgrowth endothelial cells [BOECs] (Lin et al., 2000), also referred to as endothelial colony forming cells [ECFCs] (Ingram et al., 2004) could serve as a suitable alternative cell source to study falciparum parasite adhesive interactions. BOECs are a sub-fraction of circulating rare cells (0.010 %) in peripheral mononuclear cells of healthy individuals and have differentiated matured endothelial cell phenotypes (Dauwe et al., 2016; Timmermans et al., 2007; Yoder et al., 2007; Ingram et al., 2004). Their levels are thought to be modified in some disease states such as sickle cell anaemia where high levels signify the inflamed status of the endothelium (Sakamoto et al., 2013; Strijbos et al., 2009; Solovey et al., 1997). Also in malaria, significantly raised circulating levels have been implicated in CM complications (Oduro, 2015). These cells can easily be harvested from peripheral blood, adapted to in vitro cultures with enhanced expansion capacities while maintaining a stable phenotype and are believed to be well suited for vascular regeneration (Dauwe et al., 2016; Martin-Ramirez et al., 2012; Yoon et al., 2005). The identity of BOECs is confirmed by a combination of characteristics including 160 their morphological features in cultures, expression of endothelial surface antigens and endothelial specific functional assays (Fuchs et al., 2010). They display cobblestone morphology reminiscent of endothelial cells and also express cell surface markers such as CD146, von Willebrand factor and CD31. These cells possess the ability to form capillary-like structures on Matrigel and are able to incorporate acetylated low-density lipoproteins (Ingram et al., 2004; Lin et al., 2000). BOECs have been successfully utilised to study diseases associated with endothelial perturbation and to investigate differential transcript levels of endothelial genes (Sakamoto et al., 2013; Chang et al., 2008; Fernandez et al., 2005). Thus BOECs derived from malaria patients could potentially be a practical alternative in vitro system to evaluate the adhesive properties between P. falciparum parasites and the host endothelial cells. 6.2 Hypothesis and specific objectives The study hypothesized that BOECs can be derived from the peripheral blood of malaria patients Specific objectives 1. To isolate and culture BOECs from PBMCs obtained from malaria patients 2. To profile the surface expression markers on BOECs 161 6.3 Methods 6.3.1 Sample collection and processing and isolation of peripheral blood mononuclear cells Samples for this study were collected during the 2016 malaria transmission season at the Hohoe Municipal Hospital in Ghana (see chapter four section 4.3.1). Briefly, heparinised blood samples (3 mL-8 mL) were obtained from 34 hospitalised children (< 12 years old) after written consent was given and processed for peripheral blood mononuclear cells (PBMCs) within 1 h of collection. Briefly, the blood sample was carefully layered on top of an equal volume of Ficoll-Paque solution (GE Healthcare, Sweden) in a 50 mL conical flask and centrifuged (400 g; 15 min) without brakes. Three different phases were observed after centrifugation. The topmost plasma layer was aspirated. The white blood cells containing the PBMCs formed the middle layer and that was collected into a new 50 mL falcon tube. The bottom layer contained the Ficoll and was discarded. The PBMCs were washed with RPMI 1640 with 25 mM Hepes modified (Sigma-Aldrich, Germany), supplemented with 50 µg/mL gentamicin sulfate (Sigma-Aldrich, Germany), [referred to as incomplete RPMI] and centrifuged (400 g; 10 min) with brakes and the wash process repeated again. The cells were frozen in 60 % incomplete RPMI, 30 % foetal bovine serum (FBS) [Sigma-Aldrich, Germany] and 10 % Dimethyl sulfoxide (Sigma-Aldrich, Germany) in a Mr. Frosty freeze container at -80 ⁰C freezer overnight and then transferred the next day to liquid Nitrogen. The PBMCs were subsequently transported on dry ice to Copenhagen and further stored in a freezer at -150 oC. 162 6.3.2 Thawing of PBMCs PBMCs collected from the field were removed from the -150 oC freezer on ice to the laboratory. Cells were rapidly thawed at 37 ⁰C and transferred to 12 mL round bottom tubes containing 4 mL of BOEC medium. The cell suspension was centrifuged (300 g; 5 min), resuspended in 2mL BOEC medium and seeded into 6-well plate precoated with collagen (50 µg/mL in DPBS). The plates were incubated (37 ⁰C ; 5 % CO2) and the medium was carefully aspirated to remove non-adherent and cellular debris after 48 h. Thereafter, the medium was changed thrice a week until first passage or discarded at day 30 for cells that did not produce BOECs. Cells were visualised everyday under a Leica inverted microscope (Leitz Wetzlar, Germany) and distinct BOEC colonies were observed between six and 20 days after plating. Cells that generated BOECs covering about 60 % of the surface area of the well were passaged into 25 cm2 flasks for continuous expansion. 6.3.3 Preparation of rat tail collagen type I-coated tissue culture surfaces Rat tail collagen type I (BD Biosciences, USA) was diluted in Dulbecco‟s phosphate- buffered saline (DPBS; Sigma-Aldrich, Germany) to obtain a final concentration of 50 µg/mL, filtered and stored at 4 ⁰C for one month. A volume of 2 mL of collagen I solution was aliquoted into each well of a 6-well tissue culture plate, 3 mL/25-cm2 flasks and 6 mL/75-cm2 flasks. Collagen coated plates or flasks were incubated overnight at 4 ⁰C or for 2 h at 37 ⁰C in 5 % CO2 atmosphere. Before seeding of PBMCs or BOECs, the collagen solution was removed and the coated plates or flasks were washed once with DPBS (2 mL/well for 6-well plates, 3 mL/25-cm2 flasks, and 6 mL/75-cm2 flasks). 163 6.3.4 PBMCs and BOEC cultures BOECs were generated from thawed PBMCs according to (Martin-Ramirez et al., 2012) with slight modifications. PBMCs were seeded in 6-well plates and monitored carefully until BOECs appeared. Both PBMCs and BOEC cultures were maintained at 37 ⁰C in a humidified incubator under 5 % CO2 atmosphere and in endothelial cell basal medium (EBM-2) [Lonza, USA] supplemented with 10 % FBS (Sigma-Aldrich, Germany) and a set of SingleQuotsTM kit (Lonza, USA). The EBM-2 together with supplements will now be referred to as BOEC medium. The volume of BOEC medium used for maintaining cells were 2 mL/well for 6-well plates, 8 mL/25-cm2 flasks, and 15 mL/75-cm2 flasks, unless otherwise stated. 6.3.5 Passage of cells BOECs were split at 80-90 % confluency, depending on the requirement. The culture medium was aspirated from flask and pre-warmed PBS was added and swirled around to wash attached cells. The PBS was aspirated and the wash process repeated again. To detach cells, 1-2 mL of 0.025 % trypsin-EDTA (Sigma-Aldrich, Germany) was added to the cells and incubated at 37 ⁰C for 5 min until all the cells were rounded up and lifted from the surface of the flask. The activity of trypsin was stopped by addition of 5-10 mL BOEC medium and the cell suspension transferred to a 12 mL round bottom tube and centrifuged (300 g; 5 min). The supernatant was gently decanted, cells were resuspended in BOEC medium and were usually split 1:4 for continuous expansion of culture in collagen coated 25 cm2 flasks. The cell suspension was incubated for about 2 h to allow cells to adhere and the medium exchanged to remove non-attached cells with pre-warmed fresh BOEC medium. 164 6.3.6 Freezing of BOECs BOECs at about 80 % confluency in a 75 cm2 flask were trypsinised as detailed above. Cells were then resuspended in a freeze mix comprising 60 % BOEC medium, 30 % FBS (Sigma-Aldrich, Germany) and 10 % Dimethyl sulfoxide (Sigma-Aldrich, Germany) to a concentration of 106 cells /mL which were aliquoted (1 mL each) into cryotubes. Cells were stored at -80 ⁰C freezer overnight in MR Frosty freezing container and subsequently transferred to -150 ⁰C freezer. 6.3.7 Thawing of BOECs Frozen stocks (1 mL) were transferred from the -150 ⁰C freezer on ice, thawed at 37 ⁰C and transferred to 12 mL round bottom tubes containing 4 mL of BOEC medium. The cell suspension was centrifuged (300 g; 5 min) and the supernatant gently decanted. The cells were resuspended in 8 mL BOEC medium, seeded on a collagen coated 25 cm2 flask and incubated (37 ⁰C; 5 % CO2). The medium was replaced with 8 mL BOEC medium the next day. 6.3.8 Phenotypic assessment of BOECs Flow cytometry was carried out to phenotypically characterise cultured BOECs using cells between third and sixth passage from eight different donors. Cultures showing greater than 80 % confluence in 75 cm2 flasks were used for the assay. One flask containing BOEC was stimulated with human recombinant TNF-α (10 ng/mL; Thermofisher Scientific, USA) overnight and another culture flask served as resting cells. At least, three independent experiments in duplicates were carried out for all successfully expanded BOEC cultures. Resting or stimulated BOEC cultures were detached with trypsin-EDTA as described before (section 6.3.5). BOECs were washed 165 once with PBS, resuspended in cold PBS and viable cells counted using the trypan blue (VWR, USA) exclusion method in a Neubauer chamber. BOECs were centrifuged (300 x g; 5 min) and resuspended in PBS (Thermofisher Scientific, Germany) supplemented with 3 % BSA (Saveen Werner AB, Sweden) [assay buffer] to a concentration of 1x106/mL. Hundred microlitres of resuspended BOECs was aliquoted into wells and incubated (45 min; 4 ⁰C) in the dark with primary monoclonal mouse antibodies against human CD146 (0.25 µg/mL; Biolegend, Switzerland), human CD31 (0.1 µg/mL; R&D Systems, USA), human ICAM-1 (0.1 µg/mL; R&D Systems, USA), human thrombomodulin (0.1 µg/mL; TM) [R&D Systems, USA], human CD36 (0.1 µg/mL; Beckman-Coulter, Germany) or goat polyclonal antibody against human EPCR (1.5 µg/mL; R&D Systems, USA). Cells were washed three times with assay buffer and incubated (30 min; 4 ⁰C) with fluorescein isothiocyanate (FITC) conjugated anti-mouse (1:200; Vector Laboratories, USA), FITC conjugated anti-goat secondary IgG (1:200; Vector Laboratories, USA) or FITC conjugated mouse anti-human CD133 (1:10; Miltenyi Biotec, Germany) where appropriate. Following washing, cells were resuspended in assay buffer (100 µL) and data acquired on an FC500 cytometer (Beckman-Coulter, Denmark). 