Acknowledgements First and foremost I offer my sincerest gratitude to my supervisor, Dr. Paul A. MacAry, who has supported me throughout my thesis with his patience and knowledge whilst allowing me the room to work in my own way. I attribute the level of my Doctor of Philosophy degree to his encouragement and effort and without him this thesis, too, would not have been completed or written. One simply could not wish for a better or friendlier supervisor. Next, I will like to thank my co-supervisor, Professor Ng Mah Lee, Mary for offering so much advice and insight throughout my work on Dengue. She, together with Mdm Boon had smoothen the path of my PhD journey by providing me with the necessary materials and skills that are crucial for building a strong foundation at the beginning. In my daily work I have been blessed with a friendly and cheerful group of fellow colleagues who will not hesitate to lend a helping hand. Teo En Wei, especially, has been a great learner, friend and confidante and I attribute part of this thesis to her. Life at work will not be as enjoyable and interesting without her around. The continuation of all that we had set up together and the prospect of future discovery, I now consider them to be in her capable hands. Too Chien Tei has provided me with her technical expertise in animal handling and antibody production and purification. Without her, I will not have the materials vital for the platform of my project. In the various laboratories I have been aided for many years in various experiments, I got to make many friends along the way who provided valuable insight and expertise. Dr. Brendon J. Hanson and Ms. Angeline Lim Pei Chiew from DSO National Laboratories, Dr. Wouter Schul and Mr. Andy Yip from Norvartis Institute of Tropical Diseases, Nalini Srinivasan and Emeritus Professor Chan Soh Ha from World Health Organisation Singapore, Professor Mike D. Kemeny from National University of Singapore and Wang Jin from National University Hospital all contributed to the completion of this project. To the rest of the friends in Paul A. MacAry (PAM) laboratory, thank all of you, for the good laughs that never fail to pull me out of the brink of insanity. I thank my family for letting me be, especially Adrian Lee Kok Hee in supporting me in whichever way he can. I thank my mom, Lee Geck Keng, for all the sacrifices she had made. I also thank a special someone, Alex Liau Whatt Meng, for the moral support and encouragement. Most importantly, I thank God for the strength to meet the demands of a Doctor of Philosophy degree. i Table of contents Acknowledgments i Table of contents ii Summary viii List of Tables x List of Figures xi Abbreviations xv Chapter – Introduction 1.1 Dengue virus 1.1.1 Classification 1.1.2 Epidemiology 1.1.3 Structure of dengue virions 1.1.4 Organization of the Flavivirus genome 1.1.5 Replication strategy 1.1.5.1 Receptor interaction 1.1.5.2 Viral entry and the E protein 11 1.1.5.3 Translation of the DV genome 14 1.1.5.4 Virus assembly and propagation 17 1.1.6 Phylogeny of Dengue Virus 19 1.1.7 Pathogenesis of Dengue Virus 22 1.1.8 Immune response to Dengue Virus 26 1.1.8.1 Innate immunity to DV 26 1.1.8.2 Adaptive immunity to DV 28 1.1.9 Antibody dependent enhancement 35 ii 1.2 Epstein-Barr Virus 39 1.2.1 Classification 39 1.2.2 Epidemiology 39 1.2.3 Structure of EBV virions and organization of the virus genome 40 1.2.4 EBV patterns of latency 42 1.2.5 Replication strategy 44 1.2.6 EBV latent gene products in B cell immortalization 47 1.2.6.1 EBNAs in B cell immortalization 47 1.2.6.2 LMPs in B cell immortalization 49 1.2.7 Pathogenesis of EBV 50 1.3 Somatic hypermutation during EBV-driven B cell growth 54 1.4 Dengue