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 buffer ...... 49 2.1.1.5 ELISA dilution buffer ...... 49 2.1.1.6 ELISA wash buffer ...... 49 2.1.1.9 Influenza virus growth media ...... 50 2.1.1.10 LB-kanamycin broth ...... 50 2.1.1.11 LB-kanamycin agar...... 50 2.1.1.12 Live/dead Stain for flow cytometry ...... 50 2.1.1.13 MDCK cell maintenance media ...... 51 2.1.1.14 RPMI-F10 media ...... 51 2.1.1.15 SDS-PAGE loading buffer ...... 51 2.1.1.16 SDS-PAGE buffer ...... 51 2.1.1.17 SDS-PAGE gel fixing buffer ...... 51 2.1.1.18 Tris-Acetate EDTA buffer for DNA gel electrophoresis...... 51 2.1.1.19 Trypsin-EDTA media ...... 52 2.1.2 Protein/DNA purification kits and buffers ...... 52 2.2 Methods ...... 53 2.2.1 Molecular cloning and expression of recombinant mAbs and proteins ...... 53 2.2.1.1 Construction of ferret antigen protein-coding plasmids ...... 53 2.2.1.2 Construction of ferret antibody constant chain coding plasmids ...... 53 2.2.1.3 Construction of ferret / chimeric ferret-human antibody coding plasmids ...... 54 2.2.1.4 Construction of murine antibody coding plasmids ...... 55 2.2.1.5 Restriction digestion and ligation of restriction digestion products ...... 55 2.2.1.6 Transformation and validation of antibody/protein-coding plasmids...... 56 2.2.2 Agarose gel electrophoresis ...... 56 2.2.3 Maintenance of Expi293F cells ...... 57 2.2.4 Eukaryotic expression of proteins and antibodies ...... 57 2.2.5 Purification of recombinant proteins and antibodies ...... 57 2.2.6 Denaturing SDS-PAGE protein gel electrophoresis ...... 58 2.2.7 HA-specific ELISA ...... 59 CHAPTER 3 ...... 60 DEVELOPING THE CAPACITY TO EXPRESS FERRET MONOCLONAL ANTIBODIES ...... 60 3.1 Abstract ...... 60 3.2 Introduction ...... 61 3.3 Materials and Methods ...... 64 3.3.1 Annotation of ferret immunoglobulin genes ...... 64 3.3.2 Generation of ferret immunoglobulin variable gene phylogenetic trees...... 64 3.3.3 Flow cytometric sorting of single ferret B-cells ...... 65 3.3.4 RT-PCR recovery of immunoglobulin sequences from single sorted ferret B-cells ...... 65 3.3.5 Analysis of recovered immunoglobulin sequences ...... 70 3.3.6 RNA-seq validation of ferret immunoglobulin constant region sequences ...... 70 3.3.7 Human CD16 / CD32 dimer ELISA assay to detect cross-reactive ferret IgG binding activity...... 71 3.4 Results ...... 72 3.4.1 Annotation of ferret germline variable, diversity and joining genes ...... 72 3.4.1.1 Ferret Immunoglobulin heavy chain locus ...... 72 3.4.1.2 Ferret immunoglobulin kappa chain locus ...... 75 3.4.1.3 Ferret immunoglobulin lambda chain locus ...... 78

3.4.2 Single-cell RT-PCR for recovery of ferret immunoglobulin sequences ...... 80 3.4.2.1 Recovery of ferret immunoglobulin heavy chain sequences ...... 80 3.4.2.2 Recovery of ferret immunoglobulin kappa chain sequences ...... 81 3.4.2.3 Recovery of ferret immunoglobulin lambda chain sequences ...... 82 3.4.3 Sequence validation of ferret immunoglobulin constant gene segments...... 84 3.4.4 Recombinant Expression of chimeric human/ferret antibodies ...... 87 3.5 Discussion ...... 89 3.6 Conclusion ...... 92 CHAPTER 4 ...... 93 PROOF OF CONCEPT FOR RECOVERING HA-SPECIFIC MABS FROM INFLUENZA-INFECTED FERRETS...... 93 4.1 Abstract ...... 93 4.2 Introduction ...... 94 4.3 Materials and Methods ...... 98 4.3.1 Single-cell sorting of HA-specific class-switched B-cells from infected ferrets ...... 98 4.3.2 Recovery of immunoglobulin gene transcript sequences from single sorted ferret B-cells...... 98 4.3.3 Analysis of HA-specific sequences recovered from ferret B-cells ...... 100 4.3.4 Mardin-Darby Canine Kidney (MDCK) cell culture handling and maintenance ...... 100 4.3.5 Influenza virus neutralisation assay ...... 101 4.3.6 Haemagglutination Inhibition (HAI) assay ...... 101 4.3.7 Influenza viral escape assay ...... 102 4.3.8 RT-PCR amplification and sequencing of HA genes ...... 102 4.4 Results ...... 103 4.4.1 Single-cell sorting of HA-specific ferret B-cells ...... 103 4.4.2 Genetic features of sequences recovered from HA-specific ferret B-cell populations ...... 105 4.4.3 Clonal expansion of HA specific ferret B-cells ...... 108 4.4.4 Recovering and validating ferret HA-specific mAbs from infected ferrets ...... 110 4.4.5 Viral escape mapping validation of 4A06 mAb...... 113 4.5 Discussion ...... 115 4.6 Conclusion ...... 118 CHAPTER 5 ...... 119 ISOLATING RECOMBINANT MURINE ANTI-FERRET ANTIBODIES USING SINGLE CELL-RT PCR ...... 119 5.1 Abstract ...... 119 5.2 Introduction ...... 120 5.3 Materials and methods ...... 124 5.3.1 Identification and bioinformatics validation of ferret proteins ...... 124 5.3.2 Immunisation of mice with recombinant ferret proteins ...... 125 5.3.3 ELISA validation of ferret immunogen specific serological responses ...... 125 5.3.4 Single cell sorts for ferret antigen specific murine B-cells...... 126 5.3.6 Analysis of recovered immunoglobulin sequences ...... 130 5.3.7 ELISA validation of recombinant murine antibodies ...... 130 5.3.8 Flow cytometric staining profiles of recombinant ferret specific murine antibodies ...... 131 5.4 Results ...... 132 5.4.1 Identification and expression of ferret antigens for mAb development ...... 133 5.4.1.1 CD19 ...... 133 5.4.1.2 IgD ...... 136 5.4.1.3 CD138 ...... 139 5.4.1.4 NKp46 and LAMP-1 ...... 143 5.4.2 Immunisation of C57BL/6 mice with recombinant ferret antigens ...... 149 5.4.3 IgD-CD38+ memory B-cell responses to ferret antigens ...... 151 5.4.4 RT-PCR recovery of immunoglobulin sequences from ferret CD19/IgD specific murine B-cells ...... 151 5.4.5 Analysis of recovered murine immunoglobulin transcript sequences ...... 154 5.4.5.1 Genetic features of ferret CD19 specific murine memory B-cells ...... 154 5.4.5.2 Clonal expansion of ferret CD19 specific murine memory B-cells ...... 157 5.4.5.3 Genetic features of ferret IgD specific murine memory B-cells ...... 159 5.4.5.4 Clonal expansion of ferret IgD specific murine memory B-cells ...... 162 5.4.6 ELISA validation of murine anti-ferret CD19/IgD IgG+ mAbs ...... 164 5.4.7 Flow cytometric staining profiles of CD19/IgD mAbs ...... 169 5.5 Discussion ...... 171 5.6 Conclusion ...... 174

CHAPTER 6 ...... 177 GENERAL DISCUSSION ...... 177 6.1 Overview ...... 177 6.2 In-depth ferret B-cell studies are hindered by the lack of immunological reagents and immunogenetic information ...... 178 6.3 Development of techniques and tools to recover and express ferret mAbs ...... 179 6.4 Unidentified germline immunoglobulin ferret genes in the draft copy of the ferret genome ...... 182 6.5 Recovery of antigen-specific B-cell transcripts and mAbs from ferrets ...... 183 6.6 Studying mAb recognition profiles of influenza HA ...... 184 6.7 FcR and IgG subclasses in ferrets ...... 186 6.8 Development of ferret reagents ...... 187 6.9 Cell display and NGS technologies to isolate antigen specific mAbs ...... 189 6.10 Concluding remarks ...... 190 REFERENCES ...... 191 APPENDICES ...... 213 Chapter 3 Appendix ...... 213 Appendix 3.1 Amino acid sequence alignments of ferret IGKV genes ...... 213 Appendix 3.2 Amino acid sequence alignments of ferret IGLV genes ...... 214 Appendix 3.3 Ferret germline heavy variable genes ...... 215 Appendix 3.4 Ferret germline kappa variable genes ...... 219 Appendix 3.5 Ferret germline lambda variable genes ...... 225 Chapter 4 Appendix ...... 229 Appendix 4.1 Ferret Immunoglobulin heavy chain clonal families recovered from influenza infected ferrets ..... 229 Appendix 4.2 Ferret immunoglobulin lambda chain clonal families recovered from influenza infected ferrets ... 244 Appendix 4.3 Ferret immunoglobulin kappa chain clonal families recovered from influenza infected ferrets ..... 249 Appendix 4.4 HA sequences from viral escape mapping of 4A06 mAb...... 251 Chapter 5 appendix ...... 252 Appendix 5.1 IgK+ transcripts recovered from NKp46, CD138 and LAMP specific murine memory B-cells ...... 252 Appendix 5.2 Murine immunoglobulin IgG clonal families recovered from ferret CD19 specific B-cells ...... 254 Appendix 5.3 Murine immunoglobulin IgK clonal families recovered from ferret CD19 specific B-cells ...... 259 Appendix 5.4 Murine immunoglobulin IgG clonal families recovered from ferret IgD specific B-cells ...... 262 Appendix 5.5 Murine immunoglobulin IgK clonal families recovered from ferret IgD specific B-cells ...... 266 PUBLICATIONS ...... 271

REVIEW PAPER : IMPROVING IMMUNOLOGICAL INSIGHTS INTO THE FERRET MODEL OF HUMAN VIRAL INFECTIOUS DISEASE ...... 271 RESEARCH PAPER : SEQUENCING B CELL RECEPTORS FROM FERRETS (MUSTELA PUTORIUS FURO) ...... 283

Abstract

Introduction

Influenza is a clinically significant disease, causing 24000-62000 deaths alone in the

United States during the 2019-2020 season. While annual vaccines are available, variable efficacies have been reported and annual updates are required due to antigenic drift. Ferrets are a useful model for studying human respiratory viruses and have been widely used to evaluate vaccines and transmission of influenza. Sera from ferrets infected with different influenza strains are used in HI assays as part of strain determination of seasonal influenza vaccines. A key limitation of the ferret model is the paucity of immunological reagents to characterise immune responses and a lack of knowledge regarding the ferret immune system. This PhD thesis aims to advance the ferret as an immunological model to study human respiratory viruses by developing methods and reagents which will enable in-depth interrogation of ferret B-cell responses.

Methods

While a draft copy of the ferret genome is available, immunoglobulin sequence information is not well-annotated. Hence, we first annotated the ferret genome with immunoglobulin variable, diversity, joining and constant chain genes by inferring homology using human and canine orthologs (Chapter 3). Novel PCR primers targeting 5’- leader, 3’- joining and 3’- constant chain immunoglobulin genes were derived, enabling the recovery of functional, paired heavy and light chain transcript sequences from single sorted ferret B-cells. Ferret immunoglobulin constant sequences were validated by RNA-seq, which enabled the development of ferret IgG

i expression plasmids. Using this technique, HA-specific B-cell responses were characterised for the first time in ferrets at the transcript level (Chapter 4). Candidate ferret mAbs were derived from the recovered sequences, expressed and screened for

HA binding specificity and in-vitro influenza virus neutralisation activity. We noted poor recovery of ferret HA specific mAbs and subsequently sought to improve flow cytometric panels available for ferrets. We established a methodology using previously developed murine single-cell BCR sequencing methods to recover murine anti-ferret mAbs (Chapter 5). First, coding sequences of ferret B and NK-cell reagents were identified on the ferret genome and validated by sequence and structural comparisons with other mammalian homologs. C57BL/6 mice were subsequently immunised with these antigens and candidate mAbs were recovered for examination by ELISA and flow cytometry.

Results

Ferret variable, diversity, joining and constant chain coding genes were identified on the draft copy of the ferret genome and show good sequence similarity to human and canine variants. Our novel ferret immunoglobulin specific PCR primers enabled the recovery and characterisation of germline ferret immunoglobulin genes from single sorted ferret B-cells. RNA-seq validation of ferret immunoglobulin constant chain genes subsequently enabled the construction of ferret IgG/IgL expression plasmids.

This facilitated the expression of chimeric human-ferret CR9114 IgG antibody retaining HA binding specificity. Subsequently, using previously developed trimeric HA probes, clonally expanded sequences were recovered from single sorted HA-specific

B-cells derived from infected ferrets. Screening of candidate ferret monoclonal antibodies enabled the identification of two novel antibodies, belonging to the same

ii clonal family showing HA binding specificities. Further examination by HAI and microneutralization assays revealed the ability of the mAbs to neutralise influenza virus in vitro. Viral escape mapping revealed binding epitope to previously reported Sa site of the HA head domain, showing proof of concept for mapping HA epitopes using these recombinant ferret mAbs. We next attempted to improve flow cytometric panels for ferrets which will enhance recovery of ferret immunoglobulin transcripts. As there are currently no mAbs targeting B and NK-cell markers in ferrets, we identified key markers for murine mAb development including CD19, IgD, CD138, NKp46 and

LAMP-1. We identified candidate anti-ferret CD19 and IgD mAbs which bound to cognate recombinant antigens by ELISA, validating this method for generating anti- ferret mAbs to improve panels for flow cytometry and confocal microscopy. As the mAbs in this thesis lacked the capacity to resolve ferret cell populations by flow cytometry, we identified and discussed key steps in the process which will inform future use of this approach to develop anti-ferret mAb reagents.

Conclusion

The body of work presented in this thesis forms the proof of concept of studying antigen-specific B-cell responses at the mAb level in ferrets. Future improvements in tools developed in this thesis and future development reagents will enable detailed interrogation of the ferret immune system.