6.3.9 Analysis Data on patients from which PBMCs were obtained were summarised as mean ± SD. Flow cytometry data were obtained from at least three independent experiments in duplicate wells with different donors and were analysed in Winlist 6.0 (Verity Software House). Unstained cells were used as a negative control and as a gating strategy for determination of stained and positive cells. Histograms of cell number versus logarithmic FITC fluorescence intensity were recorded. The mean fluorescence 166 intensity (MFI) for each of the tested cell surface antigen was determined and expressed as the geometric mean intensity ± SD. Graphical representations and statistical analysis of data were done in GraphPad Prism 7 software (GraphPad Software Inc., San Diego, USA). Differences between continuous variables were determined using a paired T-test with significance set at p < 0.05. 6.4 Results 6.4.1 Patients’ demographics The general characteristics of patients enrolled are found in Table 6.1. PBMCs were isolated from blood samples (3 mL-8 mL) obtained from 34 children with malaria (mean age 5.2 + 3.0 years) with similar numbers of males and females (Table 6.1). The mean age did not differ significantly between genders. 6.4.2 Success rate of BOEC colony establishment and morphological characterisation Thawed PBMCs were seeded on pre-coated collagen wells and cultures were examined daily using an inverted light microscope. A heterogeneous population consisting of round shaped cells and also distinct spindle-shaped adherent cells characteristic of early endothelial progenitor-like cells were observed between three and six days (Not shown). Most of these spindle-appearing cells died and detached within two weeks. Prominent cobblestone-like clusters typical of endothelial cells (Figure 6.1A) emerged in some cultures from day 6 to 20. Overall, 41 % (Table 6.1) of the PBMCs seeded generated colonies, while 64.3 % (Table 6.1) of these were observed within eight days after seeding. Colonies appearing in initial cultures after 167 15 days were observed to have a comparatively low proliferative capacity and could not be maintained beyond passage one. Further, 64.3 % of the generated colonies were observed in children < 5years old. Expanded BOEC colonies readily attached strongly to the collagen extracellular matrix within minutes of seeding, formed confluent monolayers (Figure 6.1A) in culture flasks within five days in 75cm2 flasks and maintained cell to cell contact. The majority (71.4 %) of observed BOECs successfully reached passage three (minimum passage number for assay; Table 6.1). BOECs pre-treated overnight with human TNF-α greatly altered their morphological appearance as they assumed elongated structures (Figure 6.1B) with no apparent increase in cell numbers when compared with resting BOECs. 168 Table 6.1: Patients profile and success rate of BOEC colony generation Figure 6.1: Morphology of expanded BOEC cultures in resting (A) and activated (B) states. BOECs readily attached to collagen upon seeding and formed confluent monolayers within three to five days of culturing. (A). Resting cells appeared cobblestone-like. (B) Overnight activation with TNF-α resulted in elongated cells (representative images). Bright round cells indicate dead floating cells. Bar indicates 100 µm. 169 6.4.3 Phenotypic assessment of resting and activated BOECs BOEC cultures were expanded and their surface antigen expression simultaneously assessed in both resting and TNF-α activated states using cells between passages three and six. The phenotype of resting and activated BOECs was assessed with specific antibodies targeting surface exposed markers including a positive endothelial marker CD146 and a negative control stem cell marker CD133 in flow cytometry experiments. Data were shown as MFI of expression of cell surface antigen markers (Figures 6.2A-6.2B, Table 6.2). Results for BOEC resting cultures showed that these cells constitutively expressed endothelial cell surface antigens CD146, CD31 and TM (Figure 6.2A, Table 6.2). In addition, cell surface adhesion molecules including ICAM-1 and EPCR were expressed (Figure 6.2A, Table 6.2). Specifically, high expression levels were recorded for CD146 and CD31, moderate levels were seen in EPCR and TM and very weak surface expression documented for ICAM-1 (Table 6.2). Conversely, BOECs tested negative for the endothelial cell marker CD36 and the immature stem cell marker CD133 (Table 6.2). The inflammatory response of these cells was assessed by overnight treatment with 10 ng/mL of TNF-α and tested in flow cytometry assays (Figure 6.2B, Table 6.2). In comparison with resting cells, TNF-α treatment induced the expression of ICAM-1 significantly (Student‟s paired t-test; p<0.001), while the expression of CD146 and CD31 were marginally increased (Student‟s paired t-test; p < 0.05). By contrast, the expression of cell surface EPCR and TM levels was considerably reduced by overnight activation with TNF-α (Student‟s paired t-test; p<0.01). Pretreatment with TNF-α did not induce the surface expression of CD36 on BOECs (Table 6.2). 170 Figure 6.2: Characteristics of cell surface antigens. (A). Phenotypic characterisation of cell surface antigens of resting BOEC cultures assessed by flow cytometry using cells between passage three and six. Representative histograms showing overlays with gray histogram representing negative control and black histograms positively stained for CD146, CD31, ICAM-1, EPCR, TM and negatively stained for CD133 and CD36. (B). Comparative cell surface antigen expression in resting and activated states. BOECs activated overnight with 10 ng/mL of TNF-α showed increased expression of CD146, CD31 and ICAM-1, and decreased EPCR and TM expression. Representative histograms showing overlays with black histogram representing resting cells and red histograms for activated cells stained for CD146, CD31, ICAM-1, EPCR and TM. 171 Table 6.2: Flow cytometry assessment of BOEC cells Quantitative comparison of BOEC in resting and activated states was done using flow cytometry. Results represent the mean MFI+SD of cells expressing surface antigens from at least three independent experiments with cells propagated from different donors (n=8) and normalised to the mean MFI values of unstained cells. Analysis done by paired Student T-test (***p < 0.001; **p < 0.01; *p < 0.05) CD: cluster of differentiation; ICAM-1: intercellular adhesion molecule 1; GP IV: Glycoprotein four; TM: thrombomodulin; EPCR: endothelial protein C receptor; PECAM-1: Platelet endothelial cell adhesion molecule 1; MCAM: melanoma cell adhesion molecule 6.5 Discussion The discovery that rare precursors of endothelial progenitor cells resident in PBMCs can be isolated and propagated in vitro to yield endothelial cell-like phenotype (Asahara et al., 1997) raised significant interest in exploiting their potential therapeutic use in vascular regeneration. Their usefulness as a research tool in studying disorders linked to the vascular endothelium was also explored (Sakamoto et al., 2013; Martin-Ramirez et al., 2012; Fuchs et al., 2010; Fernandez et al., 2005). Since this significant finding, several studies have provided evidence that progenitors circulating in peripheral blood contain a heterogeneous population of cells (Hur et al., 2004; Gulati et al., 2003; Lin et al., 2000). Among the diverse phenotypes characterised, is a unique population of cells so called BOEC or ECFC; these cells 172 display highly similar features to mature and differentiated endothelial cells in terms of their morphological appearance, surface antigen expression profile and the potential to form capillary-like networks on Matrigel (Fuchs et al., 2010; Yoder et al., 2007; Ingram et al., 2004; Lin et al., 2000). The present study evaluated the feasibility of isolating and characterising the surface antigens on BOEC from malaria patients as a potentially useful in vitro cell model to study malaria parasite adhesion properties. Overall, the study demonstrated for the first time that PBMCs from Ghanaian malaria patients, propagated on the basis of adhesion and maintenance in an endothelial selective culture medium, can yield BOEC colonies. The data further showed that BOECs can be readily established from small blood volumes and from frozen PBMC samples. Following seeding of thawed isolated PBMCs, spindle-like structures described as early circulating endothelial progenitors (Hur et al., 2004; Gulati et al., 2003) were observed in cultures between three and six days after culturing and this has been previously documented (Estes et al., 2010; Yoder et al., 2007; Ingram et al., 2004). The appearance of these structures possibly indicated that cells were appropriately frozen and viable. BOEC colonies were successfully observed in approximately 41 % of the PBMCs donors (Table 6.1). Earlier studies have recorded over 70 % success in the establishment of BOEC colonies. It is important to emphasize that these studies (Dauwe et al., 2016; Ormiston et al., 2015; Tura et al., 2013; Ingram et al., 2004) used relatively large volumes of blood (40 mL- 100 mL) compared to the volumes used in this study (3 mL-8 mL) and also some studies had similar or lower success rate of isolation in comparison with this study while using large volume (Campioni et al., 2013; Medina et al., 2013; Sakamoto et al., 2013; Mutunga et al., 2001). Moreover, most previous studies have generated BOEC from freshly harvested PBMCs whereas 173 this study shows for the first time, the generation of BOECs from frozen PBMCs. This may have contributed to the relatively low success rate. Furthermore, available data indicate that BOECs cannot be generated from some individuals and the reason for such difficulty remains to be investigated, although increased age has been suggested to be a potential confounder (Medina et al., 2013; Martin-Ramirez et al., 2012). A greater majority of the BOEC colonies (~64 %) emerged in individual cultures within eight days of seeding (Table 6.1). Previous findings have reported the appearance of colonies characteristic of BOECs from five days onwards post seeding, although most studies typically detect them between 14-21 days (hence referred to as late colony forming) (Bou et al., 2015; van Beem et al., 2009; Zhang et al., 2009; Yoder et al., 2007; Ingram et al., 2004; Lin et al., 2000). Although it is difficult to precisely explain these variations, they could possibly be related to factors such as material source, methods of propagation or genetic differences in study populations. A significant challenge in this field is the differing protocols and culture conditions employed in the derivation of these cells, resulting in high variabilities in individual