human monoclonal antibody therapeutics and vaccine 55 Chapter – Material and methods 60 2.1 Cell lines 60 2.2 EBV virus production 60 2.3 Dengue virus production 60 2.4 Cryopreservation of cells 61 2.5 Hybridoma cultures and antibody purification 61 2.6 Ascites and antibody purification 62 2.7 Primary cell culture 62 2.8 Source of primary CD22+ B cells 63 2.9 RNA extraction and RT-PCR for serotyping of patients 63 2.10 Cloning of B cells from Dengue virus-infected patients 64 2.10.1 Preparation of feeder layer 2.10.2 Serial dilution and immortalization of patients’ memory B cell 64 2.11 Plaque reduction neutralization assay 65 iii 2.12 Titration of virus stocks 66 2.13 Cytopathic effect assay 66 2.14 Captured ELISA for B cell screening 66 2.15 Captured ELISA for Antibody/Fab binding 67 2.16 RNA extraction and cDNA amplification for antibody genes 67 2.17 PCR amplification 71 2.18 Generation of single chain fragment variable of antibody 72 2.18.1 SOE PCR 72 2.18.2 Single chain fragment variable cloning into pCANTAB vector 75 2.19 Production of recombinant neutralizing dengue antibody 77 2.19.1 Amplification of heavy and light chain sequences 77 2.19.2 Purification of heavy and light chain PCR products 79 2.19.3 Gel extraction 80 2.19.4 Gel electrophoresis 81 2.19.5 Restriction enzyme digestion of purified PCR products 82 2.19.6 Preparation of IgG1, IgG3 and IgG4 framework vector 82 2.19.7 Ligation of insert and vector 84 2.19.8 Bacterial transformation of chemically competent cells 85 2.19.9 Miniprep purification of DNA from bacteria 85 2.19.10 Restriction enzyme screening of Miniprep DNA 86 2.19.11 Maxiprep purification of DNA from bacteria 86 2.19.12 Establishing FreestyleTM 293-F cells 87 2.19.13 Transfection of FreestyleTM 293-F cells 88 2.19.14 Quantification of purified recombinant IgG antibodies 89 2.19.15 SDS PAGE analysis of purified IgG antibody 90 2.20 Western blotting analysis 91 2.21 Flow Cytometry 91 iv 2.22 Co-immunoprecipitation and radioactive immunoblotting 91 2.23 Immunocytochemistry 92 2.24 Mouse infection and blood sampling 92 Chapter – Establishment and optimization of EBV immortalized human B cell lines 94 3.1 Optimization of transformation of B cells with EBV 94 3.2 Optimization of cloning of EBV immortalized B cells 95 3.3 Markers of immortalized B cells 97 3.4 Heat-killed DV neither enhanced B cell growth nor increase DV-specific neutralizing clones. 99 3.5 Maintenance of immortalized B cells 100 3.6 Optimization of ELISA for screening of B cell supernatants 101 Chapter – Screening of neutralizing clones derived from dengue Patients 4.1 Recruitment of Dengue patients from NUH cohort 103 103 4.2 Screening of EBV immortalized B cell clones for dengue specificity 104 4.3 Purification of antibodies form B cell supernatants 110 4.4 Determination of lowest antibody concentration for complete neutralization and serotype specificity 4.5 Total number of clones identified 112 115 v Chapter – Reactivation of somatic hypermutation in EBV-infected B cells 116 5.1 Loss of neutralizing activity of supernatant from identified clones over time 116 5.2 Sequences of IgG genes extracted from EBV-infected B cells suggest reactivation of SHM 5.3 Upregulation of AID in EBV infected B cells 117 119 Chapter – Production of neutralizing recombinant human monoclonal antibody 121 6.1 Generation of single chain variable fragment 121 6.2 Generation of recombinant human IgG (whole antibody) 123 Chapter – Characterization of neutralizing 14C10 recombinant mAb 7.1 Sequence analysis of neutralizing antibody 130 130 7.2 Serotype specificity of generated fully human recombinant 14C10 antibody 131 7.3 14C10 mAb neutralizes both mammalian and insect derived DV 134 7.4 