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Declaration

This is to certify that:

1. This thesis comprises only my original work towards the PhD except where

indicated in the Preface.

2. Due acknowledgements have been made in the text to all other material used.

3. This thesis is less than 100,000 words in length, exclusive of tables, maps,

bibliographies and appendices

Preface

Experimental work for this PhD thesis was performed in the Kent Laboratory,

Department of Microbiology and Immunology, University of Melbourne (Peter Doherty

Institute for Infection and Immunity), Victoria, Australia. This was conducted under the primary supervision of Professor Stephen Kent and co-supervised by Dr Adam

Wheatley. Funding for the PhD programme was provided by the University of

Melbourne (Melbourne Research Scholarship) and CSIRO (PhD top-up scholarship).

Due to the COVID-19 outbreak that took place in 2020, disruption of laboratory work occurred from 26th March 2020.

Parts of chapter 1 (Literature Review) and chapter 6 (General discussion) was published in the peer-reviewed article:

Wong, J., Layton, D., Wheatley, A.K. and Kent, S.J., 2019, Improving immunological insights into the ferret model of human viral infectious disease. Influenza and Other

Respiratory Viruses 13, 535-546.

Ferret immunoglobulin sequences from chapter 3 are available on Genbank under the following accession numbers:

MT330121- MT330243

Parts of chapters 3 and 4 was published as a peer-reviewed article:

Wong, J., Tai M.C., Hurt A.C., Tan H.X., Kent, S.J., Wheatley, A.K., 2020, Sequencing

B cell receptors from ferrets (Mustela putorius furo). PLOSone, 15(5): e0233794

Parts of Chapters 3 and 4 were presented at an international conference below:

Wong, J., Wheatley, A.K. and Kent, S.J., 2019.’Annotation and recovery of ferret specific immunoglobulin sequences’ Options X for the control of influenza. Singapore,

28 Aug – 1 Sep.

The contributions to this thesis by the following individuals are as follows

Dr Adam Wheatley Provided pdm2009 H1N1 viruses, HA probes and

antibody/protein cloning plasmids and molecular biology

reagents.

Dr Jennifer Juno Advice and help with flow cytometry

Dr Hyon Xhi Tan Immunised mice to generate ferret specific mAbs

Dr Heidi Peck Performed HAI assays to validate ferret mAbs

Dr Paul Whitney Providing ferret splenocytes/lymph nodes

Wenbo Jiang Processing and cryopreservation of ferret tissues

Xiao Xiao Jia Provision of MDCK cells for neutralisation and viral escape

assays

Acknowledgements

First and foremost, I would like to thank my supervisors Professor Stephen Kent and

Dr Adam Wheatley for taking me on as a PhD student to work on a such a challenging yet meaningful project. I would like to say a very big thank you to Adam, for providing the necessary ingredients for a good start in the lab.

I gratefully acknowledge the funding and resources received towards my PhD from the University of Melbourne (Melbourne Research Scholarship) and CSIRO (Top-up research scholarship). Thank you to Professor Stephen Kent for recruiting me into his lab. I am immensely grateful for his generosity in supporting my PhD financially by providing opportunities to attend conferences. The completion of this thesis would not be possible if not for his generosity, as well as feedback and knowledge in the field of infectious diseases and immunology.

To Dr Adam Wheatley, thank you for your willingness to take on an inexperienced student such as myself and providing the tools to ensure the completion of this project.

Thank you for giving me space and allowing me to make decisions regarding the project. It is also definite to say that most of this work would not have been completed if not for your tireless supervision. Although many technical challenges were faced to establish novel assays and reagents to study B-cell responses in ferrets, you have taught me to push hard, having confidence and believing in yourself while in pursuit of knowledge.

To members of the Kent Lab who helped in various ways during this candidature, thank you so much. Thank you for organising after-work drinks and outings which

vii makes life in the lab much more fun! To Hyon Xhi, thanks for introducing and guiding me through mouse work for the development of novel reagents for ferrets. Without your guidance, it would have been much more difficult! To Jennifer, thank you for pointing me in the right direction when help was needed and help with flow cytometry.

To the RAs in the laboratory, thank you for keeping the laboratory in order while everyone is busy trying the uncover the mysteries nature has in place for discovery!

My sincerest appreciation also goes to my PhD committee members, Professor Jason

MacKenzie, Dr Aeron Hurt and Dr Steven Rockman for useful feedback during the annual review meetings. Thank you, Dr Aeron Hurt, for generously providing ferret organs from the WHO collaboration centre and Dr Steven Rockman for his knowledge in monoclonal antibodies and resources from Seqirus, Australia. It was a pleasure to have had the opportunity to learn so much about hybridomas from your lab members.

I would also like to especially thank Heidi Peck from the WHO collaboration centre for helping with the HAI assays and validating our ferret mAbs.

Last, but not least, I would like to show my deepest gratitude to friends and family in

Melbourne and Singapore. To Alon, thank you for providing me with emotional support through my PhD studies and the COVID-19 crisis we are now facing. I’m lucky to have met you and you’ll always have a place in my heart. To my best friend in Melbourne,

Dedi, thanks for being a listening ear always and taking me out for meals to ensure

I’m well fed. To my parents and brother back in Singapore, thanks for always making sure that I’m fine and checking up on me. Thank you all for your support that I have received in this pursuit.

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Publications from this thesis

Wong, J., Layton, D., Wheatley, A.K. and Kent, S.J., 2019, Improving immunological insights into the ferret model of human viral infectious disease. Influenza and Other

Respiratory Viruses 13, 535-546.

Wong, J. 2019.’Annotation and recovery of ferret specific immunoglobulin sequences’

Options X for the control of influenza. Singapore, 28 Aug – 1 Sep.

Wong, J., Tai M.C., Hurt A.C., Tan H.X., Kent, S.J., Wheatley, A.K., 2020, Sequencing

B cell receptors from ferrets (Mustela putorius furo). PLOSone, 15(5): e0233794

List of Tables

Chapter 1

Table 1-1 Comparison of mAb generation techniques

Table 1-2 List of currently available reagents available for studying ferret immune cells

Chapter 2

Table 2-1 Protein/DNA purification kits and buffers

Table 2-2 IgG binding profiles of Protein A and Protein G

Chapter 3

Table 3-1 Ferret immunoglobulin specific primers

Appendix 3.3 Ferret germline heavy variable genes

Appendix 3.4 Ferret germline kappa variable genes

Appendix 3.5 Ferret germline lambda variable genes

Chapter 4

Table 4-1 Amino acid sequences of expressed candidate HA-specific mAbs

Appendix 4.1 Ferret Immunoglobulin heavy chain clonal families recovered from influenza-infected ferrets

Appendix 4.2 Ferret immunoglobulin lambda chain clonal families recovered from influenza-infected ferrets

Appendix 4.3 Ferret immunoglobulin kappa chain clonal families recovered from influenza-infected ferrets

Chapter 5

Table 5-1 Mouse immunoglobulin specific primers

Table 5-2 Summary of mAb development for ferret B-cell and NK-cell antigens

Table 5-3 Amino acid sequences of expressed murine anti-ferret CD19/IgD mAbs

Appendix 5.1 IgK+ transcripts recovered from CD138, NKp46 and LAMP specific B-cells

Appendix 5.2 Murine immunoglobulin IgG clonal families recovered from ferret CD19 specific B-cells

Appendix 5.3 Murine immunoglobulin IgK clonal families recovered from ferret CD19 specific B-cells

Appendix 5.4 Murine immunoglobulin IgG clonal families recovered from ferret IgD specific B-cells

Appendix 5.5 Murine immunoglobulin IgK clonal families recovered from ferret IgD specific B-cells

List of figures

Chapter 1

Figure 1-1 Structure of influenza virus

Figure 1-2 Replication cycle of influenza

Figure 1-3 Developmental stages of B-cells

Figure 1-4 Early B-2 cell responses against influenza

Figure 1-5 Geminal centre reaction to generate high affinity antibodies

Chapter 2

Figure 2-1 Construction of recombinant ferret-Fc fusion plasmids

Figure 2-2 Construction of ferret-Fc plasmids

Figure 2-3 Construction of murine/ferret mAb coding plasmids

Figure 2-4 Purification and expression of ferret proteins / mouse mAbs / ferret mAbs.

Chapter 3

Figure 3-1` Establishing tools for recovering ferret mAbs.

Figure 3-2 Ferret immunoglobulin germline heavy variable, diversity and joining genes

Figure 3-3 Ferret immunoglobulin germline kappa variable and joining genes

Figure 3-4 Ferret immunoglobulin germline lambda variable and joining genes

Figure 3-5 Genetic features of recovered ferret germline immunoglobulin sequences

Figure 3-6 Alignments of transcript and artificially spliced ferret immunoglobulin constant regions

Figure 3-7 Expression and recovery of chimeric ferret antibody expressing human CR9114 variable regions

Appendix 3.1 Amino acid sequence alignments of ferret IGKV genes

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Appendix 3.2 Amino acid sequence alignments of ferret IGLV genes

Chapter 4

Figure 4-1 Single-cell recovery of HA-specific mAbs from influenza-infected ferrets

Figure 4-2 Recovery of full-length heavy chain ferret immunoglobulin transcripts

Figure 4-3 Gating scheme to recover HA-specific sequences from infected ferrets

Figure 4-4 Genetic features of ferret HA-specific immunoglobulin heavy and light chain sequences

Figure 4-5. Clonal expansion of HA positive B-cell transcripts

Figure 4-6 Validation of recovered HA-specific ferret mAbs

Figure 4-7 Viral escape mapping of 4A06 mAb

Appendix 4.4 HA sequences from viral escape mapping of 4A06 mAb

Chapter 5

Figure 5-1 Single-cell recovery of ferret antigen-specific mAbs

Figure 5-2 Characterization and recombinant expression of the extracellular domain (ECD) of ferret CD19 for mAb development

Figure 5-3 Characterization and recombinant expression of the CH1 domain of ferret IgD

Figure 5-4 Characterization and recombinant expression of ferret CD138

Figure 5-5 Characterization and recombinant expression of ferret NKp46

Figure 5-6 Characterization and recombinant expression of ferret LAMP

Figure 5-7 Immunisation and serological responses against ferret antigens in C57BL/6 mice

Figure 5-8 Memory B-cell responses in ferret antigen immunised mice

Figure 5-9 Genetic features of ferret CD19 specific murine immunoglobulin sequences

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Figure 5-10 Clonal expansion of CD19 specific immunoglobulin transcripts

Figure 5-11 Genetic features of ferret IgD specific murine immunoglobulin sequences

Figure 5-12 Clonal expansion of ferret IgD-specific murine immunoglobulin sequences

Figure 5-13 ELISA validation of CD19/IgD specific mAbs

Figure 5-14 Flow cytometric staining profiles of CD19/IgD mAbs

Figure 5-15 Workflow for generating ferret specific mAbs using murine single cell-RT-PCR.

Chapter 6

Figure 6-1 Thesis overview

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List of Abbreviations

ADCC Antibody dependent cellular cytotoxicity APC Antigen presenting cell ARDS Acute respiratory distress syndrome ARG Arginine BCR B-cell receptor BSA Bovine serum albumin CCL CC chemokine ligand CCR CC chemokine receptor cDNA complementary DNA CH Constant heavy domain COVID-19 Coronavirus Disease (2019) CRM1 Exportin 1 protein CXCR CXC chemokine receptor D Diversity gene DMEM Dulbecco’s modified eagle medium DMSO Dimethylsulfoxide D.P.I Days post infection DNA Deoxyribonucleic acid DC Dendritic cell ELISA Enzyme Linked Immunosorbent Assay EVD Ebola virus disease FBS Fetal bovine serum Fc Immunoglobulin constant region GC Geminal centre GTP Guanosine Triphosphate Glu Glutamine GLY Glycine HA Haemagglutinin HAI Haemagglutinin inhibition assay HIC High income country HIS Histidine

HMPV Human metapneumovirus HRSV Human Respiratory Syncytial virus IAV Influenza virus A IFN Interferon IG Immunoglobulin IGH Immunoglobulin heavy locus IGK Immunoglobulin kappa locus IGL Immunoglobulin lambda locus Ile Isoleucine IL Interleukin IRF Interferon Regulatory Factor ISG Interferon stimulated gene ISGF Interferon stimulated gene factor IVIG Intravenous immunoglobulin J Joining gene JAK-STAT Janus kinase – signal transucer and activator of transcription protein family LMIC Low-and-middle income country M1 Influenza Matrix protein M2 Influenza ion channel protein mAb Monoclonal antibody MDCK Mardin-Dharby Canine Kidney cells MHC Major Histocompatibility Complex mRNA messenger RNA NA Neuraminidase NAI Neuraminidase inhibition assay NF-B Nuclear factor kappa-light-chain enhancer of activated B-cells NGS Next-generation sequencing NHP Non-human primate NP Nucleoprotein NP-40 Nonidet-40 NS Influenza non-structural protein

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OAS Oligoadenylate synthase PA/PB Influenza polymerase acidic/basic subunit PAMP Pathogen associated molecular patterns PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline RT-PCR Reverse-transcription-polymerase chain reaction PRR Pattern recognition receptors PSG Penicillin-Streptomycin-Glutamine QIV Quadrivalent Inactivated influenza vaccine RAG Recombination activated gene RdRp RNA dependent RNA polymerase RPMI Rosewell park memorial institute medium SARs Servere acute respiratory syndrome SDS-PAGE Sodium dodecyl sulphate- polyacrylamide gel electrophoresis SHM Somatic hypermutation SNP Single nucleotide polymorphism ssRNA Single stranded RNA TAE Tris-Acetate-EDTA buffer TCR T-cell receptor TdT Terminal deoxynucleotidyl transferase TIV Trivalent inactivate influenza vaccine TLR Toll like receptors TRIM Tripartite motif-containing 22 Tyr Tyrosine U Units (Enzyme) V Variable gene vRNP Viral ribonucleoprotein complex RNA Ribonucleic acid vRNA Viral RNA

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Specific aims of thesis

Aim 1

To annotate and derive ferret immunoglobulin specific primers and plasmids to facilitate recovery and expression of ferret mAbs

Aim 2

To recover HA-specific ferret B-cell sequences for repertoire analyses and recover ferret HA-specific mAbs for HA epitope mapping studies

Aim 3

To identify murine mAbs suitable for resolving ferret B and NK cells on flow cytometry using recombinant ferret antigens.