findings (Gumina and Su, 2017; Fujinaga et al., 2016; Glynn and Hinds, 2014). Most studies characterising BOECs have obtained samples from individuals of non–African origin, unlike this study where donors were of African origin. BOEC cultures were expanded and phenotypically characterised based on their morphological appearance in culture and expression of specific endothelial cell surface antigens (Ingram et al., 2004). Characterisation of the cell surface antigens showed that despite donor variations, BOEC abundantly expressed CD146 and CD31 which are characteristics of endothelial cells (Figure 6.2; Table 6.2). They also expressed the anti-thrombogenic antigens TM and EPCR, whereas the proinflammatory inducible marker ICAM-1 was scarcely expressed on their surface 174 (Table 6.2). These phenotypes observed on resting cells have been repeatedly documented in many studies (Wang et al., 2013b; Ahmann et al., 2011; Yoder et al., 2007; Lin et al., 2002; Lin et al., 2000). Resting BOECs were negative for the endothelial cell-specific antigen CD36 and the immature stem cell marker CD133 (Figure 6.2; Table 6.2). Few studies have described CD36 expression on some population of BOECs (Sakamoto et al., 2013; Lin et al., 2000) and in these studies, an enrichment-based surface marker isolation method was employed. Thus, their method of isolation highly differed from this study; moreover, there is evidence showing that isolation and propagation methods employed have a remarkable influence on the type of BOECs generated (Glynn and Hinds, 2014; Delorme et al., 2005). In addition, it is well-recognised that there is considerable heterogeneity in the expression of surface antigens and adhesion molecules on endothelial cells from different vascular beds (Aird, 2007; Galley and Webster, 2004). The absence of CD133 expression demonstrated that these cells were derived from non-haematopoietic progenitors (Tura et al., 2013; Wang et al., 2013b; Timmermans et al., 2007). Taken together, the combined surface expression of several antigens gave credence to their endothelial phenotypic identity (Sakamoto et al., 2013; Wang et al., 2013b; van Beem et al., 2009; Ingram et al., 2004; Lin et al., 2000). Endothelial cells play diverse roles during inflammation, increasing their expression of adhesion molecules and secreting several cytokines (Galley and Webster, 2004; Cines et al., 1998). The present study exposed BOECs to the pro-inflammatory cytokine TNF-α overnight. A profound alteration in the morphology was observed in TNF-α activated cells, notably, they appeared elongated rather than cobblestone-like when observed under the microscope (Figure 6.1). The altered cobblestone morphology has been observed before in human endothelial cells and that was 175 attributed to the redistribution of surface expression markers like CD31 resulting from TNF-α stimulation (Romer et al., 1995). Characterisation of the cell surface antigens of these TNF-α activated cells showed a marked induction in the expression of surface ICAM-1 in contrast to the scarce expression of this adhesion molecule on resting cells (Figure 6.2; Table 6.2). The surface expression levels of CD146 and CD31 were slightly elevated (Figure 6.2; Table 6.2). By contrast, TM and EPCR expression levels were noticeably decreased and no change was observed in the expression levels of CD36 which was negative on resting cells (Figure 6.2; Table 6.2). The marked alteration in the expression of these surface antigens in response to the cytokine TNF- α, documented in this study, are in accordance with many investigations (Sakamoto et al., 2013; Ahmann et al., 2011; van Beem et al., 2009; Zhang et al., 2009; Lin et al., 2000). Infection by P. falciparum is often characterised by sequestration resulting from cytoadhesion of IEs to endothelial receptors in vital organs (Miller et al., 2013). Thus investigations attempting to understand in detail this deadly adhesion process remain critical for discovering therapeutic interventions against severe disease development. However, research into this process has been greatly hampered by the lack of appropriate animal models. The scarcity of autopsy samples with its related ethical concerns has also created an additional level of complication (El-Assaad et al., 2014; Taylor et al., 2004). Other clinically relevant endothelial cell sources including vascular-derived and adipose-derived microvascular endothelial cells have been investigated, however procedures for obtaining these cells are invasive, which is an undesirable process for the donor and hence not an optimal material source (Wassmer et al., 2011; Muanza et al., 1996; Swerlick et al., 1992). In vitro endothelial cell models still remain indispensable in advancing our understanding on cytoadhesion 176 processes. A model system that naturally expresses the receptors that are known to be involved in malaria pathology can provide details on the processes and also generate hypotheses for further investigations. In falciparum cytoadhesion interactions, clinically important parasite receptors on the host endothelium include EPCR and ICAM-1 (Lennartz et al., 2017; Turner et al., 2013; Turner et al., 1994). Indeed, data from chapter three showed that simultaneous adhesion to EPCR and ICAM-1 by IEs was strongly associated with CM complications. The BOECs isolated from this study expressed these molecules on their surface; even more, their surface expression was altered in response to the proinflammatory cytokine TNF-α. This cytokine has often been implicated in malaria pathogenesis; particularly high levels have been recorded in CM patients (Hunt and Grau, 2003; Kwiatkowski et al., 1990; Grau et al., 1989), thus they could serve as useful in vitro model in probing IE sequestration in CM. An obvious concern for considering BOECs as a study tool is that they are primary cell lines, as such are prone to lose their characteristics with many passages (age), differentiate into a non-endothelial phenotype or become senescent, thus restricting their usage to early passages (Medina et al., 2013; Medina et al., 2010; van Beem et al., 2009; Au et al., 2008). However, it is worthwhile to mention that these cells have been shown to exhibit a stable phenotype over many passages (Szoke et al., 2016; Medina et al., 2010; Fuchs et al., 2006; Lin et al., 2002). Moreover, it is possible to obtain high yield at low passages which can be stored for future use (Medina et al., 2013; Medina et al., 2010; Zhang et al., 2009; Lin et al., 2002). Furthermore, BOECs also have a significant advantage over other primary cells since they can be easily accessed. There is no specific marker(s) that unequivocally identifies mature endothelial cells particularly BOECs, as such, it is recommended that a detailed combination of molecular and functional assessments are carried out. In keeping with 177 this practice, in future, functional assays including their capacity to form capillary-like networks in vitro, internalisation of acetylated low density lipoproteins and pattern of localisation of cell surface antigens would be done to confirm their endothelial phenotype (Medina et al., 2013; Yoder et al., 2007; Bompais et al., 2004). Although this study did not provide evidence that these cells can support malaria parasite adhesion, preliminary data from our laboratory using BOECs derived from healthy European donors showed that they support the adhesion of erythrocytes infected with P. falciparum (Dr. Yvonne Adams, personal communication, University of Copenhagen). Moreover, the expression of putative parasite receptors on these cells suggests their utility and experiments are ongoing to demonstrate their adhesive properties. These cells can be easily and conveniently obtained from any consenting malaria patient since blood is usually taken minimally-invasively for standard clinical management, thus placing no extra burden on the patient. The procedures utilised in the isolation and establishment of BOECs described in this study are simple and can be routinely performed successfully. Notably, PBMCs can be easily isolated in field settings, frozen down, transported and subsequently cultured when convenient without loss of cell viability. Furthermore, these cells are culture adaptable, grow rapidly and can progressively be passaged to obtain large amounts. These remarkable characteristics make them attractive candidates as well as a physiologically appropriate cell and practical alternative in vitro parasite sequestration system. In summary, this study shows that BOECs can be successfully isolated and culture- expanded from paediatric malaria patients and thus offers the feasibility of using patient-derived BOECs to study parasite adhesive interactions and also test therapies aimed at improving endothelial function. 178 CHAPTER SEVEN FREQUENCY OF IGM BINDING PFEMP1 VARIANTS IN THE GENOME OF P. FALCIPARUM 7.1 Introduction The mechanisms underpinning SM development remain largely unexplained. However, cytoadhesion of IEs to an array of receptors on the host endothelium, to uninfected erythrocytes in a process known as rosetting, and to two or more IEs to form clumps via platelet binding are well-recognised pathophysiological features (reviewed in Rowe et al., 2009). These adhesion processes culminating in the sequestration of the mature forms of IEs in deep microvascular beds is largely responsible for organ-specific disease pathology including CM and PAM (Milner, Jr. et al., 2014; Taylor et al., 2004; Salanti et al., 2003; Fried and Duffy, 1996). The polymorphic high molecular weight adhesive protein family known as PfEMP1 is thought to be responsible for IE sequestration (Howard et al., 1988; Leech et al., 1984b). PfEMP1 are composed of variable adhesive domains of Duffy binding-like (DBLα, β, γ, δ, ε, δ and x) and cysteine-rich interdomain regions [CIDRα, β, γ and δ] (reviewed in Hviid and Jensen, 2015) allowing the parasite to bind numerous host receptors. There is compelling evidence showing that the ability of some IEs expressing PfEMP1 subtypes to mediate adhesion requires support from soluble proteins present in human plasma (Stevenson et al., 2015a; Stevenson et al., 2015b; Clough et al., 1998; Scholander et al., 1996). Although several plasma proteins have been implicated, only the pentameric form of non-immune IgM has been extensively described (Stevenson et al., 2015a; Barfod et al., 2011; Scholander et al., 1998). Notably, IEs responsible for rosette formation in children and CSA binding associated with