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Advancing the Ferret As an Immunological Model to Study B-Cell Responses

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Advancing the Ferret As an Immunological Model to Study B-Cell Responses Advancing the ferret as an immunological model to study B-cell responses Julius Wong ORCID: 0000-0003-0253-6151 Submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy July 2020 Department of Microbiology and Immunology Faculty of Medicine, Dentistry and Health Sciences The University of Melbourne Table of Contents ABSTRACT ................................................................................................................................................... I DECLARATION ........................................................................................................................................... IV PREFACE ..................................................................................................................................................... V ACKNOWLEDGEMENTS ......................................................................................................................... VII PUBLICATIONS FROM THIS THESIS ..................................................................................................... IX LIST OF TABLES......................................................................................................................................... X LIST OF FIGURES ..................................................................................................................................... XII LIST OF ABBREVIATIONS ...................................................................................................................... XV SPECIFIC AIMS OF THESIS ................................................................................................................. XVIII CHAPTER 1 ...................................................................................................................................................1 LITERATURE REVIEW ......................................................................................................................................... 1 1.1 Global Burden of Influenza ...................................................................................................... 1 1.2 Influenza virus ............................................................................................................................ 4 1.2.1 Virus structure and replication ................................................................................................................... 4 1.2.2 Gross morphology and genomic organisation of Influenza Viruses ........................................................... 4 1.2.3 Influenza types ........................................................................................................................................... 7 1.2.4 Viral replication cycle ................................................................................................................................. 8 1.3 Antigenic drift and shift of influenza viruses ................................................................... 11 1.3.1 Antigenic drift........................................................................................................................................... 11 1.3.2 Antigenic shift .......................................................................................................................................... 12 1.4 Anti-influenza drugs and vaccines ...................................................................................... 12 1.4.1 M2 Ion channel inhibitors ........................................................................................................................ 12 1.4.2 NA inhibitors ............................................................................................................................................ 13 1.4.3 Need for broadly protective influenza vaccines ....................................................................................... 14 1.4.4 Future therapeutic agents for influenza................................................................................................... 15 1.5 Immunity to influenza ............................................................................................................. 16 1.5.1 Innate immune responses ........................................................................................................................ 16 1.5.2 Adaptive immune responses .................................................................................................................... 19 1.5.2.1 B and T-cells are important for protection against influenza ........................................................ 19 1.5.2.2 Somatic recombination of immunoglobulin genes results in highly diverse antibody/BCR repertories ......................................................................................................................................................... 19 1.5.2.3 Early B-cell responses against influenza ......................................................................................... 23 1.5.2.4 Somatic hypermutation and high-affinity B-cell responses against influenza ............................ 23 1.6 Technologies for generating mAbs ..................................................................................... 28 1.6.1 Hybridoma technology ............................................................................................................................. 28 1.6.2 Cell display technology generation of antibody libraries for screening ................................................... 28 1.6.3 Single-cell-recovery of mAb sequences .................................................................................................... 29 1.7 Animal models of human influenza infection .................................................................. 31 1.7.1 Mice ......................................................................................................................................................... 31 1.7.2 Non-Human Primates (NHP) .................................................................................................................... 31 1.7.3 Ferrets ...................................................................................................................................................... 32 1.8 Ferrets: an important animal model for influenza ........................................................... 32 1.8.1 Ferrets as an influenza pathogenesis and transmission model ................................................................ 32 1.8.2 Ferrets for influenza surveillance and vaccine development ................................................................... 33 1.9 Ferrets as an immunological model for viral infectious diseases .............................. 34 1.9.1 Ferrets as an immunological to study influenza viruses ........................................................................... 34 1.9.2 Ferrets as an immunological model for other emerging viral diseases .................................................... 41 1.9.2.1 Pathogenic Coronaviruses......................................................................................................... 41 1.9.2.2 Henipavirus .................................................................................................................................. 42 1.9.2.3 Respiratory Syncytial Virus and Metapneumovirus................................................................ 42 1.9.2.4 Ebola virus ................................................................................................................................... 43 1.10 Knowledge gaps that have to be bridged to improve ferrets as an immunological model 44 1.10.1 Immunogenetics....................................................................................................................................... 44 1.10.2 Antigenic recognition of major influenza proteins ................................................................................... 45 1.10.3 Future T-cell specific reagents for ferrets ................................................................................................ 46 1.10.4 Current and Future markers for ferret myeloid lineage cells ........................................................................ 47 1.10.5 Future B-cell specific reagents for ferrets ................................................................................................ 47 CHAPTER 2 ................................................................................................................................................. 49 GENERAL MATERIALS AND METHODS ............................................................................................................. 49 2.1 Materials ..................................................................................................................................... 49 2.1.1 Media and Buffers .................................................................................................................................... 49 2.1.1.1 1% Agarose gel ................................................................................................................................... 49 2.1.1.2 Cryopreservation media for E.coli .................................................................................................... 49 2.1.1.3 Cryopreservation media for mammalian cells ................................................................................. 49 2.1.1.4 ELISA blocking
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