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Chapter 1

Literature Review

1.1 Global Burden of Influenza

Acute respiratory disease is an important condition contributing to the global burden of respiratory disease and the leading cause of mortality in developing countries [1]. A potential complication leading to death in such cases is acute respiratory distress syndrome (ARDS). This condition is characterised by pulmonary inflammation and oedema which may eventually lead to hypoxemia. An important disease that has been reported to lead to ARDS is influenza, where the overall incidence is estimated at 2.7 cases per 100000 person-years and accounts for 4% of all hospitalizations [2]. Risk factors predisposing influenza-infected patients to ARDS include age (<5 years old)

[3], as well as individuals with pre-existing conditions such as pneumonia [4], congenital heart disease [3] and a compromised immune system [3].

Influenza is one of the top 10 leading causes of mortality worldwide, with 14.3 deaths per 100000 U.S. standard population alone [5]. Since the 1918 H1N1 Spanish influenza pandemic which caused 50 - 100 million deaths, there have been three other major outbreaks in 1957 (H2N2), 1968 (H3N2) and 2009 (H1N1pdm). These strains that orginally caused widespread pandemics can subsequently circulate in the human population in between major pandemics Since the re-emergence of H1N1 viruses in

1977 [6, 7], H1 and H3 viruses have been circulating in humans as seasonal influenza, with H2 strains disappearing shortly after the emergence of H3 strains in 1968 likely due to reassortment with avian-like H3 HA [8]. Such seasonal strains also cause a

1 significant burden all year round, especially in developing countries. A meta-analysis of low and middle-income countries (LMIC) and high-income countries (HIC) showed that children (<5 years old) in an LMIC were at an increased risk of severe disease and neurological complications [9] than individuals from HIC. Despite broad availability of seasonal influenza vaccines, the economic burden on healthcare systems and society is significant, even in developed countries. A recent study in the U.S. estimates the average annual total economic burden to be about USD 11.2 billion, with 20.1 million days of productivity lost [10] despite the presence of vaccination efforts.

A key concern surrounding the development of effective vaccines against influenza is the high variability of Haemagglutinin (HA). Vaccination induces protective neutralising antibody responses against four defined antigenic sites surrounding the HA1 receptor- binding domain (Sa, Sb, Ca and Cb) [11], but these sites are prone to antigenic drift due to the absence of proofreading activities of viral RNA polymerases [12]. Individuals must be re-vaccinated yearly to be protected from currently circulating strains.

Seasonal influenza vaccine efficacy is highly variable and ranges from 10% (2004-

2005) to 60% (2010-2011). This can be in part attributed to (a) mismatches between the chosen vaccine strain and subsequent circulating strains, and (b) vaccine strains, even when they are reasonably well matched to circulating strains, can acquire adaptive mutations through passage in eggs that reduce immunogenicity ot circulating strains [13-15]. There is also significant variation in individuals’ responses to the vaccines even when the match appears good and is dependent on factors such as age, high-risk health conditions and immune history [16]. Egg-based vaccine production is also insufficient to meet demands during pandemic outbreaks. Cell- culture based vaccines such as Flucelvax has been introduced and approved for use

2 in humans [17]. This approach involves the use of cell culture systems such as Madin-

Darby Canine Kidney (MDCK) cells and provides more flexible production timelines as culture conditions can be adapted to produce high yields [18] as compared to egg- based vaccines which can easily take six months or more. This will also enable the generation of H3N2 vaccine candidates, of which 90% are reported not recoverable in eggs [19]. Yet viruses passaged in vitro have also been recently shown to acquire mutations in surface proteins HA and NA [20] and require careful monitoring and validation before implementation.

Another concern with current seasonal influenza vaccines is the lack in the breadth of protection against all possible strains of influenza. Influenza vaccines need to be updated annually due to the propensity of protective epitopes on HA1 to undergo amino acid substitutions [21] which abrogates protective antibody responses generated against the precedent antigen. To overcome this challenge, new strategies aredeveloped to direct immune responses against the more conserved HA2 domain

[22]. However, due to HA1 immunodominance [23], it is difficult to mount immune responses against HA2. Since there are currently no vaccines that provide a good breadth of protection against different influenza strains, and there is an urgent need for a universal influenza vaccine to provide broad and long-term protection, especially against emerging pandemic strains.

Most studies conducted to evaluate vaccines are performed in influenza-naïve rodents, which do not represent multiple influenza virus infections encountered by humans due to their relatively shorter life spans [24]. Clinically relevant strains are also difficult to evaluate in rodent models due to the need for prior adaptation, which may

3 alter characteristics of the virus [25]. Improving our understanding of immune responses to influenza in animal models with longer life- spans and the ability to be infected directly with clinically relevant strains such as ferrets will provide valuable insights into the disease which will guide us in the design of improved vaccines and therapeutics.

1.2 Influenza virus

1.2.1 Virus structure and replication

Influenza viruses belong to the Orthomyxoviridae family of negative-sense ssRNA enveloped viruses with a segmented genome. This family of viruses is comprised of four genera (Influenzavirus A, B, C & D), divided based on the total number of genomic

RNA segments [26]. Viruses with eight segments (A & B) are further classified based on differences in their nucleoprotein (NP) and matrix (M) protein [26]. Out of these genera, only three (A, B and C) are clinically relevant to humans.

1.2.2 Gross morphology and genomic organisation of Influenza Viruses

Influenza Viruses A and B are morphologically indistinguishable under the electron microscope, existing as spheres (100nm diameter) or filaments (300nm in length) [27].

HA and NA proteins project from the host-derived lipid envelope, with a comparatively lower proportion of transmembrane (M2) ion channels [28] (Figure 1-1). Underlying the envelope is the matrix (M1) protein which encloses the viral particle. Within the viral particle, internal proteins include the nuclear export protein/non-structural protein

2 (NS2) and the ribonucleoprotein complex (vRNP). The viral genome is associated with nucleoprotein (NP) and heterotrimeric RNA-dependent RNA polymerase consisting of two basic (PB1, PB2) and one acidic polymerase subunit (PA). Influenza

C viruses, on the other hand, form cordlike structures (up to 500um in length) and are 4 compositionally similar as described for influenza viruses A and B with the exception of haemagglutinin-esterase-fusion (HEF) protein (rather than HA and NA) and a minor envelope protein (CM2).

As for genome structure, both influenza viruses A and B are comprised of eight negative-sense single-stranded viral RNA (vRNA) segments, each arranged in a double helical hairpin structure [29], while influenza C virus is comprised of seven segments [30]. The segments are numbered in the order of decreasing length (1-8).

While segments 1 (PB2), 3 (PA), 4 (HA) and 5 (NP) encode a single protein per segment, segments 2 (PB1, PB1-F2), 6 (NA, NB, M2), 7 (AM1, AM2, BM1, BM2) and

8 (NS1, NS2) may encode more than one depending on the strain/type of the virus.

For example, all influenza viruses express PB1 on segment two but some strains of influenza A express an additional pro-apoptotic PB1-F2 protein [31]. Segment 6 codes for NA for both influenza A and B, with an additional matrix protein NB, expressed in an alternate reading frame for influenza B [32], corresponding to the M2 protein in influenza A. Segment 7 codes for M1 protein in both influenza A and B, with influenza

A expressing AM2 (influenza A M2 ion channel) by RNA splicing [33] and B encoding

BM2 (influenza B M2 ion channel) membrane protein in an alternate reading frame

[34, 35]. Segment 8, which is absent in influenza C codes for NS1 which an interferon antagonist as well as NS2, which is a splice variant, and involved in the nuclear of export vRNP [36, 37].

Figure 1-1 Structure of influenza virus. Influenza viruses are enveloped, with three proteins (HA, NA and M2) found on the surface of the viral particle. M1 forms a coat inside the viral envelope. Viral RNA is organised in association with NP and polymerase complex (PA, PB1 & PB2) as vRNP complexes. Note. Adapted from “Influenza A virus cell entry, replication, virion assembly and movement.” By Dou D et. al. Frontiers Immunology. July 2018. CC-BY 4.0.

1.2.3 Influenza types

Influenza A has a broad host range, including humans, pigs, horses, seals, dogs, mink

[38, 39] and ferrets [40]. This group can be classified into 18 HA and 11 NA subtypes of which 16 HA and 9 NA subtypes are known to circulate in wild aquatic birds [41,

42], the natural reservoir of most influenza A viruses. The remaining subtypes were recently isolated from bats [43-45]. Despite a large number of possible combinations, only three HA subtypes (H1, H2 and H3) and two NA subtypes (N1, N2) [46] have caused widespread disease in humans and responsible for all four major pandemics that occurred in the last century, including H1N1 (1918; H1N1pdm - 2009), H2N2

(1957) and H3N2 (1968). Of the four, H1N1 (1977) and H3N2 (replaced H2 viruses) are currently endemic in the human population. The absence of nucleotide sequence evolution indicates that the re-emergence of H1N1 in 1977 might have been due to accidental release of a laboratory strain [7]. Unfortunately, the virus had been circulating for about one year prior to detection [6], making the determination of the geographical source of introduction difficult. Influenza viruses circulate for 12 – 42 years (excluding man-made events), making it hard to predict when the next outbreak might occur; There is a need broadly protective vaccines to protect against such emerging strains of influenza viruses.

In contrast to Influenza A, Influenza B primarily infects only humans and generally do not cause pandemics due to the lack of a zoonotic reservoir, except for seals [47].

Circulating strains in humans are divided into two lineages, Victoria-like and

Yamagata-like [48], based on differences in HA gene sequences. Most studies are focused on Influenza A due to the belief that Influenza B does not pose a serious threat to public health, but there is an increasing number of studies indicating that the burden of influenza B is significant. Hospitalisations associated with influenza B was reported

7 to be 81.4 per 100000, halfway between the rates for seasonal H1N1 (55.9 per

100000) and H3N2 (99.0 per 100000) respectively [49]. Influenza B is also known to cause severe disease [50] during seasonal infections despite limited host range and lower mutation rates than influenza A [51]. Influenza C is not a major concern as antigenic drift characterised by successive variants have not been detected [52], and generally causes mild to no disease symptoms except for children (<6 years of age)

[53].

1.2.4 Viral replication cycle

The replication cycle of influenza viruses can be divided into the following: Entry into the host cell, transport of RNP complexes into the nucleus, expression and replication of the viral genome, the export of vRNP/proteins from the nucleus, virus assembly and budding at the host cell plasma membrane (Figure 1-2).

Figure 1-2 Replication cycle of influenza. Influenza infection begins by viral attachment to host sialic acid via HA. Acidification of endosomes leads to membrane fusion and the release of viral genetic material. Replication and transcription occur via host machinery. Nascent viral particles are subsequently packaged and released from host cell in a NA dependent manner. Note: Adapted with permission from “Recent advances in neuraminidase inhibitor development as anti-influenza drugs”, by Feng, E et., al. ChemMedChem. 2012. 7(9): 1527-36. CC-BY 4.0.

The key protein involved in viral entry into the host cell is HA, a homotrimeric protein found on the viral lipid membrane which recognises sialic acid residues found on the surface of the host cell membrane [54]. HA is synthesized in the endoplasmic reticulum as a precursor protein (HA0), which is cleaved into two functionally distinct subunits,

HA1 and HA2 which is required for viral infectivity [55]. HA1 contains the receptor- binding domain while HA2 contains the fusion peptide, important for viral fusion into host membranes. There are two different isoforms of sialic acids that are recognised by influenza viruses, α-2,3 or α-2,6 sialic acid receptors [54]. The distribution of these sialic acids found on membrane surfaces of the respiratory system differ between 9 species and determines the tropism of influenza viruses across host species. For example, viruses infecting humans [56] and ferrets [40] recognise α-2,6 linked sialic acids, whereas mouse and avian strains recognise the α -2,3 isomer [57]. Swine express both forms of sialic acids and was hypothesised as a mixing vessel for historical pandemic influenza viruses strains prior to 2009. Subsequent analysis[58] of the 2009 pdm H1N1 virus provided more conclusive evidence of swine origins of the virus.

Upon ligation to the host cell receptor, the viral particle undergoes receptor-mediated endocytosis, where viral particles are taken up into the cell in endosomes. The low pH of the endosome (pH 5-6) triggers the fusion of viral and host membranes via a conformational change in HA2, resulting in the exposure of the fusion peptide [59].

This peptide inserts into the endosome and brings both viral and endosomal membranes into proximity [59]. Another important event is the opening of the M2 ion channel, which enables the entry of hydrogen ions and consequent acidification of the interior of the viral core [60, 61]. The acidification of the viral core triggers the release of vRNP, which enters the cytoplasm. The presence of nuclear localisation signals on the proteins NP [62], PA1 [63], PB1 [63] and PB2 [63], which makes up the vRNP enables the transport of viral genetic material into the nucleus, where transcription and replication of the viral genome occurs.

To replicate viral negative sense RNAs, RNA dependent RNA polymerase (RdRp) initiates positive sense RNA synthesis via primary transcription [64]. Positive sense

RNA are used as templates to generate more copies of negative-sense genomic RNA.

Host cell machinery is also utilised to produce viral proteins via the translation of viral

10 proteins encoded by the positive-sense RNA. Viral mRNAs have a poly(A) tail and the

5’ methylated cap, which is important for translation initiation. The 5’ cap is acquired via a “cap-snatching” mechanism, mediated by PB2 [65]. Endonuclease activity of PB2 cleaves host mRNA about 10 to 15 nucleotides 3’ to the cap structure and is utilised to initiate viral transcription for viral protein expression. The poly(A) tail is encoded by a poly(U) track found at the 5’- end of the vRNA [66].

Following which, newly synthesized negative sense vRNPs are exported from the nucleus via GTP (Guanosine triphosphate) dependent CRM1 (Exportin 1) dependent pathway via NP [63]. Nascent viral particles bud from the host cell, acquiring the host cell membrane in the process. The selective packaging model has been proposed to incorporate the eight distinct RNPs [29]. Signals that are important for the incorporation of vRNAs have been identified on 5’ and 3’ ends of all genomic segments, but the mechanism is still not fully elucidated [67]. Finally, to leave the plasma membrane, NA cleaves and removes sialic acid residues from host glycoprotein and glycolipids [68]. These nascent particles will then initiate another cycle of infection in the host.