14C10 mAb as a reagent for immunocytochemistry 136 7.5 Mapping of 14C10 mAb binding to DV E protein 137 7.6 Binding activity and affinity determination on different genotypes of DV1 141 7.6.1Comparison of binding affinity of 14C10 mAb with hu4G2 141 7.6.2 Determination of Kaff values for both 14C10 and hu4G2 144 vi 7.7 Neutralization efficiency on different genotypes of DV1 147 7.7.1 Comparison of neutralizing efficiency of 14C10 mAb with commercial 4G2 antibody 147 7.7.2 Comparison of neutralizing efficiency of 14C10 mAb within Genotypes 150 7.8 Mechanism of 14C10 mAb 153 7.9 ADE profiles of 14C10 mAb 155 7.9.1 Development of ADE among the DV serotype 155 7.9.2 Development of ADE among the antibody subclasses 157 7.10 Protective capacity of 14C10 mAb in DV-infected mice 159 Chapter – Discussion 162 Chapter – References 178 Chapter 10 – Appendix 196 vii Summary Dengue is the most significant mosquito-borne viral disease affecting humans. At present close to 2.5 billion people living in more than 100 dengue endemic countries in the tropical/sub-tropical belt are considered to be at risk of dengue infection. Dengue diseases affect 50 million people yearly, with frequent and recurrent epidemics (Stephenson 2005). The 1990’s saw a return of dengue diseases in Singapore despite stringent mosquito controls, peaking with the largest ever outbreak in 2005. Over 80% of the reported cases were young adults. In addition to the mortality and morbidity associated with infection, dengue also imposes a considerable burden on the finances and infrastructure of the healthcare systems in Singapore and developing countries. Hence, alternatives to dengue vaccines, such as passive antibody therapies and/or antivirals are needed urgently to help control dengue-associated diseases in the immediate term. These proposed therapeutics have the potential to help large numbers of infected individuals in Singapore and elsewhere until such time that safe vaccines become available and can achieve a decent coverage in target populations in endemic countries in approximately 10-20 years. As part of the project, we have isolated a first ever fully human antibody with potential clinical utility using a clone of an immortalized human B memory lymphocyte capable of producing a human antibody with remarkable neutralizing activity to Dengue Virus Type 1in vitro and in vivo. We have isolated the genes that form the template for this antibody and successfully generated a Fab (for X-ray crystallography) and various human IgG subclasses (IgG1, IgG2, IgG3 and IgG4) of recombinant monoclonal viii antibody. Our study provided greater insights to the ADE hypothesis and demonstrated that this antibody can be a good prophylactic and therapeutic candidate in the treatment of dengue. ix List of tables Table 1.1 Flavivirus classification. Table 1.2 Pattern of EBV latency and gene expression. 42 Table 1.3 Five transcription programs with listed genes and functions. 46 Table 1.4 Disease associated with EBV. 51 Table 2.1 Nucleotide sequences and positions of upstream consensus. 64 Table 2.2 Human immunoglobulin gene PCR primers for heavy chain .68 Table 2.3 Human immunoglobulin gene PCR primers for Kappa light chain. Table 2.4 Human immunoglobulin gene PCR primers for Lamda light chain. Table 2.5 73 Primers for attachment of (Gly4Ser)3 linker onto Kappa light chain. Table 2.8 72 Primers for attachment of (Gly4Ser)3 linker onto heavy chain. Table 2.7 70 Primers for cDNA synthesis of human immunoglobulin genes. Table 2.6 69 75 Primers for attachment of (Gly4Ser)3 linker onto Lamda light chain. 75 Number of clones isolated per batch and respective target serotypes. 115 Table 6.1 The presence of heavy and light chains in clones. 