Antibody Discovery for Development of a Serotyping Dengue Virus NS1 Capture Assay Kebaneilwe Lebani Master of Biotechnology (Advanced) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Australian Institute for Bioengineering and Nanotechnology ABSTRACT Dengue virus (DENV) infections are a significant public health burden in tropical and sub-tropical regions of the world. Infections are caused by four different but antigenically related viruses which result in four DENV serotypes. The multifaceted nature of DENV pathogenesis hinders the sensitivity of assays designed for the diagnosis of infection. Different markers can be optimally detected at different stages of infection. Of particular clinical importance is the identification of acute viremia during the febrile phase of infection which is pivotal for management of infection. Non-structural protein 1 (NS1) has been identified as a good early surrogate marker of infection with possible applications in epidemiological surveillance and the development of blood screening assays. This contribution is towards using serotype-specificity to achieve specific and more sensitive diagnostic detection of DENV NS1. The general aim of this work is to isolate immune-reagents that can be used to develop an assay with improved sensitivity of DENV NS1 detection in a diagnostic setting. In this work, we sought to isolate serotype-specific antibodies that discern discreet antigenic differences in NS1 from each DENV serotype. Additionally, we also sought to isolate a pairing antibody that recognises NS1 from all four DENV serotypes (pan-reactive) for tandem capture of the DENV NS1. To achieve this, three naive, immunoglobulin gene libraries (a VH domain, a scFv and a Fab library) were interrogated for binders to recombinant NS1 antigen from all four DENV serotypes using phage display technology and various biopanning approaches. From biopanning experiments, four antibody fragments specific to NS1 from each DENV serotype as well as a panel of pan-reactive antibody fragments were isolated. The isolated antibody fragments were reformatted into fully assembled IgG1 antibodies for characterisation. In a sandwich ELISA, none of the pan-reactive antibodies were able to bind DENV NS1 in tandem with the serotype-specific antibodies. All four of the serotype-specific antibodies however, recognised I their serotype-respective recombinant and native DENV NS1. The serotype-specific immunoglobulins showed equilibrium dissociation constants that ranged from µM to nM. Used as capture reagents in a sandwich ELISA with a commercial pan-reactive antibody as the detection probe, three of the antibodies (9H2 anti DENV-1 NS1, 7G11 anti DENV-3 NS1 and 6A5 anti DENV-4 NS1) were able to achieve clinically relevant limits of DENV NS1 detection. 4C11, an antibody against DENV-2 NS1 had the poorest performance and was subjected to affinity maturation through targeted mutagenesis of VH CDR3. A new variant A4, showed marginal 2-fold improvement in performance when incorporated into a limit of detection ELISA with the commercial pan-reactive antibody. Our data suggests that the four serotype-specific antibodies, including the new variant, would be useful for inclusion in a sandwich ELISA that uses NS1 to serotype DENV infections. To develop a complete DENV NS1 serotyping assay however, the serotype-specific antibodies will need to be used in a new iteration of a biopanning campaign to isolate an appropriate pan-reactive pairing partner. II DECLARATION BY AUTHOR This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. III PUBLICATIONS DURING CANDIDATURE No publications PUBLICATIONS INCLUDED IN THIS THESIS No publications included IV CONTRIBUTIONS BY OTHERS TO THE THESIS Dr Daniel Watterson – technical assistance with Vero cell culture, virus infection and immunofluorescence. STATEMENT OF PARTS OF THE THESIS SUBMITTED TO QUALIFY FOR THE AWARD OF ANOTHER DEGREE None V ACKNOWLEDGEMENTS My mum Nduka Jongman-Lebani, and my sisters; Tawana Lebani and Ame Lebani, I dedicate this thesis to you. My friends, your support has been unwavering and invaluable. I could not have concluded this journey without you. There are too many of you to name individually but I genuinely thank you all for taking the ride of high highs and low lows with me. Sumukh Kumble, thank you so much for the most awesome illustrations! My sponsor, the Botswana International University of Science and Technology (BIUST) I am thankful for the opportunity to have embarked on this journey as your development fellow. My enrolling institution, the Australian Institute for Bioengineering and Nanotechnology I am thankful for your funding and the opportunity to undertake my research in a world class institute. The Schlumberger Foundation, I am grateful for the assistance with conference travel costs through the Fellowship for the Future Award. My supervisors Steve and Martina, I am truly thankful for your guidance and patience. It has been a most humbling experience that has forced me to confront my weaknesses. I am still a work in progress. If I had to go through this journey again, I would still pick both of you. Whether you would pick me is a completely different story! I am heavily indebted to you both for having reached this juncture. VI KEYWORDS Antibodies, Phage Display, Dengue Virus, NS1, Diagnosis, Serotyping AUSTRALIAN AND NEW ZEALAND STANDARD RESEARCH CLASSIFICATIONS (ANZSRC) ANZSRC code: 110702, Applied Immunology (including Antibody Engineering, Xenotransplantation and T-cell Therapies), 40% ANZSRC code: 110302, Clinical Chemistry (diagnostics), 40% ANZSRC code: 110804, Medical Virology, 20% FIELDS OF RESEARCH (FOR) CLASSIFICATION FoR code: 1107, Immunology, 40% FoR code: 1103, Clinical Sciences, 40% FoR code: 1108, Medical microbiology, 20% VII CONTENTS ABSTRACT…………………………………………………………………………………………..I ACKNOWLEDGEMENTS………………………………………………………………………...VI FIGURES……………………………………………………………………………………...……XI TABLES………………………………………………………………………………………..…XIII ABBREVIATIONS…………………………………………………………………….…………XIV Chapter 1 .............................................................................................................................................. 1 1.1 Humoral Immune Response .................................................................................................. 2 1.2 Phage Display Technology .................................................................................................... 5 1.3 An Overview of Dengue Virus Infections ............................................................................. 9 1.4 DENV structure and Replication ......................................................................................... 10 1.5 Diagnosis of Dengue Virus Infections ................................................................................ 12 1.6 Evaluation and Application of DENV NS1 as a Diagnostic Marker .................................. 17 1.7 Project Rationale ................................................................................................................. 21 1.8 References ........................................................................................................................... 22 Chapter 2 ............................................................................................................................................ 29 2.1 Introduction ......................................................................................................................... 30 2.2 Materials and Methods ........................................................................................................ 32 2.2.1 General Recipes, Stock Reagents and Equipment ....................................................... 32 2.2.2 Biopanning using Purified Antigen.............................................................................. 37 2.2.3 Phage Precipitation ...................................................................................................... 41 2.2.4 Phage Titre ..................................................................................................................