1.3 Antigenic drift and shift of influenza viruses

1.3.1 Antigenic drift

Circulating influenza viruses may undergo two different types of genetic changes, antigenic drift or antigenic shift [69]. Antigenic drift refers to point mutations occurring in antigenic regions of proteins such HA, enabling the escape from host immune responses primed by the precedent vaccine or circulating strains [70]. This leads to localised outbreaks of varying extents which is dependent on multiple factors such as

11 virus transmissibility and susceptibility of the population. Assessment of antigenic drift of influenza is important as it informs vaccine design. A recent retrospective study in ferrets identified genetic variants which diverged from vaccine strains [71], highlighting the need for careful surveillance to inform timely influenza vaccine updates.

1.3.2 Antigenic shift

Antigenic shift, on the other hand, is caused by the generation of novel influenza strains via reassortment of the segmented genome in hosts such as swine [72]. This process is believed to be responsible for widespread epidemics and pandemics due to the lack of immunity to such novel viral strains. There have been five major pandemics that occurred, in 1918 (Spanish influenza), 1957 (H2N2), 1968 (H3N2),

1977 (H1N1) and 2009 (pdmH1N1) [73]. The most severe and extensive pandemic was in 1918 where there were 50 million deaths globally, with most occurring in healthy adults aged 15 to 34 years; This has not occurred for any subsequent influenza A pandemic or epidemic [74]. Efforts are currently underway to develop therapeutics and prophylactics with a broader spectrum of protection against influenza viruses to increase pandemic preparedness [75].

1.4 Anti-influenza drugs and vaccines

1.4.1 M2 Ion channel inhibitors

Currently, there are two main anti-influenza drugs amantadine and rimantadine targeting M2 protein of influenza A but not influenza B [76]. These two compounds are structural analogues, sharing the same mechanism of action via by either displacing water or allosterically causing conformational changes that result in a change from an opened to closed state on influenza A M2 protein [77]. While both have similar efficacy

12 and effectiveness in relieving or treating influenza A symptoms, the likelihood of adverse, i.e. central nervous system side effects is lower in rimantadine [78]. Since

2003-2004, however, substantial rates of resistance are now present in both H1N1 and H3N2 strains [79]. Mutations resulting in resistance against both strains have also been identified and occur at several positions in the transmembrane domain of the protein lining the pore, with a mutation at position 31 (Ser31Asn) being the most common [80]. Crystal structures of this mutant revealed that this mutation directly disrupts the drug binding site by forming a hydrogen-bonded network with other proximal residues from Gly34 to His 37 [80].

1.4.2 NA inhibitors

The only licensed anti-influenza antivirals against NA are Oseltamivir (Tamiflu) [81],

Zanamivir (Relenza) [82] and Peramivir (Rapivab) [83] and inhibits sialic acid cleavage by blocking the active site of NA. These antivirals are limited by the variable nature of the influenza virus. By 2008-2009, seasonal H1N1 viruses have begun to develop resistance to Oseltamivir, which was associated with His275Tyr mutations [84]. In another study where immunocompromised patients used both Oseltamivir and

Zanamivir sequentially, other mutations occurred, such as Ile223Arg and Glu119Gly, suggesting that antiviral treatment in patients with prolonged influenza can lead to multidrug resistance [85], although resistance against zanamivir by itself is rarely reported [86]. Peramivir was recently licensed in 2014 and used for the treatment of uncomplicated acute influenza in patients >18 years of age. Detailed comparative studies have shown comparable or superior NAI activity of Peramivir as compared to

Oseltamivir or Zanamivir, and improved responses to influenza B [87]. While useful to treat severe influenza, these antivirals have limited clinical efficacies such as the need

13 for administration within the first 48 hours of infection and side effects ranging from headache, nausea and vomiting to central nervous effects [88].

1.4.3 Need for broadly protective influenza vaccines

There are currently two main influenza vaccine approaches that are licensed for use in humans, either by whole virus (inactivated) or recombinant HA. Live attenuated vaccines were previously available but were subsequently found to be less effective in 2016 [89]. Whole virus vaccines are produced in eggs or cell culture, and may include adjuvants such as MF-59 to improve immune responses [90]. Recombinant

HA vaccines such as FluBlok [91] offers comparatively higher HA content as compared to whole virus vaccines, providing improved immunogencity. These approaches,however, are limited to particular strains of influenza viruses as they only confer protective immunity when there is a near identical antigenic match between vaccine and circulating strains. Antigenic variation due to drifts or shifts in both HA and

NA are responsible for epidemic and pandemic outbreaks, and there is an urgent unmet need for universal influenza vaccines conferring protection against a wider spectrum of influenza strains.

Since HA1, which is the main target for seasonal vaccines, is prone to point mutations due to antigenic drift, vaccines must be updated annually to provide protection. The focus was shifted to the HA2 domain, which is conserved across H1 and H2 influenza

A subtypes [92]. Due to the immunodominance of HA1 over HA2, different vaccination strategies have been examined to increase responses against the HA2 domain to increase the breadth of protection conferred by vaccinations. This idea first arose from observations showing that infection or vaccination of pandemic H1N1 viruses in humans elicited a boost in stem specific antibodies [93]. Mouse studies utilising

14 recombinant chimeric HA constructs with varying HA1 domains and the same HA2 domain in sequential infections/vaccinations also showed an increased response to the HA2 domain [94]. Similar observations were noted in ferrets, and protection against pandemic H1N1 was achieved utilising similar vaccination strategies [95]. For example, sequential vaccination with LAIV with an H8 head domain/H1 stem domain chimaera and split-inactivated vaccines using an H5 head domain/H1 stem domain

[96] chimaera elicited cross-reactive antibody responses. Headless H5 stem-based immunogens have also been explored and shown to elicit cross-reactive antibody responses which protected mice from morbidity and mortality following a lethal challenge [97].

More recently, studies are showing novel functions of anti-HA stem antibodies which sterically inhibit NA activity via the Fc region, thereby preventing the release of newly formed virus particles. The first evidence was the identification of mutations in both

HA and NA, which enabled viral escape from broadly neutralising stem antibodies [98].

Infection of mice with viruses expressing a short-stalked NA and concomitant administration of anti-stem antibodies have shown to lead to attenuated disease such as increased survival rates and reduced body weight loss as compared to mice infected with viruses with longer NA stalks, suggesting the extended NA stalk prevented Fc mediated steric inhibition of NA [99].

1.4.4 Future therapeutic agents for influenza

To improve clinical outcomes of influenza disease, other drug candidates and intervention strategies are currently being explored. For example, combination therapy of ferrets with Nitaoxanide and Oseltamivir was examined and has shown to be more

15 effective than Oseltamivir monotherapy. [100]. Pulmonary surfactant lipids [101] and other novel drug candidates such as EIDD-1931 [102] have also been explored as an alternative, and have shown to be promising in humans, mice and ferrets. EIDD-1931 acts as a cytidine analog, leading to lethal viral mutagenesis in both influenza A and

B in sub-micromolar range. Efficacy of drived pro-drugs such as EIDD-2801 demonstrated low toxicity and potent efficacy showing the promise of this family of drugs for universal protection against influenza viruses [103].

Recently, NA inhibition antibodies are increasingly studied as it has been shown to contribute to protection as broadly reactive NA specific mAbs have been reported to exhibit cross-reactivity and protects against lethal infection in mice in vivo [104].

Infections readily induce high proportions of NA-reactive B-cells but not vaccinations, suggesting that optimisation of vaccination regimes are required to induce NA specific antibodies. Other conserved targets such as NP have also been investigated as candidates for universal influenza vaccines; cross-reactive T-epitopes on conserved proteins such as NS1 and PB1 have been identified and protects against influenza A,

B and C [105].

1.5 Immunity to influenza

1.5.1 Innate immune responses

Innate immunity forms the first line of defence against influenza and comprises of physical barriers (epithelium), chemical components (opsonins and complement) and the recognition of pathogen associate molecular patterns (PAMPs), thought to be 5’- triphosphate groups found on viral RNA via pattern recognition receptors (PRRs)

[106]. Activation of these receptors found on innate immune cells such as

16 macrophages and dendritic cells results in cellular signalling cascades leading to the expression of effector proteins which initiates inflammation and other anti-pathogen responses.

Toll-like receptors (TLRs) are the most extensively studied PRRs. TLR 3, 7, 8 and 9 are expressed on endosomes/lysosomes and recognises nucleic acids derived from influenza [107]. Other families of cytoplasmic PRRs such as RIG-I, NOD-like receptor proteins and melanoma differentiation-associated gene 5 recognises cytoplasmic ssRNA and transcriptional intermediates [108]. Upon receptor activation, downstream transcription factors such as NF-B [109], IRF3 and IRF7 leads to the localisation of these factors into the nucleus, where coding genes for interferons and inflammatory cytokines are transcribed and expressed [110].

Key molecules involved in innate responses against influenza include type I IFNs such as IFN and IFN [111], as well as type III IFNs such as IFN- [112]. Interaction of interferons and interferon receptors leads to the activation of the JAK-STAT pathway

[113]. This causes the formation of ISGF-3 and the subsequent transcription of ISGs

[114]. ISGs target various stages of the IAV life cycle, including viral entry, viral-host cell fusion, mRNA expression/ translocation and viral release. For example, TRIM22 targets the viral nucleoprotein for degradation [115], while Viperin restricts the release of influenza viruses by perturbing lipid rafts that makeup budding sites [116]. Influenza viruses have evolved evasion strategies to circumvent innate immune responses. The key protein involved is NS1, which is a multifunctional antagonist of innate host immunity and inhibits innate responses by limiting RIG-I signalling and IFN production

[117].

NK cells, monocytes/Macrophage and dendritic cells are key innate immune cells that help facilitate control of influenza via a variety of mechanisms. One common function these cells perform is cytokine/chemokine secretion [118], which plays important roles in immune recruitment and modulation of immune responses. Key functions of dendritic cells and monocytes/macrophages include phagocytosis [119] and antigen presentation to the adaptive immune system [120]. Antibody-dependent cellular cytotoxicity (ADCC) also play an important role bridging in the innate and adaptive immune responses protection against influenza viruses. The key cell type involved in such responses is NK-cells, whose activity is regulated by the balance of activating and inhibitory receptor signals [110]. NK cells are lymphocytes commonly defined by cell surface marker NKp46 (NCR1), which is an activation receptor [121]. Infected host cells downregulate inhibitory cell surface MHC-I molecules, leading to NK-cell mediated apoptosis of target cells via mechanisms such as degranulation of granzymes and perforin that are enhanced by type I interferons [122]. Degranulation of cytotoxic cells such as NK cells and CD8 T cells is commonly studied by the expression or LAMP-1 (CD107a) [123].

Opsonised pathogens can also be targeted for direct killing via ADCC mediated mainly by FcgR receptors CD16/32. This ADCC responses has have been widely studied for heterotypic protection against different influenza strains due to the ability of antibodies to conserved parts of external proteins (such as HA stem and M2e) as well internal proteins such as NP and M1 [124] to activate NK-cells. The study of NK cells and

ADCC responses to influenza, including degranulation, has not been widely studied outside of humans, non-human primate and mouse models owing to the limited

18 availability of immunologic markers for these cells and responses in models such as the ferret. This issue is explored further in chapter 5 of this thesis.

1.5.2 Adaptive immune responses

1.5.2.1 B and T-cells are important for protection against influenza

Both B and T cells form the humoral and cellular arms of the adaptive immune system and confer specific and sustained (memory) protection against influenza. Activated lung DCs [125] which have encountered pathogen upregulate chemokine receptor

CCR7. This leads to their migration toward local draining lymph nodes via CCL19 and

CCL21 chemokine gradients and the presentation of influenza-derived peptides via cell-surface MHC-II or MHC-I-peptide-complexes. TCR recognition of the respective

MHC-peptide complexes results in the activation of CD4+ cells and CD8+ cells respectively. The activated T-cells exert anti-influenza responses via discrete mechanisms such as cytokine production (CD4+TH1 and CD8+). IFN- secretion enhances phagocytic clearance of viruses and cytotoxic lysis of infected cells (CD8+).

T-cells also promote antigen-specific B-cell/antibody responses (CD4+TFH) [126].

Antibodies (B-cells) promote clearance of influenza viruses by neutralisation, complement activation and opsonisation which results in antibody-mediated virus clearance via phagocytosis or ADCC.

1.5.2.2 Somatic recombination of immunoglobulin genes results in highly diverse antibody/BCR repertories

A key characteristic of adaptive immune responses is the ability to recognise and mount specific responses against a wide variety of pathogens. B-cells have evolved to generate diverse antibody repertoires (>1012) via somatic recombination of the

19 constitutive variable, diversity, joining and constant gene segments which are in germline configuration [127].

Immune cell development begins in the bone marrow where there are signals in the microenvironment that drive the differentiation of haematopoietic stem cells into different immune cell types. These cells first differentiate into multipotent progenitor cells (MPPs) which express surface FLT3 [128]. Signalling through FLT3 via interactions with FLT3 ligand on stromal cells upregulates IL-7 receptor expression, which is important for cell maintenance and survival [128]. This signal also drives differentiation into common lymphoid progenitor (CLP) (Figure 1-3 A) which gives rise to the earliest B-lineage cell, the pro-B-cell (Figure 1-3 B). Pro-B-cells are defined by transcription factors E2A, which are alternatively spliced into E12, E47 and early B- cell factor (EBF) [129]. These factors are important for the induction of key proteins involved in somatic recombination of antibody coding genes, such as RAG-1 and

RAG-2.

Early Pro-B-cells begin to rearrange immunoglobulin gene segments which are still in the germline configuration, as found in all non-B-cells. The heavy chain first rearranges, where D and JH segments are joined, followed by VH to DJH rearrangement in late pro-B-cells [130]. While the D to JH rearrangements occur on both alleles, VH to DJH rearrangements only occurs first on one chromosome, if productive [131]. This is known as allelic exclusion and ensures that only one type of receptor is expressed on each B-cell. Successful rearrangements at this stage lead to the expression of a functional mu- heavy chain. Otherwise, rearrangements will occur

20 on the other chromosome, and pro-B-cells that do not produce a functional mu-chain are eliminated at this stage.

