The generation of native human monoclonal antibodies with neutralising activity for dengue virus 2

v Chapter 3 – Establishment and optimization of EBV immortalized 3.4 Heat-killed DV neither enhanced B cell growth nor increase Chapter 4 – Screening of neutralizing clones derived fro

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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

Table of contents

1.1.1 Classification 1 1.1.2 Epidemiology 3 1.1.3 Structure of dengue virions 5 1.1.4 Organization of the Flavivirus genome 6

1.1.5 Replication strategy 8

1.1.5.1 Receptor interaction 8 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

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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

2.10.1 Preparation of feeder layer

2.10.2 Serial dilution and immortalization of patients’ memory B cell 64

2.12 Titration of virus stocks 66

2.18.2 Single chain fragment variable cloning into pCANTAB vector 75

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

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Chapter 3 – Establishment and optimization of EBV immortalized

3.4 Heat-killed DV neither enhanced B cell growth nor increase

Chapter 4 – Screening of neutralizing clones derived from dengue

4.2 Screening of EBV immortalized B cell clones for dengue specificity 104

4.4 Determination of lowest antibody concentration for

Chapter 5 – Reactivation of somatic hypermutation in EBV-infected

5.1 Loss of neutralizing activity of supernatant from identified

5.2 Sequences of IgG genes extracted from EBV-infected B

Chapter 6 – Production of neutralizing recombinant human

Chapter 7 – Characterization of neutralizing 14C10

7.2 Serotype specificity of generated fully human recombinant

7.6 Binding activity and affinity determination on different

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

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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

7.9.1 Development of ADE among the DV serotype 155 7.9.2 Development of ADE among the antibody subclasses 157

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 health-care 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

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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

List of tables

Table 1.1 Flavivirus classification 2 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 69 Table 2.4 Human immunoglobulin gene PCR primers for Lamda

light chain 70 Table 2.5 Primers for cDNA synthesis of human immunoglobulin

Table 2.6 Primers for attachment of (Gly4Ser)3 linker onto heavy

Table 2.7 Primers for attachment of (Gly4Ser)3 linker onto Kappa

light chain 75 Table 2.8 Primers for attachment of (Gly4Ser)3 linker onto Lamda

light chain 75

Table 4.1 Number of clones isolated per batch and respective target

Table 6.1 The presence of heavy and light chains in clones 123 Table 6.2 Heavy and light chain sequences of 12 candidate

recombinant antibodies from clone 14C10 127

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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 8 Figure 1.4 Proposed rearrangement of E dimer in Flaviviruses upon

exposure to low pH 13 Figure 1.5 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 95 Figure 3.2 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

Figure 3.5 Microscopy picture of EBV-immortalized B cells 101 Figure 3.6 Graph of the optimization of ELISA for all four dengue

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

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 114 Figure 5.1 Graph showing decreasing neutralizing activity of

neutralizing clones over time 116 Figure 5.2 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

Figure 6.1a PRNT of increasing concentrations of 17D11 scFv 121 Figure 6.1b PRNT of increasing concentrations of 20G6 scFv 122

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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 128 Figure 6.5b ELISA plate demonstrating mAb 8 from 14C10 binding

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 132 Figure 7.4 PRNT of 14C10 mAb at increasing concentrations against

4 dengue serotypes 133 Figure 7.5 Graph representative of Figure 7.4 showing PRNT90

and PRNT50 134 Figure 7.6 Neutralizing activity against insect-derived and

mammalian-derived DV 135 Figure 7.7 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 138 Figure 7.10 Western blot and native gel of 14C10 mAb against DIII

of E protein of DV 139

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

2 Figure 7.18 PRNT to elucidate mechanism of DV 154 Figure 7.19 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 Lowest effective therapeutic dose for treatment in mice 161 Figure 8.1 Probable epitopes that antibodies bind to neutralize DV 166

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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