placental malaria 179 are recognised as having a high affinity for IgM and both phenotypes have profound correlation to development of SM (Creasey et al., 2003; Rowe et al., 2002). Although the biological consequence of this binding is less clear, it is believed that the pentameric nature of IgM facilitates the grouping of multiple IEs together in order to fortify the adhesive interactions between IEs and their receptors on erythrocytes among rosetting parasites (Akhouri et al., 2016; Stevenson et al., 2015a). In PAM however, IgM binding appears to be an escape mechanism where targeted epitopes are shielded from immune destruction without jeopardizing IEs binding capacity to CSA (Barfod et al., 2011). The FCμ4 portion of the pentameric IgM is believed to interact with DBLδ and DBLε domains of PfEMP1 molecules on the surface of IEs (Stevenson et al., 2015a; Ghumra et al., 2008; Semblat et al., 2006). Altogether, this suggests that the parasite may utilise IgM to promote sequestration and hence avoid immune clearance which is critical to its survival as well as sustaining chronic infections. Previous studies have consistently reported that some PfEMP1 domains bind non- immune IgM (Jeppesen et al., 2015; Stevenson et al., 2015a; Ghumra et al., 2008; Rasti et al., 2006; Semblat et al., 2006). Thus, it is of high importance to investigate the number of IgM binders in the parasite genome given their potential connection with severe disease phenotypes. Keeping with this notion, only one study has characterised the number of PfEMP1 proteins in one parasite genome (NF54/3D7) with high binding affinity for IgM and this provided evidence of at least five PfEMP1 variants involved in IgM binding (Jeppesen et al., 2015). Of interest, one of the identified variants was found to mediate CSA adhesion (NF54 VAR2CSA), whereas the functional roles of the others could not be elucidated (Jeppesen et al., 2015). It is possible that the frequency of PfEMP1 variants involved in IgM binding may have 180 been grossly underestimated. This study aimed at identifying and characterising additional and new PfEMP1 variants that mediate IgM binding in the NF54/3D7 genome. Such information may increase our understanding of these adhesive processes and hopefully be crucial for designing strategies for interfering with IgM binding, which will reduce parasite sequestration and ultimately allow the immune system to control the infection. 7.2 Hypothesis and specific objectives In this study, it was hypothesized that there are several IgM binding PfEMP1 molecules in the genome of the P. falciparum isolate NF54 which have not yet been characterised Specific objectives 1. To identify the number of var genes upregulated in the NF54 genome following selection for IgM binding 2. To evaluate the IgM binding property of the identified var encoding PfEMP1 proteins 7.3 Methods 7.3.1 Parasite culture The previously generated P. falciparum NF54 clone, G6 (Frank et al., 2007) was transfected with a plasmid construct pVBH that carried the gene encoding blasticidin S deaminase (BSD) under the control of a UpsC var gene promoter. The P. falciparum NF54 pVBH-transfected G6 clone was kindly donated by Dr. Ron Dzikowski. The 181 procedures for generating transfectants have been previously described (Deitsch et al., 2001a). Briefly, electroporation cuvettes (0.2 cm) were loaded with 175 µL of uninfected erythrocytes and 50 μg of plasmid DNA in incomplete cytomix solution. Parasite populations that stably maintained the plasmid were first selected on 2 μg/mL blasticidin and thereafter, high copy number transfectants were subsequently generated by culturing in 10 μg/mL blasticidin. For this particular study, the stably transfected NF54 G6 clone was maintained in RPMI 1640 with 25 mM Hepes modified (Sigma-Aldrich, Germany), 50 µg/mL gentamicin sulfate (Sigma-Aldrich, Germany), 4 mM Glutamine (Sigma-Aldrich, Germany), 0.5 % Albumax II (Invitrogen, USA) (referred to as 0.5 % Albumax II medium) and blasticidin (10 μg/mL; Life Technologies, USA) to turn off transcription of endogenous var genes and wipe out the epigenetic memory. Cultures released from selective drug pressure were maintained in 0.5 % Albumax II (Invitrogen, USA) only using previously described methods (see chapter three section 3.3.9). The parasites were routinely genotyped (Snounou et al., 1999) and absence of mycoplasma contamination was regularly tested using the MycoAlert Mycoplasma Detection Kit (Lonza, USA) according to the manufacturer‟s protocol. 7.3.2 IE selection and single-cell cloning of IgM-binding IEs Antibody selection of IEs and single cell cloning were done following previously described procedures (Jeppesen et al., 2015). The pVBH-NF54 G6 clone maintained under blasticidin pressure was released from the selection pressure and maintained for two weeks in 0.5 % Albumax II culture medium only. Late stage IEs were purified by MACS as described before. Purified IEs (2 x 106 IEs/mL) were incubated (20 min; room temp) with sterile 10 nM human IgM (Sigma-Aldrich, Germany), washed three 182 times with sterile PBS supplemented with 2 % FBS (2 % FBS) and centrifuged (500 x g 5min; room temp). Then the IEs were stained with sterile phycoerythrin-conjugated donkey anti-human IgM (Jackson Immuno- Research, USA) (1:200; 30 min; room temp) and the wash process repeated (500 x g; 5min; room temp). IgM positive parasites (diluted to 1 x 106/mL in 2 % FBS) were then single cell sorted using the fluorescence-activated cell sorter Jazz sorter (Beckton Dickson, Germany) into round- bottom 96-well plates (Thermofisher Scientific, Denmark) containing Albumax II culture medium (100 µL/ well) and uninfected erythrocytes (1 µL/well). Varying numbers of IEs (50-1000) which served as controls were placed in some wells, while the majority of the wells contained single cells. The plates were incubated (37 °C) for two weeks with media replacement carried out thrice weekly with addition of uninfected erythrocytes (1 µL/well) twice a week. Control wells containing high numbers of IEs were used to monitor parasite growth by staining prepared slides with Giemsa (Merck, Germany) and subsequently observing them under the microscope. Control wells were also used for IgM binding in flow cytometry (see section 7.3.5). Thereafter, IgM positive clones were expanded in 25cm2 culture flasks (Thermofisher Scientific, Denmark). 7.3.3 Repeated selection of IgM positive clones Late stage IEs were purified by MACS as described before (see chapter three section 3.3.12) and incubated (30 min, room temp) with 10 nM human IgM (Sigma-Aldrich, Germany). Protein A dynabeads (25 µL; Invitrogen, USA) were aliquoted into a 10 mL centrifuge tube and 4 mL of incomplete RPMI was added to wash the beads. The tube containing the beads was placed in a magnetic field; the tube was inverted a few times and then allowed to stand for about 1 min. The wash medium was then aspirated 183 and the wash process was repeated. IgM (10 nM diluted in incomplete RPMI) was added to the beads and incubated (30 min; room temp) on a rocking table. The tube containing the mixture was placed on a magnet and the wash process repeated thrice to remove unbound IgM. Mature IEs were resuspended in 2 mL of incomplete RPMI, added to the IgM coated beads and incubated for about 30 min on a rocking table. The wash process was repeated thrice to remove unbound parasites. IgM selected parasites were resuspended in 0.5 % Albumax II culture medium (5 mL) at 2 % haematocrit, the mix was transferred to a 25cm2 culture flask, gassed and maintained in an incubator (see chapter three section 3.3.9). 7.3.4 TRizol samples and var gene transcription analysis Tightly synchronised ring stage IEs (> 2 %) were collected and preserved in TRizol reagent (Thermofisher Scientific, USA). The var transcription profiles were carried out as described in the methods section of chapter three (see chapter three section 3.3.8). Briefly, RNA was extracted from 100 µL ring stage parasites dissolved in 900 µL TRizol reagent (Thermofisher Scientific, USA) and used to generate cDNA (see chapter three section 3.3.7). qPCR was carried out using the synthesized cDNA and P. falciparum 3D7 var gene-specific primers as described previously (Salanti et al., 2003), with the modifications described in (Dahlback et al., 2007). Transcript levels relative to the seryl-tRNA synthetase housekeeping gene (p90) were determined using the 2-ΔCT method (see chapter three section 3.3.8). 7.3.5 IgM reactivity to the surface of IEs Growth of IEs and IgM binding were assessed by flow cytometry on a Beckman Coulter FC500 instrument (Beckman Coulter, Denmark) as described before 184 (Jeppesen et al., 2015) using MACS-purified late stage IEs or packed IE aliquots. IEs were incubated (30 min; 4 °C) with IgM (10 nM) and subsequently stained with ethidium bromide (2 µg/mL) and FITC conjugated anti-human IgM (Sigma-Aldrich, Germany; 1:150) for 30 min at 4 °C. Each experiment was done in duplicates. 7.3.6 Data analysis Calculations to determine the transcription level for each var gene relative to the p90 control gene were performed in Microsoft Excel. Comparison of transcript profiles of individual var genes within or between different subclonal parasite populations were presented as pie charts. The var gene with the highest relative transcription level was assigned as the dominant gene. Analysis of IgM binding data was done using the Winlist 6.0 software (Verity, Topsham). 