Successful VHDJH rearrangements lead to the expression of the mu-chain (sIgM) pre-

B-cell receptor, which is associated with an invariant surrogate light chain (VpreB/5) induced by E2A and EBF [132]. This acts as the first checkpoint for the cell to proceed to the next stage of development known as the pre-B-cell stage (Figure 1-3 C).

Signalling via the pre-B-cell receptor halts rearrangement of heavy chain genes and leads to the proliferation and transition of the pro-B-cell into large pre-B-cell [133].

Signalling via this receptor also ensures allelic exclusion is enforced; Only one of the two alleles of both heavy and light chain genes are expressed per B-cell. This is promoted in three ways: Directly reducing the expression of RAG-1 and RAG-2 activities [134], indirectly increasing the degradation of RAG-2 via phosphorylation

[135] and the epigenetic reduction of access of the immunoglobulin loci to the recombinase machinery [136].

Several rounds of division of large pre-B cells lead to the increase in the population of cells with successful in-frame joins known as small pre-B-cells [137]. At this stage, there are pre-B cells with identical heavy chains which will independently rearrange the light chain locus. This takes place at only one allele at a time; Repeated rearrangements of V and J segments can occur on one chromosome before initiation of rearrangement on the other chromosome, increasing the chances of generating functional light chain. In addition to allelic exclusion, light chains on also display isotypic exclusion, and only expresses either kappa or lambda light chain [138].

Pre-B-cells with both functional rearranged heavy surface IgM (sIgM) and light chains transition into immature B-cells and undergoes negative selection to eliminate, inactivate or rescue autoreactive B-cells [137]. This is mediated by surface B-cell receptor complexes, where sIgM is associated with co-receptors Ig and Ig. The resulting B-cells enter peripheral circulation as mature naïve B-cells (Figure 1-3 D), expressing both IgD and IgM from alternatively spliced heavy chain gene transcripts

[139].

Figure 1-3. Developmental stages of B-cells. Key events at each developmental stage are highlighted. (A) Common lymphoid progenitor. (B) Pro-B-cell. (C) Pre-B-cell stage. (D) Mature-naïve B- cell stage.

These processes as described generate antibody diversity to enable the recognition of a large plethora of pathogens by (1) Combinatorial use of different germline immunoglobulin gene segments during rearrangement, and (2) Different immunoglobulin heavy and light chain combinations as a result of independent rearrangement of light chain genes in pre-B-cells. These processes as described

22 generate antibody diversity that enable the recognition of a large plethora of pathogens through: (1) Combinatorial use of different germline immunoglobulin gene segments during rearrangement, and (2) Different immunoglobulin heavy and light chain combinations as a result of independent rearrangement of light chain genes in pre-B- cells. The addition of both palindromic (P) and non-templated (N) nucleotides as a result of the action of RAG1/RAG2/Artemis and Terminal Deoxynucleotidyl

Transferase (TdT) during the recombination process further increases junctional diversity of the repertoire of antibodies generated respectively. Although of great interest, it is currently difficult to definitively identify N nucleotides as such nucleotides are added randomly. While palindromic sequences representing potential P nucleotides can be identified, such nucleotides are likely inserted during the process of N nucleotide addition [140]. In addition, previous statistical studies have shown that

P nucleotides contribute to diversity of less than 1% of sequences and attempts to identify will likely introduce errors into the analysis [140].

1.5.2.3 Early B-cell responses against influenza

During the initial stages of influenza infection, IgM+ polyreactive natural antibodies secreted by CD5+ B-1 cells found in pleural and peritoneal cavities provide early protection [141]. These antibodies are secreted constitutively in the absence of infections and can be transported to mucosal surfaces of the respiratory tract via poly

Ig-receptors [142]. These antibodies directly neutralise or lyse infected cells by complement fixation [143]. The levels and affinities of these mAbs are generally low

[144] and subsequent influenza-specific B-cell responses are often required to overcome infection.

1.5.2.4 Somatic hypermutation and high-affinity B-cell responses against influenza

The key hallmark of antigen-specific B-cell responses is the gradual increase in antibody affinities over time, which is driven by activation-induced cytidine deaminase

(AID) somatic hypermutation (SHM) of antigen-binding regions of immunoglobulin genes during GC reactions. This process culminates in the emergence of high-affinity antibody-secreting plasma cells and memory cells. Such responses involve IgD+/IgM+ mature naïve B-2 cells and begin by the recognition of antigens, which are soluble or attached to the surface of other cells including macrophages and dendritic cells via cell surface BCR receptors [145]. Upon activation, B-cells present antigen via MHC-

II-peptide complex and upregulates CCR7 which leads to the migration to B-cell-T-cell boundary zones found within secondary lymphoid organs along via chemokine gradients (CC19 and CCL21) [146]. Subsequent TCR recognition of MHC-II-peptide complexes by cognate TFH provides co-stimulatory signals such as CD40-CD40L and promotes the proliferation and migration of antigen-specific B-cells toward extrafollicular areas as short-lived plasma cells, memory cells or differentiation into

GC B-cells (Figure 1-4) [147, 148]. Short-lived plasma cells secrete immediate antigen-specific antibodies providing early protection against influenza while slower

Geminal Centre (GC) responses occur [149].

Figure 1-4 Early B-2-cell responses against influenza. After initial activation, B-cells either differentiate into early short lived memory or plasma cells to provide initial protection while B-cells destined for geminal centres undergo TFH dependent geminal centre reactions to generate high affinity antibodies.

GCs are organised into two zones which segregate B-cells undergoing proliferation/SHM (Dark zone) and selection (Light zone). The polarity is maintained by the secretion of CXCL12 by stromal cells in the dark zone and CXCL13 by follicular dendritic cells in the light zone [150] (Figure 1-5 A). Within the dark zone, the proliferation of CXCR4 expressing centroblasts occur, and the generation of DNA mismatches by AID in the variable portions of the immunoglobulin genes initiates error- prone repair [151] (Figure 1-5 B). This occurs via the deamination of cytosine residues yielding uracil which leads to a DNA mismatch, activating error prone repair pathways including base excision and mismatch repair pathways [152].This results in the induction of random point mutations and a diversity of B-cells with different BCR affinities. Following which, CXCR4 is downregulated, and CXCR5 is upregulated, enabling the migration of these cells into the light zone to receive positive selection signals [150] such as CD40-CD40L from cognate TFH cells (Figure 1-5 C). These B-

25 cells are in an activated state of apoptosis [153] and compete for survival signals provided by TFH cells, resulting in the survival of clones with the highest BCR affinities.

Subsequently, these cells with higher affinity BCRs either re-enter GCs [154] or differentiate into effector subsets such as plasma or memory cells (Figure 1-5 D) [155].

Memory-B cells provide quick and rapid recall responses to previously encountered pathogens as no innate stimulation is needed [156], while plasma cells are important for the secretion of antibodies. Some subsets of plasma cells found in the bone marrow are long-lived [157], and important for the maintenance of long lived antibody based protection against pathogens.

AID also results in class-switch recombination of immunoglobulin heavy chain constant region genes, resulting in the switch from IgM/IgD to IgA/IgG/IgE constant regions which have different effector functions [152]. IgG is the major isotype and responsible for systemic immunity against influenza, while IgA is important for mucosal protection [158].

A CXCL13

CXCL12

Light zone Dark zone

Low affinity BCRs

DCs/Macrophages

high affinity BCRs

B-cell TFH cell

D B-cell

High affinity effector B-cells (memory/plasma)

Figure 1-5 Geminal centre reaction to generate high affinity antibodies. (A) Polarity of GCs are maintained by concentration gradients of CXCL12 and CXCL13. (B) Division and somatic diversification of BCRs via AID resulting in BCR pools with varying antigen binding affinities. (C) Selection of GC-B- cell with high BCR affinities. (D) High affinity effector B-cell pool.

1.6 Technologies for generating mAbs

1.6.1 Hybridoma technology

The earliest use of antibodies to treat infectious diseases is in the early 20th century, where the passive transfer of serum from immune patients protected immunologically naïve patients from the same infection for diseases such as diphtheria [159]. This was gradually replaced by antibodies purified from pooled sera, known as IVIG [160]. No significant progress was made in the generation of antibodies until the development of hybridoma technology to generate monoclonal antibodies in 1975 by Kohler and

Milstein [161]. Monoclonal antibodies refer to antibodies generated from a single B- cell clone targeting a specific epitope on an antigen. Isolation of mAbs using hybridomas involves the limiting dilution of fused myeloma and splenocytes derived from immunised mice and the subsequent screening of cell supernatants for antigen binding. This technique is well established and is used extensively to isolate mAbs for both therapy [162] and basic research [163]. A key disadvantage of using murine based mAbs is the generation of human anti-murine responses which decreases efficacies of therapeutic murine mAbs [164]. With the advent of recombinant mAb technology, chimeric mouse/human mAbs were eventually developed to minimise such responses [165].

1.6.2 Cell display technology generation of antibody libraries for screening

With advances in molecular biology, the use of cell display to generate protein libraries was first reported by Smith in 1985 [166]. This method has since been adapted to recover antibodies for screening [167]. This process initially involves the isolation and amplification of immunoglobulin mRNA sequences from B-cells from mice immunised with the antigen of interest. These gene segments are subsequently expressed on the

28 cell surface of bacteria, yeast or mammalian cells as displayed libraries for biopanning to select candidates with the highest affinity to target antigens [168]. The most established technology is phage display [169], where candidate antibody coding genes are cloned into bacteriophages proximal to bacteriophage coat gene (PIII). This enables the infection and expression of E.coli to generate a large library of as many as 1010 cells in the presence of helper phages for screening, though the cognate heavy and light chain pairing is lost, resulting in the need to screen candidates that do not exist in the antibody repertoire. Other emerging cell display technologies in development include mammalian cell and cell-free based display technologies [167].

1.6.3 Single-cell-recovery of mAb sequences

With a wide range of flow cytometric reagents and immune gene receptor sequences available, single-cell based recovery of immunoglobulin sequences from B-cells enables the recovery of antibody sequences from different B cell populations. This technique has been developed in humans [170] and animal models such as mice [171] and guinea pigs [172]. Recovery of antibody sequences allows characterisation of the expressed antibody repertoire for the selection of candidate mAbs for expression. This reduces labour intensiveness as compared to both hybridoma and phage display based methods by targeting rare antigen-specific B-cell populations via antigen baiting on flow cytometry [173]. Cognate heavy and light chain pairs constituting expressed antibodies of interest can be recovered and expressed for further characterisation.

Comparisons of these three techniques of mAb generation are shown in Table 1-1.

Table 1-1 Comparison of mAb generation techniques

Advantages Disadvantages

Hybridoma Low costs Efficiency of fusion is low

Readily available reagents and Only available for mouse

cell lines

Cell display Enables generation of entire Loss of cognate antibody heavy

technology antibody library for screening and light chain pairing

information

Use of microorganisms enables

large number of candidates to Can be expensive

screen

Single cell BCR Cognate heavy and light chain Variable recovery efficiencies of

sequencing antibody pairs are retained immunoglobulin transcript

sequences

Antigen specific B-cells can be

isolated to recover mAbs Can be expensive

efficiently

1.7 Animal models of human influenza infection

1.7.1 Mice

Mice (Mus musculus) are widely used in influenza research due to relatively low costs, wide availability, small size and the ease of handling/housing. In addition, many inbred models such as C57BL/6 and BALB/c mice are established and are useful for reducing the effects of genetic variation on experimental results. Many genetically modified strains such also available, which allows the study of specific effector functions in the context of influenza infections such as B-cell kinetics via modification of the mb-1 allele to be regulated via Cre and tamoxifen administration [174]. A key issue with the mouse model is the need to use mouse-adapted viruses [175], as most human variants cannot infect mice. While mouse-adapted strains such as PR8 [176],

WSN [177] are available, they do not necessarily represent circulating and clinically relevant strains of influenza virus. The clinical signs of disease in mice also differ from that in humans; Upon infection, mice typically show signs such as weight loss, hunching and fur ruffling and hypothermia rather than a febrile response [178]. The sneeze reflex is also absent in mice, leading to the need for animal models such as ferrets for transmission studies.

1.7.2 Non-Human Primates (NHP)

The close phylogenetic relationship between NHP and humans makes them useful for studying human disease due to similarities in physiology [179]. This also makes NHPs a critical animal model in translational research projects for the development of therapeutics aimed at curing or ameliorating human diseases [180], such as the use of mini stem only HA constructs to broaden influenza-specific responses [181]. Serum transfer experiments from immunised NHP to mice demonstrate that antibodies

31 induced by stem-HA provided heterosubtypic protection against influenza as compared to TIV [181]. Key limitations of this animal model, however, are high costs, increased husbandry requirements and ethical issues and the lack of statistical power during analysis due to low sample sizes.

1.7.3 Ferrets

Ferrets are widely used in influenza research due to the ability to be infected directly with human influenza strains. This was first demonstrated by the inoculation of filtered nasal washes from a human patient [182], similar to NHPs. Inoculated ferret showed similar signs as humans three d.p.i, characterised by fever, sneezing, nasal discharge and weight loss for up to 10 days. Since then, ferrets have played an important role in increasing our understanding of influenza infections. The following section (section

1.8) describes and evaluates the ferret as an important animal model for influenza and other emerging human respiratory diseases in greater detail.

1.8 Ferrets: an important animal model for influenza

1.8.1 Ferrets as an influenza pathogenesis and transmission model

Influenza infection of ferrets recapitulates key hallmarks of human clinical disease, such as an increase in body temperature by 2 – 3oC, as well as respiratory symptoms such as rhinorrhea and sternutation [183, 184]. The shared susceptibility to influenza infection is based on similarities in respiratory tract physiology, where a predominance of α2,6-linked sialic acid receptors in the upper respiratory tract of ferrets mimics that of humans [185-189], unlike α2,3 forms prevalent in other species such as mice.

Ferrets are an extremely valuable model for studies on influenza pathogenesis [190,

191] and both direct and aerosol transmission [192, 193]. For example, using viruses

32 with destabilising HA mutations, it was demonstrated that HA stability is critical for the retention of infectivity in airborne droplets in ferrets [194]. The site of viral shedding has also recently been identified on the nasal respiratory epithelium on ferrets, and further studies will enable the understanding of the airborne transmission of influenza viruses [195]. Critically, ferrets are a susceptible host for highly pathogenic avian strains of influenza with pandemic potential, such as H5N1 and H7N9, although disease severity in infected ferrets is somewhat variable [190, 196-198]. Similar variability in pathogenesis has been reported for some seasonal strains such as recent

H3N2 isolates [199, 200], which display a range of disease severity in humans [201] but generally remain mild in ferrets [197, 202].