123 Table 6.2 Heavy and light chain sequences of 12 candidate Table 4.1 recombinant antibodies from clone 14C10. 127 x List of Figures Figure 1.1 Global prevalence of DF and DHF as shown by WHO. Figure 1.2 Ribbon drawing of E protein. Figure 1.3 Schematic representation of the polyprotein processing for flaviviruses. Figure 1.4 Proposed rearrangement of E dimer in Flaviviruses upon exposure to low pH. Figure 1.5 13 Schematic diagram of E glycoprotein in neutral and the proposed acidic pH conformation. 13 Figure 1.6 Life cycle of dengue virus. 18 Figure 1.7 Course of dengue infection and timing of diagnosis 24 Figure 1.8 Type I Interferon transduction pathway and putative inhibition by flavivirus. 28 Figure 1.9 Immunopathogenesis of dengue virus infection. 33 Figure 1.10 Location and transcription of the EBV virus genes on the doubled stranded viral genome. 41 Figure 2.1 Human IgG1, IgG3 and IgG4 framework vector. 83 Figure 3.1 EBV immortalized B cell numbers versus time (days) in culture with addition of CpG and EBV. Figure 3.2 95 EBV immortalized B cell numbers versus time (days) in culture with addition of IL-2 and IL-4. 96 Figure 3.3 Markers on EBV immortalized B cells. 98 Figure 3.4 EBV immortalized B cell numbers versus time (days) in culture with addition of heat-killed dengue virus. 99 xi Figure 3.5 Microscopy picture of EBV-immortalized B cells Figure 3.6 Graph of the optimization of ELISA for all four dengue 101 serotypes. 102 Figure 4.1 Graph of number of patients versus dengue serotypes 104 Figure 4.2 Pie-chart of the distribution of neutralizing and non-neutralizing clones. 105 Figure 4.3 PRNT of screening of B cell clones. 106 Figure 4.4 Graph of CPE assay with ranking order of fluorescence intensity. 108 Figure 4.5a PRNT screening of B cell clones identified by CPE assay. 109 Figure 4.5b Graph of percentage neutralization versus B cell clones. 110 Figure 4.6 SDS PAGE of antibody in supernatant of Clone 14C10. 111 Figure 4.7 PRNT of 14C10 antibody at increasing concentrations. 112 Figure 4.8 Graph showing serotype specificity of 14C10 antibody. 113 Figure 4.9 PRNT of 14C10 antibody at different concentrations to show serotype specificity. Figure 5.1 Graph showing decreasing neutralizing activity of neutralizing clones over time. Figure 5.2 114 116 Mutations in DNA sequence of the IgG heavy chain of Clone 20G6. 118 Figure 5.3 Upregulation of AID in EBV-infected cells. 119 Figure 5.4 Densitometry graph of AID with β – actin as internal control. 120 Figure 6.1a PRNT of increasing concentrations of 17D11 scFv. 121 Figure 6.1b PRNT of increasing concentrations of 20G6 scFv. 122 xii Figure 6.2 Gel photo of cloned and amplified DNA sequence of 124 heavy and light chains of Clone 14C10 Figure 6.3 A schematic diagram of the in-house p-CMV human IgG expression vector. 124 Figure 6.4 Gel photo of human IgG expression vector after digestion 125 Figure 6.5a ELISA data on the 12 recombinant antibodies mAb derived from 14C10 against DV1. Figure 6.5b 128 ELISA plate demonstrating mAb from 14C10 binding to DV. 129 Figure 7.1 Sequence of DV neutralizing recombinant 14C10 mAb. 130 Figure 7.2 Serotype specificity of 14C10 mAb. 131 Figure 7.3 Serotype specificity of 14C10 mAb at different concentrations. Figure 7.4 PRNT of 14C10 mAb at increasing concentrations against dengue serotypes. Figure 7.5 134 Neutralizing activity against insect-derived and mammalian-derived DV. Figure 7.7 133 Graph representative of Figure 7.4 showing PRNT 90 and PRNT50. Figure 7.6 132 135 Immunocytochemistry of 14C10 mAb detecting DV in DV-infected BHK 136 Figure 7.8 Immunoprecipitation of 14C10 mAb to E protein of DV1. 