7.4 Results 7.4.1 var transcription profile of NF54 G6 IEs propagated with or without blasticidin The study made use of the previously derived P. falciparum NF54 G6 clone to determine the frequency of PfEMP1 molecules involved in IgM binding. Briefly, the P. falciparum NF54-derived G6 clone which almost exclusively transcribes the group C var gene PFD1015c was transfected with a blasticidin resistance gene pVBH controlled by a var promoter (Fastman et al., 2012; Dzikowski and Deitsch, 2008; Frank et al., 2007). Transfectants were subsequently selected on 10 µg/mL blasticidin to ensure the carriage of high copy numbers of the plasmids. Maintaining this stably transfected parasite population in 10 µg/mL of blasticidin effectively halts the 185 transcription of the dominant var gene PFD1015c and results in the deletion of the epigenetic memory by the competing episomes, thus selecting for a parasite population that essentially expressed only the BSD gene. In contrast, exclusion of the blasticidin pressure allows the parasite population to begin to shed the pVBH plasmids resulting in the expression of essentially all var genes by the parasite population over a period of time in a random fashion (Fastman et al., 2012; Dzikowski and Deitsch, 2008). Taking advantage of this elegant technology, this study evaluated the transcription profile of var genes that were overexpressed following the removal of blasticidin pressure from the pVBH-NF54 G6 clone and subsequently selected these parasites for IgM binding. To verify that the BSD selected parasites did not express any var genes, RNA extracted from ring stage IEs maintained in blasticidin, were analysed by qPCR using var gene specific primers (Dahlback et al., 2007; Salanti et al., 2003). As expected, the relative levels of all var genes including the previous prominently transcribed PFD1005c var gene were effectively silenced (Figure 7.1). To investigate the onset of var transcription and switching, the blasticidin pressure was removed for two weeks to allow activation of var genes in the parasite population. The var gene expression profile showed the random transcription of almost all var groups at very low levels relative to the housekeeping gene (p90) [Figure 7.1] suggesting the induction of active transcription of endogenous var genes in the parasite population in the absence of blasticidin. 186 Figure 7.1: var gene transcription profile of 3D7/NF54 with or without blasticidin. The relative distribution of each var mRNA transcript in the parasite genome is represented as bar charts. Transcript levels were measured by qPCR using a specific set of primers targeting each of the var genes present in the 3D7 genome (Salanti et al., 2003; Dahlback et al., 2007). All values are presented as relative transcript number to the housekeeping gene, seryl-tRNA synthetase (p90). Different culturing conditions (under bsd (blue) or off bsd #1.1 (green), 1.2 (red)) are annotated at the topmost right of bar graphs. Off bsd#1.1 (green) and off bsd #1.2 (red) represent two rounds of sorting after release from blasticidin pressure. 7.4.2 IgM selection and var gene profile of IgM positive IEs Following the activation of var transcription after drug pressure removal, IEs were stained with IgM and sorted into 96-well plates using fluorescence-activated cell sorting to select for IgM positive cells which were then individually cloned. In vitro culturing and expansion resulted in 21 growing cultures arising from the single sorted IEs and one growing culture from a control well that received 1000 sorted IEs (subline). The var expression patterns were assayed and the results revealed the transcription of at least nine distinct var genes from the IgM positive IEs (Figure 7.2). All subclonal isolates showed the presence of several sub-population of var genes resulting from transcriptional switching, although, dominant transcripts could be 187 detected in some subclones. The relative transcript levels of some of the var gene encoded PfEMP1 implicated in IgM binding in the different subclonal isolates are represented in figure 7.2 (representative images). Notably, eight of the IgM-positive subclonal isolates (G6. 1.14, G6. 5.13, G6. 2.28, G6. 4.80, G6. 3.30, G6. 1.46, G6. 2.72, G6. 3.50) transcribed a particular var gene, two transcribed two dominant var genes (G6. 3.11, G6. 3.81) at similar levels and the remaining subclones (G6. 3.41, G6. 5.22, G6. 1.90, G6. 3.61, G6. 5.72) and one subline (G6. 3.15) displayed variable expression of multiple var genes at very low proportions. With respect to the subtypes of PfEMP1 positive for IgM binding, three subclones solely transcribed PFL0030c (G6 1.14, G6 5.13, G6 2.28), and the following individual subclones: PFD0995c (G6. 3.30), MAL6P1.316 (G6 4.80), PFL2665c (G6 2.72), PF08_0107 (G6 3.50) and PFA0005w (G6 1.46), prominently expressed a unique var gene. Also, one subclone showed high transcription for both MAL6P1.1 and PF13_0001 (G6 3.11), whereas another subclone co-expressed PFL2665c and PFL0020w (G6 3.81). 188 Figure 7.2: var gene profiling of IgM positive P. falciparum NF54 following subcloning and in vitro expansion. Expression of var genes was measured by qPCR and the values presented as relative transcript level to p90. Pies represent the relative distribution of var transcripts in the subclonal isolates (identified by a code on the top of each pie chart). Representative images of the major identified transcripts and the corresponding percentage in the various subclonal isolates are indicated on the pie charts. Colour coding and classification of var gene groups were assigned as previously described by Fastman et al., 2012 and Lavstsen et al., 2012 respectively: group A (gray), group B/A (orange), group B (blue), group B/C (green), group C (red) and VAR2CSA white The domain architecture of the PfEMP1 encoding the dominant var genes (Figure 7.3) identified variable numbers of DBLs and CIDRs domains. However, all the PfEMP1 could be placed into two semi-conserved structurally distinct groups. One group which comprised (MAL6P1.316, PFL0020w, PF07_0139 and PFL0030c) contained a similar subtype of DBLε domain with a variable number of this domain in the different var gene subtypes (Figure 7.3), which is consistent with a previous finding (Jeppesen et al., 2015). Within the DBLε subtype group, two var gene types 189 (MAL6P1.316 and PFL0020w) additionally encoded a DBLδ domain similar to the one described in the HB3VAR06 rosetting parasite isolate which is known to bind IgM (Stevenson et al., 2015a). The other semi-conserved group shared a common DBLδ subtype (MAL6P1.1, PFL2665c, PF13_0001, PFD0995c, PF08_0107 and PFA0005w) similar to one of the domains observed in PF07_0139 var subtype (Figure 7.3). Thus, the results suggest different PfEMP1 subtypes to be involved in non- immune IgM binding. Figure 7.3: Schematic of the extracellular domain organisation of the PfEMP1 proteins encoded by major var gene transcripts in identified subclonal IEs. Annotation for the new var IgM binders was obtained from the VarDom 1.0 server (http://www.cbs.dtu.dk/services/VarDom/). The domain numbers are indicated in italics along the top of the panel, the names of the PfEMP1 and the var promoter groups are indicated at the beginning of each domain structure. The identified IgM binding candidates in this study were PFL0030c, MAL6P1.316, PF07_0139 (Jeppesen et al., 2015) and the new var encoded PfEMP1 candidates with IgM binding potential (MAL6P1.1, PFL2665c, PF13_0001, PFD0995c, PF08_0107, PFA0005w). The underlined domains have been shown to mediate IgM binding. 190 7.4.3 Binding of IgM positive PfEMP1 IEs The ability of IgM positive IEs transcribing some of the identified var genes to mediate IgM binding was tested in flow cytometry experiments. The data showed differential levels of IgM binding of the expected var genes in the identified subclones (Figure 7.4). Figure 7.4: IgM binding of IEs expressing PfEMP1. IEs were incubated with 10 nM IgM (30 min; 4 °C) and subsequently stained with 2 µg/mL ethidium bromide and FITC conjugated anti- human IgM (1:150; 30 min; 4 °C) for flow cytometry analysis. Representative histograms showing overlays with gray histogram representing unstained negative control IEs and brown histograms for IEs stained positive for IgM. 191 7.5 Discussion Earlier demonstration that rosetting and placental IEs expressing PfEMP1 (Rasti et al., 2006; Creasey et al., 2003; Clough et al., 1998; Scholander et al., 1996) share dual binding affinity for the Fc region of IgM has generated considerable interest to unravel the contributions made by IgM to disease severity (Creasey et al., 2003; Rowe et al., 2002). To date, several PfEMP1 domains have been reported to bind IgM (Jeppesen et al., 2015; Stevenson et al., 2015a; Ghumra et al., 2008; Semblat et al., 2006). Recent emerging data revealed that non-CSA binding and non-rosetting PfEMP1 subtypes that bind IgM do exist, further complicating the exact consequence of this interaction (Jeppesen et al., 2015). Besides, very little is known about the frequency of these Fc-mediated IgM-binding PfEMP1 phenotypes in the P. falciparum genome and it is pertinent to investigate given the potential clinical implications. This study utilised the pVBH-NF54 G6 transfected parasites in which var gene epigenetic mechanism can be reversibly manipulated in a controlled manner. The pVBH-NF54 G6 transfected parasites were initially released of the drug pressure and the transcriptional results showed the expression of nearly all var genes (Figure 7.1), an observation which has previously been documented (Jeppesen et al., 2015; Fastman et al., 2012; Dzikowski and Deitsch, 2008). Following the onset of transcription and the ensuing var switching, the parasite population was selected by IgM and positive clones were single sorted and propagated. The var transcription profile on expanded IgM positive IEs identified nine different var genes among the subclonal isolates as potential candidates for the IgM-binding phenotype (Figure 7.2). The molecular interactions between PfEMP1 and IgM have been well described. Notably, PfEMP1 domains of the DBLϵ and DBLδ subtypes (interacting with the cµ3 192 and cµ4 domains of the Fc portion of IgM) have been implicated (Jeppesen et al., 2015; Stevenson et al., 2015a; Ghumra et al., 2008; Rasti et al., 2006; Semblat et al., 2006). The domain structure of the encoded forms of PfEMP1 identified in this study revealed two semi-conserved structural groups. Members belonging to one group shared a similar DBLε domain structure present at the C-terminal end (PF07_0139, MAL6P1.316, PFL0020w and PFL0030w) with variable representations, whereas the other group had a semi-conserved C-terminal DBLδ domain structure [PFL2665c, PF13_0001, PF08_0107, MAL6P1.1, PFD0995c and PFA0005w] (Figure 7.3). In the DBLϵ-related domain group is the var subtype PFL0030c encoding VAR2CSA in the 3D7 clone that has been strongly connected with pregnancy-associated SM (Salanti et al., 2004; Salanti et al., 2003). The Fc-mediated IgM binding has been mapped to the C-terminal DBLϵ domains of VAR2CSA expressing IEs (Jeppesen et al., 2015; Barfod et al., 2011; Rasti et al., 2006). The other three non-VAR2CSA var gene types with a similar DBLε subtype (MAL6P1.316, PFL0020w, PFL07_0139) discovered in this work were recently identified as IgM binders in a study that characterised IgM binding PfEMP1 in the NF54 parasite (Jeppesen et al., 2015). Thus, the results obtained from this study further corroborated their IgM binding identity. Furthermore, two of the