1.8.2 Ferrets for influenza surveillance and vaccine development

Ferrets play a critical role in the annual seasonal influenza vaccine strain selection.

Antigenic drift in circulating strains is monitored primarily using HI assays on serum from ferrets infected with recently circulating human viral isolates [203, 204]. In both ferrets and humans, HI titres are a marker of protection from acquiring infection and currently the key immunological correlate for assessing potential vaccine effectiveness. Currently licensed seasonal vaccines, historically trivalent (TIV) but increasingly quadrivalent inactivated vaccines (QIV), can protect both ferrets [205,

206] and humans [207, 208] from infection and disease. Protection is mediated through neutralizing antibodies targeting a cluster of epitopes surrounding the viral receptor-binding domain (RBD) within the highly variable hemagglutinin (HA) head domain (HA1) [209, 210]. Inactivated influenza vaccines significantly reduce mortality rates in children [211, 212] and severe disease in adults [207, 213]. However, vaccine

33 protection is notoriously strain-specific, and mismatches between vaccine and circulating strains leads to low vaccine efficacy [214-216].

There are global efforts to increase the breadth of protection of influenza vaccines, with an eventual goal of universal protection [217], and most strategies have been evaluated in ferret models. A non-exhaustive list of strategies to induce heterosubtypic immunity against influenza evaluated in ferrets include: HA stem vaccination [218-

220]; prime-boost with chimeric HA-based vaccines[95]; use of conserved influenza proteins such as nucleoprotein (NP) [221-223], matrix-1 (M1) [222, 224], matrix-2 (M2)

[223, 225], and RNA polymerase subunit B1 (PB1) [222]; replication-deficient viruses

[226-229]; live-attenuated formulations [230-232]; the use of potent adjuvants such as

Protollin [233], Glucopyranosyl lipid adjuvant – aqueous formulation (GLA-AF) [234]

CoVaccine HT [235], cationic adjuvant formulation [236], Poly-g-glutamic/chitosan nanogel [237]; E.coli derived vaccines [238]; DNA, mRNA and viral vector vaccines[223, 239-241]; COBRA vaccines [242] and the use of virus-like particles

(VLP) [224]. Additional examples of important ferret studies include (but are not limited to) evaluating the influence of changing the route of influenza inoculation on subsequent immunity [243] and the use of neuraminidase (NA) inhibitors as prophylaxis [244].

1.9 Ferrets as an immunological model for viral infectious diseases

1.9.1 Ferrets as an immunological to study influenza viruses

The utility of ferrets for incisive immunological studies is hampered by limited reagents to study ferret immunity and a paucity of background knowledge about the ferret immune system. Some insights into ferrets immunological responses to influenza have

34 been gained by indirect measurements of immune gene expression such as quantitative RT-PCR (qRT-PCR), transcriptome analysis or oligonucleotide microarrays [245-250]. For example, assessing the differential expression levels of innate and adaptive immune genes in the lungs following primary or secondary 2009

H1N1pdm infection revealed upregulation of interferon-stimulated genes involved in antiviral responses such as C-X-C motif chemokine 10 (CXCL10), 2’-5’ oligoadenylate synthase 1 (OAS1), interferon regulatory factor 1 (IRF1) and radical S-adenosyl methionine domain containing 2 (RSAD2) as well as chemokines such as CXCL16,

C-C motif chemokines 3, 4 and 5 (CCL3, CCL4 and CCL5) [248]. Similarly, the degree of disease severity, virus shedding and transmission in ferrets has been associated with tumour necrosis factor (TNF) and interleukin-6 (IL-6) mRNA expression in the upper respiratory tract [246]. However, mRNA levels can correlate poorly with protein levels [251], and techniques such as flow cytometry, bead arrays and immunohistochemistry would facilitate direct measurement of immune marker expression. To date, flow cytometric or microscopy techniques have been limited in ferrets by the lack of suitable antibodies specific for ferret immune cell markers.

Screening for cross-species reactivity has identified antibody clones recognising ferret

T-cell markers such as CD3 and CD8, and an intracellular B-cell marker CD79β [252-

254] (summarized in Table 1-2). The utility of such cross-reactive reagents has been shown experimentally. For example, prime-boost immunisation using DNA and adenoviral based influenza vaccines provided effective protection in experimentally challenged ferrets, with protection correlating to the capacity of CD3+ T-cells to express interferon-gamma (IFN-γ) following in-vitro stimulation on peripheral blood mononuclear cells (PBMCs) with HA peptide pools [240]. Caution should be taken

35 when using antibodies developed for other species in ferret experiments, as there may be subsets of cells displaying variable reactivity to the antibodies [255, 256]. In addition, currently available anti-ferret B cell antibodies such as CD79 target intracellular epitopes, requiring fixation and permeabilization. This limits downstream applications such as RT-PCR or the recovery of antigen-specific immunoglobulin sequences from sorted B-cells. In the absence of cross-reactive clones, several groups have generated novel monoclonal antibodies specific for ferret cellular markers. For example, novel anti-ferret CD4, CD8 and CD5 specific antibodies were derived by immunising mice with the CD4 ectodomain [257], whole CD4 protein [258] or ferret thymocytes [231]. Efforts to develop novel antibody-based reagents to define various ferret immune cell subpopulations are accelerating, particularly through the

Centers of Excellence for Influenza Research and Surveillance (CEIRS) network

[259], and are reviewed in detail further below

Table 1-2 List of currently available ferret antibody reagents

Antigen Species Clonality-Clone Cell type Reference(s) CD20 Human Polyclonal- RB-9013-P B [258, 260, 261] CD32 Human Monoclonal 2E1 B [252] CD79α Human Monoclonal - HM47/HM57 B [258, 260] CD79β Human Monoclonal – ZL9-2 B [252] IgA Ferret Polyclonal- NBP-72747 B [96] IgA Canine Polyclonal B [252] IgA/G/M Ferret Polyclonal B [258] IgG Ferret Polyclonal B commercial IgG Mink Polyclonal B [252, 262] IgM Ferret Polyclonal B commercial IgM Human Polyclonal B [252] Kappa Ferret Monoclonal – multiple B [263] Lambda Ferret Monoclonal – multiple B [263] Immunoglobulin Heavy chain Ferret Monoclonal – multiple B [263] CD11b Mouse Monoclonal - M1/70 innate [253, 258] CD14 Human Monoclonal – Tuk40 innate [252] CD172a Human Monoclonal – DH59B innate [252] CD163 Swine Monoclonal- 2A10/11 innate [261] MAC387 Human Monoclonal- M0747 innate [261] CD88 Human Monoclonal – S5/1 innate [252] SWC3 Swine Monoclonal – BA1C11 innate [260] CD43 Mouse Monoclonal- S7 Pan-leucocyte [253] LFA-1 Mouse Monoclonal – 2D7 Pan-leucocyte [253] Ly6C Mouse Monoclonal – AL-21 Pan-leucocyte [258] TNF Mouse Monoclonal MP6-XT22 Pan-leucocyte [253] MHC-II Human L243 T [258] CD3 Human Polyclonal - IS503 T [260, 261] CD103 Mouse Monoclonal - M290 T [253] HLA-DR Human Monoclonal -TAL.1B5 T [261] CD25 Human Monoclonal- B1.49.9 T [252] CD4 ferret CL3.1.5 T [258] CD4 ferret Monoclonal T [257] CD8 Human Monoclonal – OKT8 T [252, 253, 258] CD8 Ferret Polyclonal – 60001RPO2 T/NK [260] IL-4 Bovine Monoclonal – CC303 T [253] Thy1.1 Mouse Monoclonal – OX-7 T [253] IFN- Bovine Monoclonal – CC302; T/NK [253] XMG1.2

The influenza protein specificity of the T cell response to influenza is beginning to be explored in ferrets using IFN-γ expression assays and other standardized protocols

[258, 264]. DiPiazza, Richards [258] found that the majority of bulk memory CD4+ T- cell responses were specific for the M1 protein whereas non-structural protein (NS) was mainly the target for CD8+ T-cell responses, and hierarchal responses were found to change over time without preferential retention of immunodominant specificities

[265]. In comparison, cross-reactive ferret CD4+ T-cells recognise HA and NA epitopes preferentially whereas CD8+ T-cells mount immune responses toward M1,

NS2 and RNA polymerase subunit A (PA), with NP as a significant antigenic target

[254]. Interrogation of ferret T cell responses with improved reagents will increase our understanding of immunodominance hierarchies analogous to studies performed in other animal models and humans [266].

Markers targeting B-cell antigens such as CD79α, CD20, and surface immunoglobulin also allow ferret B-cells to be examined by immunohistochemistry and flow cytometry in ferret tissues [258, 263, 267, 268]. Circulating B-cell frequencies are also transiently decreased two d.p.i [269, 270] after infection, with a corresponding increase in secondary lymphoid organs 2-5 d.p.i [270]. The number of major histocompatibility complex (MHC-II) expressing cells also increased, with no significant difference in surface immunoglobulin positive cells by ten d.p.i. [258]. Influenza specific antibody- secreting cells (ASCs) was increased by 37 d.p.i, highlighting the role of B-cells in the resolution of infection [231]. These observations suggest changes in the maturation status of B-cells after activation and mirrors observations in humans and mice [126].

MHC-II is upregulated in B-cells to induce GC formation through cognate interactions with T follicular helper cells (TFH). This has been demonstrated in mice, where the

38 ablation of MHC-II expression in mice led to a decrease in influenza-specific IgG and

IgA titres and decreased survival rates [271]. Surface immunoglobulin expression in antibody-secreting plasma cells (ASCs) is also downregulated, consistent with the decrease in the number of surface immunoglobulin expressing HA reactive GC cells by 14 d.p.i [272].

Similar to T-cells, B-cell HA immunodominance hierarchies have been studied widely

[273]. A key target for broader antibody responses is the conserved stem domain

(HA2) of HA; however, immunodominant responses against HA1 often limit antibody responses to variable regions, leading to escape from host responses [23]. Different vaccination strategies to induce broadly protective antibody responses have been studied in ferrets [95, 96]. Using purified ferret immunoglobulins and cross-reactive polyclonal immunoglobulin antisera from mink, goat, canine and rabbits, a ferret immunoglobulin class-specific ELISA was developed [252]. By exposing ferrets to recombinant HA constructs with exotic HA head domains via infections and vaccinations (H9/H8/H5 head domain with H1 stem domain), immunologically subdominant anti-HA stem responses were induced as measured by ELISA [96].

Polyclonal stem reactive antibodies were detected serologically, and protected ferrets against pH1N1 challenge in the presence of low neutralization activities as measured by microneutralization assays. This is consistent with studies in other mammalian models and humans, suggesting that Fc mediated functions such as ADCC are important for HA2 mediated protection [274].

The effects of prior infection on host susceptibility to re-infection, i.e. viral interference have also been studied in ferrets [275]. Ferrets sequentially challenged with B/Victoria

39 and B/Yamagata viruses display decreased virus shedding, which correlated with the induction of high frequencies of cross-reactive IFN-γ-expressing T-cell responses between initial infection and heterologous challenge [276]. Infection with

A(H1N1)pdm09 was also shown to prevent HRSV infections in ferrets, though no IFN-

γ responses or cross-reactive serological responses were observed, suggesting different underlying mechanisms driving viral interference between unrelated viruses

[277]. This observation mirrors epidemiological studies in humans, where peak incidences of HRSV infections in humans were delayed by influenza A outbreaks

[277].

Several key questions remain unanswered in ferrets with regards to influenza-specific immune responses. First, chemotactic signals important for the spatiotemporal distribution of immune cells are still largely unknown in ferrets; for example, influenza- infected epithelial cells secrete CCL-2 which recruits monocytes to the lungs during early infection and may be associated with acute lung injury [278] during severe infections. Secondly, different innate immune subsets such as natural killer (NK) cells, dendritic cells (DCs), monocyte/macrophages and granulocytes have also yet to be studied in detail, and development of reagents to allow the delineation of these cell populations [279-282] will enable a more detailed picture of early immune responses in ferrets. Thirdly, while adaptive immune responses have been studied in ferrets, markers to delineate B/T cell subpopulations will be useful to study long term protection against influenza infection [283]. Examples include CD62L and CD44 for naïve and memory T-cells; IgD and CD27 for naïve and memory B-cells respectively.

40 1.9.2 Ferrets as an immunological model for other emerging viral diseases

In addition to influenza, the ferret serves as a critical model for other important human pathogens such as SARs-CoV, pneumoviruses (HRSV and HMPV), Ebola virus and henipaviruses. While these infections remain less characterized in comparison to influenza, the ferret provides a platform to examine disease pathogenesis, transmission and to evaluate potential vaccine efficacy. However, like influenza, evaluation of host immune responses in ferrets is commonly restricted to gene expression analyses.

1.9.2.1 Pathogenic Coronaviruses

SARS-CoV infection causes acute respiratory distress in humans, with mortality rates of up to 10% [284]. Ferrets display clinical signs of infection such as elevated body temperatures, sneezing, increase in lymphocyte counts, and lesions in the respiratory tract and alveolar oedema [285] and are therefore a good mammalian model to study the pathogenesis of SARS-CoV [285, 286] and evaluate vaccines [287-290]. In terms of immunity, ferrets exhibit strong antiviral interferon responses after infection and vaccination as measured by interferon response gene expression levels [291, 292].

However, leukocyte counts and interferon related gene expression were decreased upon reinfection [291] suggesting that innate immune dysregulation is a possible mechanism of pathogenesis, although a protective antibody response was also evident during attempts to re-infect ferrets [293]. Ferrets have shown to be a valid model of COVID-19 showing fever and loss of appetite 10-12 d.p.i.. SARS-CoV-2 predominantly infects the upper respiratory and digestive tracts of ferrets, although transmission is somewhat inefficient [294]. Major pathological hallmarks include inflammation within alveoli as well as submucosal eosinophilic foci and collagen

41 degeneration within bronchi [295].. Nevertheless, recent transcriptional studies in ferrets have shown muted immune responses against COVID-19 virus, such as lowered Type I/III interferons and other chemokines which may explain high morbidity and mortality rates in the elderly [296]. Vaccines are required urgently, as this virus is highly transmissible and has placed incredible strain on the economy and healthcare systems around the world. Efforts are underway to develop vaccines globally [297] to minimise the impact of the virus.