137 Figure 7.9 Western blot of 14C10 mAb binds to E protein of DV1. Figure 7.10 Western blot and native gel of 14C10 mAb against DIII of E protein of DV. 138 139 xiii Figure 7.11 Graph of binding affinities of 14C10 Fab, 14C10 mAb and Hu 4G2. 140 Figure 7.12 PRNT of 14C10 Fab at increasing concentrations. 140 Figure 7.13 Comparison of binding affinities of 14C10 to 4G2 in various genotypes of DV1. 141 Figure 7.14a Kaff graph of 14C10 mAb. 145 Figure 7.14b Kaff graph of 4G2 antibody. 146 Figure 7.15a Graph of percentage neutralization versus 14C10 mAb concentration against seven different genotypes of DV1. 148 Figure 7.15b Graph of percentage neutralization versus 4G2 antibody concentration against seven different genotypes of DV1. 149 Figure 7.16 Sequence alignment of various genotypes of DV1. 151 Figure 7.17 Neutralization efficiency of 14C10 mAb against individual genotypes of DV. 152 Figure 7.18 PRNT to elucidate mechanism of DV. Figure 7.19 ADE profiles of 14C10 mAb and humanized 4G2 on 154 all DV serotypes. 156 Figure 7.20 ADE profile of 14C10 mAb subclasses on DV1. 158 Figure 7.21 Effect of prophylactic and therapeutic treatment in mice. 160 Figure 7.22 Lowest effective therapeutic dose for treatment in mice. 161 Figure 8.1 Probable epitopes that antibodies bind to neutralize DV. 166 xiv Abbreviations ADE Antibody Dependent Enhancement AID Activation-Induced cytidine Deaminase BL Burkitt’s Lymphoma BLCL B lymphocyte cell line bp base pair C Core protein CDR Complementarity Determining Region CLEC-5 C-type Lectin domain family 5, member A CRD Carbohydrate Recognition Domain CPE Cytopathic Effect DC Dendritic Cell DC-SIGN DC-Specific ICAM-3-Grabbing Nonintegrin DF Dengue Fever DHF Dengue Hemorrhagic Fever DV Dengue Virus DSS Dengue Shock Syndrome E Envelope protein EBER Epstein-Barr virus Encoded RNAs EBNA EBV Nuclear Antigens EBV Epstein-Barr Virus ER Endoplasmic Reticulum Fab Fragment, Antigen Binding xv g gram HLA Human Leukocyte Antigens HRP Horse Radish Peroxidase IFN Interferon IL Interleukin IM Infectious Mononucleosis JEV Japanese Encephalitis Virus kD kilo Daltons Lat Latency LCL Lymphoblastoid Cell Lines LMP Latent Membrane Proteins M Membrane protein mAb Monoclonal Antibody MHC Major Histocompatibility Complex minute ml mililitre NC Nucleocapsid core NPC Nasopharyngeal Carcinoma NS Non-structural protein OD Optical Density PBMC Peripheral Blood Mononuclear Cell PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction RNA RiboNucleic Acid prM Pre-membrane Protein xvi PRNT Plaque Reduction Neutralizing Test scFv Single Chain Variable Fragment SHM Somatic HyperMutation TNF Tumor Necrosis Factor UTR Untranslated Region WHO World Health Organization WNV West Nile Virus YFV Yellow Fever Virus µg microgram µl microliter xvii [...]... activity of neutralizing clones over time Figure 5 .2 114 116 Mutations in DNA sequence of the IgG heavy chain of Clone 20 G6 118 Figure 5.3 Upregulation of AID in EBV-infected cells 119 Figure 5.4 Densitometry graph of AID with β – actin as internal control 120 Figure 6.1a PRNT of increasing concentrations of 17D11 scFv 121 Figure 6.1b PRNT of increasing concentrations of 20 G6 scFv 122 xii Figure 6 .2 Gel... alignment of various genotypes of DV1 151 Figure 7.17 Neutralization efficiency of 14C10 mAb against individual genotypes of DV 1 52 2 Figure 7.18 PRNT to elucidate mechanism of DV Figure 7.19 154 ADE profiles of 14C10 mAb and humanized 4G2 on all 4 DV serotypes 156 Figure 7 .20 ADE profile of 14C10 mAb subclasses on DV1 158 Figure 7 .21 Effect of prophylactic and therapeutic treatment in mice 160 Figure 7 .22 ... conformation 13 Figure 1.6 Life cycle of dengue virus 18 Figure 1.7 Course of dengue infection and timing of diagnosis 24 Figure 1.8 Type I Interferon transduction pathway and putative inhibition by flavivirus 28 Figure 1.9 Immunopathogenesis of dengue virus infection 33 Figure 1.10 Location and transcription of the EBV virus genes on the doubled stranded viral genome 41 Figure 2. 