identified non-VAR2CSA subtypes (MAL6P1.316, PFL0020w) additionally harboured a DBLδ domain similar to the one found in the well-characterised rosetting parasite isolates TM284VAR1 and HB3VAR06 which are also known to mediate IgM binding (Stevenson et al., 2015a; Ghumra et al., 2012; Scholander et al., 1996). Based on their structural resemblance to HB3VAR06, the ability of MAL6P1.316 and PFL0020w encoding PfEMP1 variants to mediate formation of rosette was investigated in a previous study that first identified them as IgM binding phenotypes. Results showed that these PfEMP1 variants did not mediate 193 formation of rosettes, indicating that PfEMP1 variants involved in IgM binding were not peculiarly limited to rosette-forming parasites but yet to be discovered phenotypic roles (Jeppesen et al., 2015). The results presented in this work indicate that both DBLδ and DBLε at the C–terminal end are important mediators of IgM binding and even more, the data confirmed findings of earlier studies (Jeppesen et al., 2015; Stevenson et al., 2015a; Ghumra et al., 2012). The other candidates identified in this study had a shared DBLδ structural domain (PFL2665c, PF13_0001, MAL6P1.1, PFD0995c, PF08_0107, PFA0005w) and these have not been previously reported as IgM binding PfEMP1 molecules (Figure 7.3). Furthermore, these putative PfEMP1 molecules did not possess the classical domains that were already known to mediate IgM binding. Notwithstanding, the structurally related C-terminal DBLδ domain found among these members have previously been observed in some well-studied IgM binding PfEMP1 (HB3VAR06, PF07_0139, MAL6P1.4). Thus, it can be speculated that this DBLδ might be an additional domain that can mediate IgM binding. A detailed characterisation of the newly reported PfEMP1 candidates with potential affinity for IgM was not investigated in this study; as such future studies would have to demonstrate that they are indeed localised on the surface of IEs and that they do mediate IgM binding. Such experiments would involve selecting NF54 IEs with specific antibodies raised against the newly identified PfEMP1 and then subsequently testing the PfEMP1 expressed by the selected IEs for IgM binding as well as assessing the var gene transcript profiles of the selected IEs. Furthermore, the exact domains (PfEMP1 and IgM) involved, as well as the affinity of the interactions would have to be determined. Additionally, functional assays (such as rosette formation, 194 cytoadhesion) to evaluate the potential relevance of IgM binding are needed in order to confirm their phenotypic relevance. The fact that IgM binding PfEMP1 phenotypes are observed suggest that they may have important roles that promote the survival of the parasite. Indeed, several pathogenic microorganisms have evolved mechanisms that limit their detection by the host immune system, including binding to human immunoglobulins (Lin et al., 2004; Medina et al., 1999; Bjorck and Kronvall, 1984). It is therefore important that the frequency of IgM binding PfEMP1 in other falciparum genomes is characterised in future. Furthermore, other parasite-derived variant surface antigens, including RIFINs which have been implicated in SM complications (Goel et al., 2015) need to be investigated to determine if they are co-selected for IgM binding. Understanding the interaction between the parasite-encoded proteins and IgM would undoubtedly contribute new insights into strategies that can block this adhesion process. In summary, this study was able to identify IgM binders consistent with previous studies. More importantly, we have provided evidence for the existence of several PfEMP1 variants that potentially mediate IgM binding in the genome of the NF54 parasite, that were previously unreported. 195 CHAPTER EIGHT GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS 8.1 General discussion It is widely acknowledged that malaria-related deaths have markedly reduced globally in the last decade by approximately 40 %. This has been attributed to an increase in control interventions including better testing diagnostics, vector-based interventions and treatment with ACTs (WHO, 2016). Despite such remarkable achievements and intensified efforts, childhood deaths due to P. falciparum remain significantly high. Notably, CM, the most deadly form of SM, has a case fatality rate of approximately 20 % (reviewed in Birbeck et al., 2010) suggesting that current efforts are still inadequate. Thus, additional measures, including more effective drugs and vaccines will remain paramount for malaria control and a global health priority (reviewed in Crompton et al., 2010). Obviously, this can be facilitated by an in-depth understanding of the intricate host-parasite relationship. The marked sequence diversity found within the PfEMP1 molecules has frustrated efforts in identifying conserved functional regions that could be linked to specific SM syndromes such as CM. Nevertheless, the discovery that VAR2CSA specifically interacts with placental CSA during PAM demonstrates (Salanti et al., 2004; Salanti et al., 2003) the existence of some conserved PfEMP1 determinants involved in organ- specific severe disease. Although the mechanisms leading to CM complications are only partially understood, cytoadhesion, mediated by the virulent parasite protein PfEMP1 to the ICAM-1 receptor on brain endothelial cells, has been implicated (Bengtsson et al., 2013b). Thus, a major objective of this thesis was to dissect the molecular details characterising ICAM-1 binding by group A PfEMP1, by using sequence information to define a sequence motif that can be used to identify ICAM-1- 196 binding DBLβ domains. Furthermore, it was investigated whether the expression of these DBLβ domains involved in ICAM-1 binding is linked with CM development. Thus, in chapter three, sequence information and data from structural analysis (from collaborators) of group A DBLβ PfEMP1 proteins binding to ICAM-1 were used to identify a highly conserved sequence motif (the ICAM-1 interaction site). An in silico analysis using the motif sequence identified a broad set of motif-containing DBLβ of group A PfEMP1 proteins that mediated ICAM-1 binding. These PfEMP1 proteins also contained an immediate upstream CIDRα domain involved in EPCR binding and thus suggested the ability of these proteins to adhere to both receptors. Indeed, we demonstrated that IEs expressing PfEMP1 containing the motif have a dual binding affinity for the host proteins EPCR and ICAM-1. This binding property allows for increased cytoadhesion and that constitutes an important risk factor for CM development. These findings were further supported by transcriptional data that showed higher transcription of the motif-containing PfEMP1 in CM patients compared with patients with other forms of malaria. The sequences defining the motif were exceptionally conserved, giving an indication that they could trigger cross- reactive responses. Significantly, antibodies from plasma samples of asymptomatic individuals in malaria-endemic regions affinity purified on either the motif-containing DBLβ domain or the motif peptide were able to inhibit ICAM-1 binding of both homologous and heterologous motif-containing DBLβ domains and IEs, indicating that such strain-independent immune responses occur in natural infections and may be involved in acquired protective immunity. Furthermore, purified rat antibodies raised against the motif peptide effectively abolished the interaction of IEs that mediate ICAM-1 binding in vitro. 197 The extreme intra-strain variations found within var genes encoding PfEMP1 present a major limitation in considering PfEMP1 as potential vaccines (Trimnell et al., 2006). This challenge was highlighted in a previous study that dissected the interaction between PfEMP1 containing CIDRα1 domains and EPCR, a phenotype that has been strongly linked with SM development (Turner et al., 2013; Lavstsen et al., 2012). A crystal structure of CIDRα1-EPCR complex was used to identify conserved features that could be targeted for therapeutics against SM (Lau et al., 2015). The study showed that the overall organisational structure of the CIDRα1 domain of PfEMP1 was preserved to mediate interaction with its receptor, however, the residues that directly interacted with EPCR were highly variable (Lau et al., 2015). Thus, the sequence diversity observed may hamper the possibility of generating extensive cross-reactive natural responses against these CDIRα1 residues that directly interact with EPCR. In the present study, using sequence analysis combined with complementary crystal structure data of a group A PfEMP1 binding to ICAM-1 (Lennartz et al., 2017) from our collaborators (Professor Matthew Higgins laboratory, Oxford University), we show that the amino acid sequences defining the motif (the residues that directly interact with ICAM-1) were exceptionally conserved. To the best of our knowledge, this is the first report identifying such a conserved motif sequence in a group A PfEMP1 molecule, thus providing a strong rationale for considering the ICAM-1 binding motif as a new target for vaccine development against CM. In order to make good arguments for vaccine consideration, this study investigated (chapter four) whether plasma antibodies from malaria patients or rat antibodies raised against the motif–containing DBLβ domain or the motif peptide, were cross-reactive and capable of blocking the ICAM-1 interaction with a large selection of DBLβ domain constructs or IEs. The data generated showed that antibodies against motif- 198 containing DBLβ were acquired in an age-dependent manner. No significant difference in antibody reactivity was detected between SM and UM in motif- containing DBLβ domains. Possible explanations for this observation may be that our sample size was too small to detect any differences. Moreover, CM cases were not detected during the sampling year, partially limiting the analysis that could be done. A plasma pool generated from the enrolled children and affinity purified on the motif- containing DBLβ domain was capable of inhibiting ICAM-1 binding of a heterologous motif-containing DBLβ domain, which further validated our initial observation (chapter three). To show that this inhibition was solely mediated by motif- specific antibodies with biological relevance, rat immunisations were carried out using either motif peptides (the ICAM-1 interaction site) or the motif DBLβ domains. Both the motif-containing DBLβ rat antisera and IgG affinity purified