1.9.2.2 Henipavirus

Emerging viruses belonging to the Paramyxoviridae family (Henipaviruses) can cause severe respiratory illness and/or encephalitis in humans. Ferrets infected with henipaviruses exhibit similar symptoms as humans including respiratory signs such as cough, nasal discharge, neural signs such as depression [298], and high mortality rates with experimentally infected ferrets succumbing within one week [299]. While the virus is detected in pharyngeal and rectal secretions, it is currently unclear if ferrets could serve as a transmission model for the disease [298, 299]. Ferrets infected intranasally with henipaviruses similarly display clinical illness [299, 300]. Assessment of immune gene expression by Leon, Borisevich [299] in both lungs and brain tissues of infected ferrets revealed upregulation of macrophage markers such as CD40 and

CD80 in both lung and brain tissues, whereas lymphocytic markers were unchanged in the lungs.

1.9.2.3 Respiratory Syncytial Virus and Metapneumovirus

RSV and HMPV cause severe respiratory disease in young children, the elderly and immunocompromised patients. Both RSV and HMPV readily infect ferrets but in

42 general, do not exhibit signs of the disease [301-303]. Nevertheless, ferrets have proven to be a useful model to study RSV. Several groups have successfully infected ferrets with a wild type strain of human RSV and demonstrated efficient replication in both the upper and lower respiratory tracts of adult ferrets [302, 303], consistent with humans where the infection is often limited to the upper respiratory tract [304].

Immunocompromised ferrets, induced by oral administration of immunosuppressive drug mycophenolate mofetil (MMF), demonstrate prolonged RSV shedding and effective contact transmission to both immunocompetent and immunocompromised ferrets [305], confirming antiviral immunity in the ferret can curtail viral replication. An assessment of lung immune gene expression in ferrets infected with RSV demonstrated an upregulation of proinflammatory cytokines such as interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β) by five d.p.i which coincided with maximum levels of RSV mRNA, while levels of other cytokines such as interferon-alpha (IFN-α) and IFN-γ remained unchanged [303]. In terms of humoral responses, increased serum titres of fusion (F) glycoprotein antibodies were seen by 15 d.p.i [303] that were protective against re-infection.

1.9.2.4 Ebola virus

Ebola virus disease (EVD) is caused by a zoonotic virus from the Filoviridae family of viruses [306]. This disease can transmit from human to human and causes acute and often fatal disease. Ferrets can be directly infected with the Zaire, Bundibugyo and

Sudan Ebola strains [247, 307], which have previously caused major human outbreaks. Ferrets display hallmarks of pathological processes of lethal human infections such as petechial rashes, reticulated pallor of the liver and splenomegaly

[307, 308]. The transmission of the virus has also been reported in ferrets [309]. As

43 for immunological studies, Transcriptomic sequencing in ferrets infected with lethal doses (1000 plaque-forming units (PFU)) of the Makona variant of Zaire ebolavirus revealed upregulation of proinflammatory related genes such as interferon activation,

Toll-like receptor signalling, interleukin-1/6 responses and coagulation cascades by five d.p.i.[310].

1.10 Knowledge gaps that have to be bridged to improve ferrets as an

immunological model

1.10.1 Immunogenetics

There is a lack of well-annotated, ferret genomic sequence information to characterise immune responses, limiting the scope of molecular analyses that can be performed.

While ferret TCRB locus has been recently sequenced [311], ferret B cell receptor repertoire analysis is currently not possible. Next-generation sequencing (NGS) has become increasingly important for immunological research and has led to the generation of huge amounts of data and the development of tools for data extraction and analysis. An important aspect of T and B-cell research is the immune cell receptor repertoire during an infection and the effects of allelic variation of important immunological molecules such as Major Histocompatibility Complex (MHC) on host immune responses. A draft copy of the ferret genome is available [211], but genes coding for B- or T-cell receptors have yet to be fully annotated and validated.

Genomic sequencing and assembly of closely related species such as minks [312] are also far from complete, though several similarities such as genome size and relative abundance of repeat elements have been found. In comparison, high-quality draft genome assemblies for dogs and cats are available and have been used for genome-

44 wide association studies [313] and identification of single nucleotide polymorphisms

(SNPs) [314]. The identification of SNPs in immunoglobulin genes is useful for distinguishing between somatically mutated B-cell receptor sequences and germline variants in affinity matured antigen-specific B-cell populations. There are currently databases of immunoglobulin sequences for well-established animal models such as those found in the international ImMunoGeneTics information system (IMGT) database [315] and have been useful for identifying somatic hypermutations in immunoglobulin sequences [316]. A curated and annotated database of immune gene sequences is a prerequisite for PCR primer design and post-sequencing data analysis used to recover and express antibodies from single sorted B-cells [170] and recombinant T Cell receptors [317]. Chapter 3 addresses this knowledge gap by the identification of ferret immunoglobulin genes in the draft copy of the ferret genome, which was critical for the subsequent development of a novel single-cell RT-PCR based-method to recover ferret antibody sequences. This method was utilised to recover germline heavy, kappa and lambda light chain immunoglobulin transcript sequences from single sorted ferret B-cells. RNA-seq validation of immunoglobulin constant chain genes led to the development of ferret IgG/IgL coding plasmids, which enabled the expression of ferret mAbs.

1.10.2 Antigenic recognition of major influenza proteins

Epitope mapping studies using either human and murine monoclonal antibodies have greatly increased our understanding of influenza viral evolution and allowed the identification of major HA epitopes and pathways of immune escape. While factors that determine the dominant escape mutants are still unknown, such studies have the potential to improve the process of influenza vaccine design. The current inability to

45 isolate monoclonal antibodies from ferret B-cells has limited studies into the antigenic recognition of influenza proteins in ferrets. This requires the resolution and sorting of rare HA-specific ferret B-cell populations and the screening of transcript sequences coding for HA-specific mAbs.

Confirming epitope-specific recognition of HA at the monoclonal antibody level in ferrets is critical, as there have been several reports that human and ferret serum antibodies can display variable antigenic recognition [318, 319]. This is critically important as anti-sera from infected ferrets is widely used in HI assays as part of the strain determination process for influenza vaccines [320, 321]. In chapter 4 of this thesis, we recovered and analysed immunoglobulin transcript sequences from influenza-infected ferrets. Sequences were clonally expanded, showing the activation and proliferation of HA-specific B-cells in response to an infection. While the recovery of ferret mAbs is currently inefficient, reconstitution of fully ferret mAbs using the developed ferret IgG/IgL coding plasmids enabled the identification of HA binding mAbs belonging to a single clonal family which retained the capacity to neutralise influenza viruses in-vitro. Neutralisation epitopes were mapped by viral escape assays, showing proof of concept for performing ferret HA epitope mapping studies.

1.10.3 Future T-cell specific reagents for ferrets

Future development of markers to delineate more T-cell subsets will increase the utility of the ferret as an immunological model; a recent report listed several important ferret

T-cell specific antibodies to be in production at the CEIRS: CD4, CCR7, CD3e, CD40,

CD40L, CD44, CD62L, CD69, CD103, PD-1, CXCR3, CXCR5, IL-7R, IL-15R [259].

Particular reagents that would be extremely useful but are not yet available include

46 PD1 and CXCR5 which are markers for TFH. These will ultimately be critical for the evaluation of T-cell help for antibody responses.

1.10.4 Current and Future markers for ferret myeloid lineage cells

Several markers defining innate cell populations in mice and humans such as CD11b

[253, 258] and CD14 [252] have also been reported to cross-react with ferret leukocytes and have been utilised to characterise ferret innate immune responses.

However, these markers have also been found to be expressed in non-myeloid lineages in humans [322, 323], and other markers such as CD16 [324] and CD66

[325], will be required to better define myeloid cell populations.

1.10.5 Future B-cell specific reagents for ferrets

To increase our understanding of antibody responses in ferrets, flow cytometric reagents that can delineate B-cell subsets are required. Important ferret B-cell specific antibodies that are in production at the CEIRS include CD83, CD86, CD95, CD19,

CD20, CD25, CD27, CD38, CD138, and FcR [259]. While polyclonal reagents targeting surface ferret immunoglobulins are readily available, mAbs targeting pan-B- cell lineage surface marker CD19 is ideal since currently available cross-reactive

CD20/CD79α/CD79β specific mAbs target intracellular epitopes, which limits downstream PCR analyses due to the requirement for sample fixation and permeabilization. In chapter 5 of this thesis, we established the capacity to isolate ferret antigen murine recombinant mAbs using currently available single-cell RT-PCR methods and recombinant murine mAb coding plasmids. While our candidate anti- ferret mAbs in this chapter lacked the capacity to resolve ferret cell populations by flow cytometry, cognate recombinant ferret antigen binding was present, validating this

47 method to recover antigen specific murine mAbs. We discuss potential improvements in the workflow presented in this chapter to enable the recovery of effective ferret mAb reagents. This method will enable the isolation of potential ferret marker specific mAbs to increase the utility of the ferret as an immunological model by improving flow cytometric and confocal microscopy panels.

Together, this thesis advances the use of ferrets for studying B-cell repertoires by enabling the recovery of antibody sequences from single ferret B-cells and demonstrating proof of concept for recovering HA-specific ferret mAbs. Increasing the efficiency of ferret HA-specific mAb recovery via improvements in flow cytometric panels will enable larger panels of HA-specific mAbs and detailed evaluation of antibody effector functions in ferrets.

48 Chapter 2

General materials and methods

2.1 Materials

2.1.1 Media and Buffers

2.1.1.1 1% Agarose gel

1% (w/v agarose gel) was prepared by dissolving 1 g molecular biology grade

agarose (Promega) in 1 X TAE. SYBR safe gel stain was added to a final

concentration of 1X to enable visualisation of DNA following electrophoresis.

2.1.1.2 Cryopreservation media for E.coli

10% (v/v) DMSO in LB.

2.1.1.3 Cryopreservation media for mammalian cells

10% (v/v) DMSO in HI-FBS.

2.1.1.4 ELISA blocking buffer

2.5% (w/v) BSA (Sigma) in 1 X PBS

2.1.1.5 ELISA dilution buffer

2.5% (w/v) BSA (Sigma), 0.05% Tween-20 and in 1 X PBS

2.1.1.6 ELISA wash buffer

2.5% (w/v) BSA (Sigma), 0.2% (v/v) NP-40 and 0.05% (v/v) Tween-in 1 X PBS

49 2.1.1.7 FACS wash buffer

1mM molecular biology grade EDTA (Sigma) and 10% FBS (v/v) in 1X PBS

2.1.1.8 FACS fixing buffer

1% (v/v) para-formaldehyde in FACS wash buffer

2.1.1.9 Influenza virus growth media

0.3% bovine serum albumin (Sigma), 1 g/mL TPCK treated trypsin (Sigma) in

DMEM media (Thermofisher) for influenza virus based in-vitro assays.

2.1.1.10 LB-kanamycin broth

10 g tryptone, 10 g NaCl, 5 g yeast extract in 1L milliQ water (pH 7.0) (LB).

Kanamycin was added to a final concentration of 30 g/mL after autoclaving LB media.

2.1.1.11 LB-kanamycin agar

1.5% (w/v) agarose was added to LB broth. Kanamycin was added to a final concentration of 30 g/mL after autoclaving media and set in sterile Petri dishes and kept at 4oC before use

2.1.1.12 Live/dead Stain for flow cytometry

5L Live/Dead fixable Aqua (Thermofisher) was diluted in 1 mL of 1 X PBS to prepare 20 stains (50L each).

50 2.1.1.13 MDCK cell maintenance media

10% HI-FBS and 1X PSG (100 units penicillin, 100g streptomycin, 0.292 mg/mL L-glutamine) in DMEM media (Thermofisher) for maintenance of MDCK cells.

2.1.1.14 RPMI-F10 media

10% heat-inactivated FBS and 1X PSG (100 units penicillin, 100g streptomycin, 0.292 mg/mL L-glutamine) in RPMI 1640 media (Thermofisher).

2.1.1.15 SDS-PAGE loading buffer

1 X SDS loading buffer (Biorad) containing 5% v/v -mercaptoethanol (Biorad)

2.1.1.16 SDS-PAGE buffer

One part 10x Tris/Glycine/SDS buffer (250 mM Tris, 1920 mM glycine, 1 %

SDS, pH 8.3) in 49 parts of Milli-Q water to obtain 1X SDS-PAGE buffer for denaturing protein gel electrophoresis.

2.1.1.17 SDS-PAGE gel fixing buffer

10% (v/v) acetic acid and 40% (v/v) ethanol in MilliQ water

2.1.1.18 Tris-Acetate EDTA buffer for DNA gel electrophoresis.

One part 40x TAE buffer (Promega; containing 1.6 M Tris-acetate, 40mM

EDTA, pH 8.2) was dissolved in 39 parts of Mili-Q water to obtain 1x TAE buffer for DNA electrophoresis.

51 2.1.1.19 Trypsin-EDTA media

0.1% trypsin, 0.02% EDTA, 0.05% glucose and Phenol red in Dulbecco’s

PBS.

2.1.2 Protein/DNA purification kits and buffers

DNA/Protein purifications were carried out using commercial kits and reagents

according to the manufacturer’s instructions (Table 2-1)

Table 2-1 Protein/DNA purification kits and buffers

Manufacturer

Pierce Protein A

agarose beads

Pierce Protein A

binding buffer

Pierce Protein G Thermoscientific agarose beads

Pierce Protein G

binding buffer

Pierce IgG elution

buffer

Plasmid midi kit Qiagen Plasmid maxi kit

52 2.2 Methods

2.2.1 Molecular cloning and expression of recombinant mAbs and proteins

2.2.1.1 Construction of ferret antigen protein-coding plasmids

Validated DNA coding sequences of ferret CD19, IgD, CD138, NKp46 and LAMP were synthesized as a recombinant construct (Geneart) with 5’ IL-2 signal sequence tag to facilitate purification from transfection supernatants. These constructs were flanked with 5’ XbaI (5’- TCTAGA) and 3’ BamHI (5’-AGATCT) cut sites (Figure 2-1) to facilitate cloning into human IgG1 (3’- purification tag) plasmids (see section 2.2.1.5).