1 Human IgG1, IgG3 and IgG4 framework... Graph of the optimization of ELISA for all four dengue serotypes 1 02 Figure 4.1 Graph of number of patients versus dengue serotypes 104 Figure 4 .2 Pie-chart of the distribution of neutralizing and non-neutralizing clones 105 Figure 4.3 PRNT of screening of B cell clones 106 Figure 4.4 Graph of CPE assay with ranking order of fluorescence intensity 108 Figure 4.5a PRNT screening of B cell clones identified... 1 32 135 Immunocytochemistry of 14C10 mAb detecting DV in DV-infected BHK 136 Figure 7.8 Immunoprecipitation of 14C10 mAb to E protein of DV1 137 Figure 7.9 Western blot of 14C10 mAb binds to E protein of DV1 Figure 7.10 Western blot and native gel of 14C10 mAb against DIII of E protein of DV 138 139 xiii Figure 7.11 Graph of binding affinities of 14C10 Fab, 14C10 mAb and Hu 4G2 140 Figure 7. 12 PRNT of. .. 122 xii Figure 6 .2 Gel photo of cloned and amplified DNA sequence of 124 heavy and light chains of Clone 14C10 Figure 6.3 A schematic diagram of the in-house p-CMV human IgG expression vector 124 Figure 6.4 Gel photo of human IgG expression vector after digestion 125 Figure 6.5a ELISA data on the 12 recombinant antibodies mAb derived from 14C10 against DV1 Figure 6.5b 128 ELISA plate demonstrating... culture with addition of CpG and EBV Figure 3 .2 95 EBV immortalized B cell numbers versus time (days) in culture with addition of IL -2 and IL-4 96 Figure 3.3 Markers on EBV immortalized B cells 98 Figure 3.4 EBV immortalized B cell numbers versus time (days) in culture with addition of heat-killed dengue virus 99 xi Figure 3.5 Microscopy picture of EBV-immortalized B cells Figure 3.6 101 Graph of the optimization...List of Figures Figure 1.1 Global prevalence of DF and DHF as shown by WHO 4 Figure 1 .2 Ribbon drawing of E protein 6 Figure 1.3 Schematic representation of the polyprotein processing for flaviviruses Figure 1.4 Proposed rearrangement of E dimer in Flaviviruses upon exposure to low pH Figure 1.5 8 13 Schematic diagram of E glycoprotein in neutral and the proposed acidic pH conformation 13 Figure... Comparison of binding affinities of 14C10 to 4G2 in various genotypes of DV1 141 Figure 7.14a Kaff graph of 14C10 mAb 145 Figure 7.14b Kaff graph of 4G2 antibody 146 Figure 7.15a Graph of percentage neutralization versus 14C10 mAb concentration against seven different genotypes of DV1 148 Figure 7.15b Graph of percentage neutralization versus 4G2 antibody concentration against seven different genotypes of. .. DV 129 Figure 7.1 Sequence of DV neutralizing recombinant 14C10 mAb 130 Figure 7 .2 Serotype specificity of 14C10 mAb 131 Figure 7.3 Serotype specificity of 14C10 mAb at different concentrations Figure 7.4 PRNT of 14C10 mAb at increasing concentrations against 4 dengue serotypes Figure 7.5 134 Neutralizing activity against insect-derived and mammalian-derived DV Figure 7.7 133 Graph representative of . Translation of the DV genome 14 1.1.5.4 Virus assembly and propagation 17 1.1.6 Phylogeny of Dengue Virus 19 1.1.7 Pathogenesis of Dengue Virus 22 1.1.8 Immune response to Dengue Virus 26 1.1.8.1. 61 2. 6 Ascites and antibody purification 62 2. 7 Primary cell culture 62 2. 8 Source of primary CD 22 + B cells 63 2. 9 RNA extraction and RT-PCR for serotyping of patients 63 2. 10 Cloning of. Professor Ng Mah Lee, Mary for offering so much advice and insight throughout my work on Dengue. She, together with Mdm Boon had smoothen the path of my PhD journey by providing me with the