on the motif peptide demonstrated binding inhibition against several heterologous motif-containing ICAM- 1-binding DBLβ domains at comparable levels, in addition to interfering with the adhesion of IEs to ICAM-1. More importantly, antisera generated from only two motif-specific peptides were effective at significantly abolishing adhesion of ICAM-1- binding falciparum parasites from paediatric SM patients including CM. Altogether, the data demonstrated that broadly reactive cross-inhibitory antibodies against the motif can be acquired following P. falciparum natural infections. Additionally, these antibodies could be experimentally induced. Such cross-reactive antibodies interfered with ICAM-1 binding of recombinant motif-containing DBLβ domains and IEs from four out of five paediatric CM patients. Cross-reactive antibody responses raised against specific PfEMP1 domains or minimal binding regions have been reported before particularly in studies involving VAR2CSA (Bordbar et al., 2012; Magistrado et al., 2011; Nielsen et al., 2009; 199 Andersen et al., 2008), which is the only PfEMP1 based vaccine candidate targeting PAM (Fried and Duffy, 2015). Notably, antibodies generated against the DBL4ε domain demonstrated strain transcending adhesion inhibition activity (Magistrado et al., 2011; Nielsen et al., 2009). However, antibodies generated against peptides covering epitopes targeted by DBL4ε-specific adhesion blocking antibodies did not recognise the surface of IEs expressing VAR2CSA as well as not mediate blocking of parasite binding to CSA (Ditlev et al., 2012). Thus, till now, no single VAR2CSA epitope that could serve as ideal vaccine target has been conclusively identified. By contrast, this study identified a novel conserved linear motif which is the ICAM-1 interaction epitope. Taken together, the results indicate the feasibility of producing an epitope focussed vaccine which aims at eliciting protective immune responses specifically towards the motif. With the global reduction in malaria deaths as well as the rapid fall in malaria transmission intensity, there have been documented reports of redistribution of malaria cases from younger children to older ones (reviewed in Nkumama et al., 2017; Griffin et al., 2014). For example, reports from a Gambian study showed a dramatic decline in the number of malaria-related admissions between 2003 and 2007. This was further supported by a significant shift in the mean age of children admitted for malaria from 3.9 years to 5.6 years (Ceesay et al., 2008). This observation may possibly be linked to a slower pace in the build-up of protective immunity, although the factors contributing to that remain unclear. A decline in malaria transmission may lead to a reduction in the total mortality which is in line with efforts to eliminate and hopefully eradicate malaria (reviewed in Nkumama et al., 2017). Paradoxically, this may increase the risk of resurgent malaria with severe consequences due to the emergence of a population of individuals with little or non-existent naturally-acquired 200 protective immunity. An effective vaccine against malaria remains the best long-term cost effective and preventive measure. RTS,S is currently the most advanced malaria vaccine (RTS,S, 2015), however, it is well acknowledged that the protection offered by this vaccine is inadequate. This reinforces the need for continued search for efficacious vaccines to accelerate malaria control and eradication efforts. Data from this study raise the optimism of using the motif peptide (the ICAM-1 interaction site) to produce a stand-alone vaccine construct to prevent CM or as part of a multi- component vaccine strategy against SM. Some additional experiments would have to be done in order to validate the biological importance of the identified ICAM-1 binding motif. Future work using the same experimental procedure described here could be carried out using plasma samples and parasite isolates from patients living in different geographical locations. Notably in Asia, where adult CM is very common and disease phenotypes differ remarkably from paediatric CM. Additionally, prospective longitudinal studies in naturally exposed population are needed to determine the frequency of antibody responses to the ICAM- 1 binding motif. This information would be crucial for establishing the involvement of the anti-motif antibodies in the development of immunity and could also serve as a useful marker in sero-epidemiological studies. Furthermore, monoclonal antibodies raised against the motif can be generated and tested for its adhesion blocking activity against IEs. It is known that SM pathology is complex comprising both parasite cytoadhesion and host inflammatory processes; with both processes occurring concurrently and reinforcing each other (reviewed in Storm and Craig, 2014; Cunnington et al., 2013b). In this study, markers of inflammation and endothelial activation (sICAM-1, sEPCR, Ang-2, HMGB1) were investigated in children with SM and UM and also their levels 201 following resolution of infection (Chapter five). Overall, the data showed higer levels of these soluble markers in malaria patients in comparison with healthy controls. These levels declined significantly in convalescent samples except for HMGB1 which remained high to a similar degree as observed during the infection phase. To the best of our knowledge, this is the first report of raised levels of HMGB1 in convalescent malaria patients which is similar to what has been previously observed in sepsis patients (Sunden-Cullberg et al., 2005). Since plasma HMGB1 was measured at only two time points, this study cannot rule out if these levels decline after that. Then, it would be interesting if future studies could determine when these levels decline and whether these elevated levels in convalescent samples have important implications for malaria pathogenesis in endemic populations where multiple infections or reinfections are very common. Overall, the study did not detect differences in the levels of soluble markers between SM and UM, though a trend of higher levels in severe cases was observed. The non-significant differences between both malaria groups could be attributed to our study population. Prior investigations have shown that levels of soluble markers of endothelial activation are highest in CM patients (Lovegrove et al., 2009). This study was unable to include enough CM cases possibly due to the rapidly changing malaria epidemiology of our study site where only five CM cases have been recruited in the past three years. Notwithstanding, the data indicate that endothelial activation is a central component of malaria pathogenesis. Although a highly conserved motif with vaccine potential was discovered (Chapter three), it cannot be considered the magic bullet. There is a consensus that a holistic approach is needed in order to totally eradicate malaria. Thus in future, targeting important immune responses combined with our newly discovered PfEMP1 motif 202 could unravel new mechanisms of protection from cerebral IE sequestration including the development of therapeutic interventions. In the absence of suitable animal models (Craig et al., 2012; White et al., 2010), in vitro endothelial cell models still remain fundamental in understanding malaria cytoadhesion processes. Most studies have utilised several cell sources of non-human origin, cancer derived cells from humans or cells from pooled donors usually of non- African origin (Rogerson et al., 1995; Hasler et al., 1993; Swerlick et al., 1992; Dorovini-Zis et al., 1991; Schmidt et al., 1982). Most of these cells may not reflect the vascular beds of malaria-exposed individuals, and moreover, the procedures in obtaining some of them are time consuming and invasive. This study proposed BOECs from malaria-exposed indivduals as suitable practical cell source to study falciparum parasite adhesive interactions. BOECs form part of the circulating rare cell population in peripheral blood with matured endothelial cell phenotypes (Ingram et al., 2004; Lin et al., 2000). Recently, high circulating levels of these cells have been implicated in CM complications in Ghanaian children (Oduro, 2015). During this study, PBMCs were purified from small volumes of blood obtained from malaria patients to generate BOECs and the surface receptors expressed by these cells were characterised (Chapter six). These cells were shown to express several endothelial markers including ICAM-1 and EPCR and their surface expression was altered in response to the pro-inflammatory cytokine TNF-α. Of interest, this study provided evidence that the simultaneous adhesion to EPCR and ICAM-1 by IEs is strongly associated with CM complications (Chapter three). Future studies using BOECs could advance our understanding of cerebral cytoadhesion involving both receptors and also serve as a model for assessing potential adjunctive therapies. Indeed data from our laboratory using BOEC derived from European donors 203 demonstrated that they supported malaria parasite cytoadhesion (Dr. Yvonne Adams, University of Copenhagen, personal communication). Although, prior investigations have isolated and characterised BOECs from healthy individuals as well as individuals with inflammatory vascular disorders (Martin-Ramirez et al., 2014; Sakamoto et al., 2013; Ingram et al., 2004), this study was the first to show that BOECs can be isolated from malaria patients, readily established from small blood volumes and frozen PBMC samples and adapted to in vitro culture. Studies are still ongoing to further characterise these cells. Furthermore, malaria patient-derived BOECs would be used to study parasite ligands and the endothelial cell receptors as well as changes in receptor level expression during parasite-adhesive interactions. This offers the possiblility of using endothelial cells derived from malaria-exposed donors of suitable ages and geographic locations and thus provide better physiologically relevant insights into cytoadhesion processes. Consistent with that, a similar approach reported here, could be used to establish BOECs from other geographic regions where SM in non- immune adults is very common. The receptor expression levels of the BOECs generated, including responses to inflammatory cytokines and parasite adhesion could be investigated. The results of these measurements between paediatric malaria and adult malaria patients as well as healthy individuals could also be compared. Such studies may increase our understanding of malaria pathogenesis and may hopefully lead to the identification of novel interventions. Finally, this study also investigated other PfEMP1 phenotypes that have been implicated in severe disease. Previous findings demonstrated that rosetting and placental IEs expressing PfEMP1 share dual binding affinity for the Fc region of non- immune IgM, both phenotypes associated with severe disease (Barfod et al., 2011; Rowe et al., 2002). However, very little is known about the frequency of these Fc- 204 mediated IgM-binding PfEMP1 phenotypes in the P. falciparum genome and it is important to investigate given the potential clinical implications. Here, at least six new var/PfEMP1 candidates that can potentially mediate binding to IgM in the 3D7 genome are reported, suggesting that PfEMP1-specific IgM binders may have some relevance to the parasite survival (Chapter seven). This study however, was unable to provide evidence that these new variants are indeed localised on the surface of IE. Additionally, no detailed functional characterisation was carried out due to time constraints. Notwithstanding, there is ongoing work to further investigate these observations and additionally determine the frequency of these IgM binders in the other falciparum genomes. Such information will be vital for understanding disease mechanisms and may result in better disease management. This study has highlighted the contributions made by both host and parasite factors to SM pathogenesis. Detailed understanding of these important factors remains paramount for effective management of malaria. Of interest, this study identified a novel epitope targeted by protective antibodies that can be used to develop a broadly reactive vaccine against CM. 8.2 Conclusions 1. A highly conserved motif was identified using sequence information and that was used to predict a broad set of motif-containing DBLβ of group A PfEMP1 that mediated ICAM-1 binding. IEs expressing PfEMP1 containing the motif have dual binding affinity for the host proteins EPCR and ICAM-1. This 205 binding property allows for increased cytoadhesion and that constitutes an important risk factor for CM development. 2. Cross-reactive antibody responses against an array of group A PfEMP1 with the motif-containing ICAM-1 recombinant proteins were acquired following natural infections by P. falciparum parasites or could be experimentally induced. Such cross-reactive antibodies interfered with ICAM-1 binding of recombinant motif-containing DBLβ domains and IEs from paediatric CM patients. 3. Endothelial activation, characterised by high levels of sICAM-1, sEPCR, plasma Ang-2 and plasma HMGB1 were observed in children with acute malaria. 4. BOECs derived from malaria patients may potentially serve as an easily accessible and physiologically relevant cell source for the study of falciparum parasite-adhesive interactions 5. New var/PfEMP1 candidates that can potentially mediate binding to IgM were identified in the 3D7/NF54 genome. 8.3 Recommendations This study identified a PfEMP1 motif involved in CM. Prospective longitudinal studies in naturally exposed population are needed to determine the frequency of motif var transcripts and motif-specific antibody responses and correlate these observations with disease outcomes. Additional experiments are highly recommended to optimize motif peptides to induce more broadly neutralising antibody responses. 206 High levels of markers of endothelial activation were observed in children with acute malaria. Future investigations that concurrently look at markers of endothelial activation and the newly identified PfEMP1 motif could provide useful insights into SM pathogenesis. The procedures used in this study to characterise patient-derived BOECs can be used in future to isolate BOECs from different categories of malaria patients. These cells can be used to investigate malaria parasite adhesive interactions with the endothelium and responsiveness to cytokines and thus allow comparative analysis of endothelial activation among different individuals and also between clinical categories. Such information may offer clues on why some individuals succumb to life-threatening SM as well as provide new treatment options. This study identified new var/PfEMP1 candidates that can potentially mediate binding to IgM. It is necessary that future studies provide proof that these PfEMP1 variants are indeed expressed in NF54 selected parasites, localised on the surface of IEs and also determine their functionality. 207 REFERENCES Abbott,N.J., Ronnback,L., and Hansson,E. (2006). Astrocyte-endothelial interactions at the blood-brain barrier. Nat. Rev. Neurosci. 7, 41-53. 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Physiol 296, H1675-H1682. 254 APPENDICES Appendix A Ethical Clearance 255 Appendix B Primers for amplification of PfEMP1-specific DNA sequences and production of recombinant proteins Recombinant protein in E. coli Gene ID Geno Primers (F: Forward; R: Reverse) me DBL_D4 PFD1235w/PF3D7_0 3D7 Published in Bengtsson et al. 2013b (b3) 425800 DBL_D5 PFD1235w/PF3D7_0 3D7 Published in Bengtsson et al. 2013b (b3) 425800 Motif PFD1235w/PF3D7_0 3D7 F: 5´-GCGGATCCTTATACGCAAAAGCACG-3 ́ 425800 R: 5´-CTAgctagcttaGGGTGTTGTATCATCAG-3 ́ D5_motif PFD1235w/PF3D7_0 3D7 PCR1 425800 F: 5´-GAGGATTTATACGCAAAAGCACGA-3´ R: 5´- TGCAGCAGTAGAATACACACTATACACAGT GGGTGTTGT-3 ́ PCR2 F: 5´-GAAGATCT AATCCGTGTGCTG -3 ́ R: 5´-TCGTGCTTTTGCGTATAAATCCTC-3 ́ PCR3 F: 5´- ACAACACCCACTGTGTATAGTGTGTATTCTA CTGCTGCA-3 ́ R: 5´-CTAgctagcttaGCAATCACACGC-3´ PCR4 F: 5´-GAAGATCTAATCCGTGTGCTG-3´ 256 R: 5´- TGCAGCAGTAGAATACACACTATACACAGT GGGTGTTGT-3 ́ PCR5 F: 5´-GAGGATTTATACGCAAAAGCACGA-3´ R: 5´-CTAgctagcttaGCAATCACACGC-3´ PCR6 F: 5´-GAAGATCTAATCCGTGTGCTG -3 ́ R: 5´-CTAgctagcttaGCAATCACACGC-3´ DBL_D4 PF11_0521/ 3D7 F: 5´-GAAGATCTAACCCGTGTGCTAAACCTC- (b3) PF3D7_1150400 3 ́ R: 5´- CTAGCTAGCttaGCATTTACATGCTGTATC-3´ DBL_D4 JF712900 BM02 Published in Bengtsson et al. 2013b (b3) 1 DBL_D4 JF712901 BM02 Published in Bengtsson et al. 2013b (b3) 8 DBL_D4 JF712902 BM04 Published in Bengtsson et al. 2013b (b3) 8 DBL_D4 JF712903 BM06 Published in Bengtsson et al. 2013b (b3) 6 DBL_D4 JN037695 BM05 Published in Bengtsson et al. 2013b (b3) 7 DBL_D4 Dd2var32 KOB85388 Dd2 Published in Bengtsson et al. 2013b (b1) DBL_D4 DD2var25 Dd2 F: 5´-CTAgctagcAACCCGTGTGTTAAACCTCG- (b11) 3 ́ R: 5´- CTAGCTAGCttaACAACCACACGCACCATC-3 ́ 257 DBL_D4 DD2var52 Dd2 F: 5´-GCGGATCCAATCCCTGCGTTACTGGTG- (b7) 3 ́ R: 5´- CTAGCTAGCttaACAAGTACACTGATCTTTAT A-3 ́ DBL_D4 HB3var03 HB3 F: 5´- (b3) KOB63865 GCGGATCCAACTCGTGTGGAAAGAACAC-3 ́ R: 5´-CTAgctagcttaGCAACGTAACGCCTCATC- 3 ́ DBL_D4 HB3var01 HB3 F: 5´-GCGGATCCAATCCGTGTGTCAATGGTG- (b7) 3 ́ R: 5´- GCGGATCCttaACAAATACACGCCTCATC-3´ DBL_D4 HB3var1csa HB3 5´-CTAgctagcAACCCGTGTGTTAAACCTCG-3´ (b11) 5´-CTAGCTAGCttaACAACCACACGCACCATC- 3 ́ DBL_D4 AFJ66668 BT191 F: 5´- (b1) 4 GAAGATCTAACCCGTGTGGAAAGAACAC-3 ́ R: 5´- GAAGATCTttaACATGCACACGGCGTTTCAT- 3 ́ DBL_D4 KC608962 pap1 Synthetic gene produced by Eurofins (bx) DBL_D4 KF984156 MN06 F: 5´- (b1) 2 GCGGATCCAACCCGTGTGTAAAAAACAATA ATG-3 ́ R: 5´- CTAGCTAGCttaGCAACGTAACGCCTCATC-3 ́ DBL_D4 KJ866957 MN03 F: 5´- (b3) 5 GCGGATCCAACCCCTGTGCAAAGAACACTA C-3 ́ R: CTAGCTAGCttaACATTCGCAAGCTTTTTC- 258 3 ́ DBL_D4 KJ866958 - F: 5´- (b3) GCGGATCCAATCCTTGTGGACATAAAAGTG- 3 ́ R: 5´- CTAGCTAGCttaACAACCTAACGCATAATC-3 ́ DBL_D4 KM364031 MN05 F: 5´- (b1) 6 GCGGATCCAACCCCTGTGCAAAGAACAC-3´ R: 5´- CTAGCTAGCttaACAACCACACGCCTCATC-3 ́ DBL_D4 KM364033 - F: 5´- (b3) GCGGATCCAATCCTTGTGGACATAAAAGTG- 3 ́ R: 5´- CTAGCTAGCttaACAACCTAACGCATAATC-3 ́ DBL_D4 JQ691646 BT198 F: 5´- (b12) 3 CTAgctagcAACCCCTGTGGAAAAAAGAAT-3´ R: 5´-CTAgctagcttaACACTTACACGCCTCATC- 3 ́ DBL_D4 JQ691647 BT198 F: 5´-GCGGATCCAATCCATGCGTTAATGGTC- (b3) 3 3 ́ R: 5´-CTAgctagcttaACACGTACATTTCGCATG- 3 ́ DBL_D4 JQ691649 BT198 F: 5´-GCGGATCCAATCCCTGCGTTAATGGTC- (b6) 3 3 ́ R: 5´- GCGGATCCttaACATTTACATGCCACATC-3´ DBL_D4 KJ866959 - F: 5´- (b3) GCGGATCCAATCCTTGTGGACATAAAAGTG- 3 ́ R: 5´- 259 ctaGCTAGCttaACAACCTAACGCATAATC-3´ DBL_D4 KM364034 MN03 F: 5´- (b6) 5 GCGGATCCAATCCGTGTGTAAACACCACTG- 3 ́ R: 5´- CTAGCTAGCttaACACGTACATTTCGCATG-3´ DBL_D4 IT4var13 IT4 F: 5´-GCGGATCCAATCCCTGTGTAGTTGGA-3 ́ (b5) R: 5´- CTAGCTAGCttaACATTTACATGCATTAGC-3´ CIDR_D3 PFD1235w/PF3D7_0 3D7 F: 5´- (a1.6)- 425800 GCGGATCCCCTGAATGTGGAGTCCAATG-3´ DBL_D4 R: 5´CTAgctagcttaACACTTACACGCCTCAAC (b3) Recombinant protein in insect cells CIDR_D6 PF11_0008/PF3D7_1 3D7 F: 5´- (b4) 100200 GAATTCAAAAAACAAGAAAAACTATAT-3´ R: 5´- TGCGGCCGCTACATGGATTTGCTGGAACA-3 ́ DBL_D2 PF11_0521/ 3D7 F: 5´- (a1.7)- PF3D7_1150400 CTCTAGAAATGGGGAATGCAATACCAGCGA CIDR_D3 CTCCGGAT-3 ́ (a1.4)- R: 5´- DBL_D4 CTGCGGCCGCTGCATTTACATGCTGTATCAT (b3) GATCATGTGG-3´ DBL_D2 Dd2var32 KOB85388 Dd2 F: 5´-CTCGAATTCatggggggaaattcttcaaaaggtgctcct- (a1.7)- 3 ́ CIDR_D3 R: 5´- (a1.4)- CTGCGGCCGCTccacctgattatgaaaaagcttgcgaatgt-3 ́ DBL_D4 (b1) 260 Full length IT4var13 IT4 Published in Turner et al. 2013 Other primers used M3637 - - F: 5´-TgctNKtaatggtggtcc-3 ́ M3638 - - R: 5´-TGCCACCATTTTGTA-3 ́ DBLα2/1.1/2 − - F: 5'- /4/7) GATTAYGTBCCTCAATTTTTAMGWTGGT R: 5' TACAATCATATCCATTAWGACTACAA R: 5'-TCACAATCGCATCCATTATGACTACAA Q0183 - - F: 5´-AATTNHTGCTNKTAATGGTGGTCCTG-3 ́ Q0186 - - 5´-GGACCAAGTATGCCACCATTTTGTA-3 ́ 261 Appendix C Recombinant PfEMP1 proteins used in this study Domain sub- Genome PfEMP1 type Gene source 3D7 PF11_05213/PF3D7_1150400 DBLb3_D4 BM048 JF712902 DBLb3_D4 BM066 JF712903 DBLb3_D4 BM021 JF712900 DBLb3_D4 BM057 JN037695 DBLb3_D4 3D7 PFD1235w/PF3D7_0425800 DBLb3_D4 MN35 KJ866957 DBLb3_D4 HB3 VAR03 DBLb3_D4 Dd2 VAR32/KOB85388 DBLb1_D4 MN56 KM364031 DBLb1_D4 A4395 KJ866958 DBLb3 1914 AFJ66668 DBLb1_D4 BM028 JF712901 DBLb3_D4 - KM364033 DBLb3 MN062 KF984156 DBLb1_D4 CDO62031 Synthetic gene (https://www.eurofinsgenomics.eu/) CDO61797 Synthetic gene (https://www.eurofinsgenomics.eu/) CDO63496 Synthetic gene (https://www.eurofinsgenomics.eu/) Dd2 VAR25 DBLb11_D4 HB3 VAR1CSA DBLb11_D4 3D7 PF13_0003 DBLb9_D8 A4393 KJ866959 DBLb3 IT4 IT4VAR13/ABM88750 DBLb3_D4 1983 JQ691647 DBLb3_D4 MN35 KM364034 DBLb6 Dd2 VAR52 DBLb7_D4 HB3 VAR01 DBLb7_D4 1983 JQ691649 DBLb6_D4 IT4 IT4VAR16/AAS89259 DBLb5_D4 262 Appendix D Levels of inflammation and endothelial activation markers and age dependency in malaria patients. Symbols indicate individual soluble levels in the different age categories. Multiple variables were analysed using the Kruskal-Wallis Rank Sum Test with Dunn‟s Multiple Comparison Test. SM= severe malaria; UM= uncomplicated malaria 263 Appendix E Publications 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321