Figure 2-1 Construction of recombinant ferret-Fc fusion plasmids. Coding sequences of ferret CD19, CD138, NKp46, LAMP-1 and IgD were flanked with XbaI and BamHI restriction sites to facilitate cloning into human IgG1 Fc coding plasmids.

2.2.1.2 Construction of ferret antibody constant chain coding plasmids

Validated ferret IgG, and lambda constant domain sequences (1 g each) were synthesised (Geneart) and cloned into established human IgG1 and lambda chain eukaryotic expression vectors (1 g each) [170] using flanking SalI (5’-

53 GTCGAC)/BamHI (5’- GGATCC) (IgG1) and XhoI (5’-GTCGAG)/BamHI(5’-

GGATCC)(Lambda) restriction enzymes (NEB). (Figure 2-2; See section 2.2.1.5)

Figure 2-2 Construction of ferret-Fc plasmids. Coding sequences of ferret IgG and lambda chain constant regions were flanked with SalI/BamHI and XhoI/BamHI restriction sites respectively to facilitate cloning into human IgG1 Fc and human Lambda chain Fc coding plasmids.

2.2.1.3 Construction of ferret / chimeric ferret-human antibody coding plasmids

Recombined heavy chain (VDJ) and lambda chain (VJ) sequences (1 g each) of human anti-HA antibody CR9114 (1 g) [326] or HA-specific ferret antibodies were synthesised (Geneart) and subsequently cloned into ferret IgG1 and ferret lambda chain expression vectors (Figure 2-3) using flanking AgeI (5’-ACCGGT) /SalI (5’-

GTCGAC) (IgG) and AgeI (5’-ACCGGT) /XhoI (5’-GTCGAG) (lambda) restriction enzymes (NEB). (See section 2.2.1.5)

54 2.2.1.4 Construction of murine antibody coding plasmids

Recombined heavy chain (VDJ) and lambda chain (VJ) sequences (1 g each) of murine antibodies were synthesised (Geneart) and cloned into plasmids (1 g each) based on previously reported murine IgG1 and kappa chain expression vectors

(Figure 2-3) [171] using flanking AgeI (5’-ACCGGT) /SalI (5’-GTCGAC) (IgG) and

AgeI/BsiWI (5’-CGTACG) (kappa) restriction enzymes (NEB). (See section 2.2.1.5)

Figure 2-3 Construction of murine/ferret mAb coding plasmids. V-regions of heavy or light chain mAbs were cloned into the respective Fc coding plasmids using the indicated restriction enzymes as shown in the figure.

2.2.1.5 Restriction digestion and ligation of restriction digestion products

Restriction digestion of vectors (1 g) and inserts (1 g) were carried out respectively in 1 x cutsmart buffer (NEB) with 10 U each of the respective restriction enzymes at

37oC for 1 h in a total reaction volume of 40 L. Double digested inserts and vectors were resolved by agarose gel electrophoresis. Bands of interest were subsequently

55 excised and gel purified. Gel purification of resolved vectors and inserts was performed using Qiagen gel purification kit according to the manufacturer’s instructions. Purified restriction digested fragments were eluted in 20 L buffer EB

(10mM Tris-HCl, pH 8.5). Purified inserts were ligated into linearized vectors at a molar ratio of 3:1 in 1 X T4 DNA ligase buffer and 1 U DNA ligase (Promega) in a total reaction volume of 20 L at 25oC for 1 h.

2.2.1.6 Transformation and validation of antibody/protein-coding plasmids

4 L unpurified ligation products was added to 15 L freshly thawed competent DH5

E.coli (Thermofisher) and incubated on ice for 1 h. Transformation of ligated constructs was performed by heat shock at 37 oC for 40 s. Transformed cells were recovered for

1 h at 37oC with the addition of SOC media (Thermofisher). Cells were subsequently plated on LB-Kanamycin (30 g / mL) plates at 37oC for 16 h. Individual colonies were grown up in 2mL LB-Kanamycin media (30 g / mL) on an orbital shaker (500 rpm) at

37oC for 16 h. Minipreps (Promega) of cloned plasmids were subsequently prepared according to the manufacturers’ instructions. Plasmids were resuspended in nuclease- free water and sequenced using 1012F sequencing primer to validate the presence of insert in the cloned constructs. Validated E.coli clones were cryopreserved at -20oC in

E.coli cryopreservation buffer.

2.2.2 Agarose gel electrophoresis

DNA samples were resuspended with 1 X Loading buffer and added to wells of 1% agarose gel. Gels were run at 100 V for 15 min (RT-PCR recovery of immunoglobulin genes) or 1 h (molecular cloning). DNA bands were illuminated using LED transilluminator (509 nm) and excised as required.

56 2.2.3 Maintenance of Expi293F cells

1 x 107 Cryopreserved Expi293F cells (Life Technologies) were thawed and washed twice in Expi293 transfection medium to remove DMSO present in cryopreservation media. Thawed cells were recovered in Expi293 media (Life Technologies) and checked via trypan blue exclusion to ensure 90-95% viability 4 days post-thaw. Cells were grown up to 3-5 x 106 cells/mL and sub-cultured every 3 – 4 days. All cultures

o were maintained in an orbital shaker (125 rpm) maintained at 37 C with 8% CO2.

2.2.4 Eukaryotic expression of proteins and antibodies

Transformed E. coli clones with validated recombinant protein or antibody coding plasmids were grown up in 200 mL LB-Kanamycin media on an orbital shaker at 37oC for 16 h. Midipreps/Maxipreps (Qiagen) were prepared according to the manufacturer’s instructions and resuspended in TE buffer for transfections. Expi293F cells were grown to a density of 2.5 x 106 cells/mL and transfected with 200 g of plasmid preparations complexed with 270 L of Expifectamine in 200 mL cultures

(Figure 2- 4 A). For co-transfections of antibodies, 100 g each of heavy chain and light chain coding plasmids was complexed with Expifectamine. After the addition of transfection enhancers 16 hours post-transfection, culture supernatants were harvested 5 days post-transfection (Figure 2-4 A).

2.2.5 Purification of recombinant proteins and antibodies

Transfection supernatants were clarified by high-speed centrifugation (5000 g, 4oC,

15 min) and filtration (0.22M). Antibodies or proteins were purified (Figure 2-4 B) using either Pierce Protein A or Protein G agarose (Thermofisher) (Table 2-1). Briefly, supernatants were equilibrated with Protein A or Protein G IgG binding buffer and

57 loaded into purification columns packed with 0.2mL of equilibrated Protein A or Protein

G agarose (Thermofisher). Bound protein was washed using 4 column volumes of

Protein A or Protein IgG binding buffer (Thermofisher), eluted with 15 mL IgG elution buffer (Thermofisher) and neutralised with 1.5mL 1M TE buffer pH 8.0 (Merck).

Proteins were concentrated via centrifugation (100 kDa Amicon; Merck Millipore) and resuspended in PBS. Protein integrity was confirmed by SDS-PAGE, and concentrations were determined using OD280/260 measurements. Proteins and antibodies were subsequently analysed by SDS-PAGE.

Table 2-2: IgG Binding profiles of Protein A and Protein G

Antibody/Purification Tag Purification beads/buffer

Human IgG1 Protein A Ferret IgG

Murine IgG1 Protein G

2.2.6 Denaturing SDS-PAGE protein gel electrophoresis

10 g of purified protein was resuspended in SDS loading buffer (total volume of 20

L). Proteins were incubated at 98oC for 15 min to reduce proteins on a block heater.

Proteins were resolved on 4 – 20% Mini-PROTEAN Precast protein gels (Biorad) in 1

X SDS-PAGE buffer (Biorad) at 120 V for 1 h (Figure 2-4 C). Resolved gels were subsequently washed in distilled water and fixed in SDS-PAGE Fixing buffer for 10 minutes. Fixed gels were subsequently stained with colloidal coomassie blue (Biorad) for 1 h with agitation to visualise resolved proteins.

Figure 2-4 Purification and expression of ferret proteins / mouse mAbs / ferret mAbs. (A) Expi293 cells were utilised to express the proteins/mAbs presented in this thesis. (B) Proteins and mAbs were purfieid by using either protein G/ protein A agarose beads by gravity flow. (C) Purified proteins were analysed using SDS-PAGE.

2.2.7 HA-specific ELISA

96-well ELISA plates were coated with 400ng per well of full length recombinant CA09

HA in 1 X PBS at 4OC overnight. Wells were blocked with ELISA blocking buffer for 1h at room temperature and washed with ELISA wash buffer. Four-fold serial dilutions of chimeric human/ferret CR9114 or fully ferret anti-HA antibody (starting at 0.01mg / mL) and serum samples (starting at 1 in 100 dilution) from immunologically naïve ferrets and ferrets infected with 1000 TCID50 of H1N1 A/California/07/2009 were prepared in ELISA dilution buffer and incubated in the coated plates at room temperature for 1 h. Detection was performed by sequential staining with donkey anti- ferret IgG (Rockland cat. 618-101-012) and goat anti-donkey-HRP (Abcam cat.

Ab6988) at 1:2000 and 1:1000 dilutions in ELISA blocking buffer respectively for 30 min each. OD630 readings were obtained after addition of Sureblue TMB peroxidase substrate (Seracare) and TMB BlueSTOP Solution (Seracare).

59 Chapter 3

Developing the capacity to express ferret monoclonal

antibodies

3.1 Abstract

The domestic ferret (Mustela putorius furo) provides a critical animal model to study human respiratory diseases. However, immunological insights are restricted due to a lack of ferret-specific reagents and limited genetic information about ferret B and T cell receptors. In this chapter, we aimed to address this lack of knowledge regarding ferret

BCRs and antibody coding genes by first annotating ferret antibody coding genes and deriving methods and tools to recover ferret mAbs. Putative germline genes from heavy, kappa and lambda chain loci were first identified on the draft copy of the ferret genome using conserved immunoglobulin gene sequence orthologues from humans and canines. These sequences subsequently enabled the design of PCR primers flanking the 5’-leader and 3’- constant regions of ferret immunoglobulin transcripts which enabled the recovery of paired heavy and light chain antibody germline transcripts from single sorted ferret B-cells. Plasmids coding for ferret IgG/IgL constant regions were constructed, which subsequently enabled expression and purification of chimeric human/ferret CR9114 mAb that retained HA-specificity. These tools advance the ferret model by enabling the recovery of ferret mAbs for in-depth studies of B-cell responses.

60 3.2 Introduction

Effective humoral immunity is contingent upon the phenomenal diversity of antibodies, which in mammals is derived via genetic recombination of numerous variable (V), diversity (D) and joining (J) gene segments localised to heavy, kappa and lambda immunoglobulin loci. In recent years, the capacity to clone and express antibodies from single B cells has proved a powerful tool to study antibody repertoires in a variety of infectious disease settings in humans [170, 327-329], and important animal models such as mice [171, 330] and non-human primates [331, 332]. These approaches have subsequently been extended using next-generation sequencing platforms [333, 334], allowing unprecedented depth in the characterisation of anti-pathogen antibody responses.

The domestic ferret (Mustela putorius furo) is a critical mammalian model to study pathogenesis and evaluate vaccines against a variety of human respiratory pathogens

[335], most critically influenza. However, the majority of influenza research using ferrets is focused upon viral transmission and/or pathogenesis, with in-depth immunological studies limited by a critical lack of ferret-specific reagents and limited understanding of the ferret immune system. A key knowledge gap surrounds the immunogenetics of ferret immunoglobulins. While the ferret genome was recently sequenced [211], accurate annotation of germline immunoglobulin genes is currently incomplete. This has hindered the ability to sequence ferret B cell receptors and allow the recovery of ferret mAbs, limiting detailed interrogation of ferret serological responses that inform current influenza vaccine strain selection efforts.

61 In this chapter, we sought to increase the utility of ferrets for studying humoral immunity. Ferret heavy, kappa and lambda immunoglobulin loci were annotated using available genomic sequences (Figure 3-1 A), allowing the design of a novel set of multiplex PCR primers flanking recombined ferret immunoglobulin genes.

Recombined B cell receptor sequences were recovered from single sorted ferret B cells (Figure 3-1 B), partially confirming our initial gene segment annotation and allowing identification of potential novel germlines. Ferret immunoglobulin constant gene sequences were confirmed by the de-novo assembly of RNA-seq transcripts, allowing the design of expression plasmids and the recombinant production of ferret

IgG monoclonal antibodies (Figure 3-1 C).

Figure 3-1 Establishing tools for recovering ferret mAbs. (A) Sequences coding for V, D, J and C segments of ferret antibodies were identified on the draft copy of the ferret genome. (B) Based on the annotated sequences, primers targeting ferret immunoglobulin transcripts were designed, which enabled recovery of heavy and light chain transcripts from single ferret B-cells. (C) Immunoglobulin constant region sequences were validated by RNAseq and plasmids coding for ferret IgG and IgL were constructed.

63 3.3 Materials and Methods

3.3.1 Annotation of ferret immunoglobulin genes

Ferret genomic contigs containing potential immunoglobulin genes were retrieved from e!Ensembl (http://www.ensembl.org). (Immunoglobulin Heavy - GL897360.1,

GL897427.1, GL897453.1, GL897498.1, GL897556.1, GL897558.1, GL897564.1,

GL897795.1, GL898421.1; Immunoglobulin Kappa - GL896905.1; Immunoglobulin

Lambda - GL897406.1, AEYP01111698.1, GL896906.1, AEYP011112098.1,

GL897285.1, GL897406.1, GL897344.1, GL897565.1, GL897418.1,

AEYP01110728.1, AEYP01108526.1, GL897638.1 GL897285.1, GL897484.1,

GL897019.1, GL897400.1). Iterative BLAST searches using human, canine and ferret immunoglobulin gene segments were used to identify and annotate putative germlines. Sequences with nonsense mutations and/or non-functional regulatory elements were considered pseudogenes.

3.3.2 Generation of ferret immunoglobulin variable gene phylogenetic trees

Ferret immunoglobulin variable gene sequences were analysed with reference to human, mouse or canine databases using IMGT/V-Quest [315] and assigned to mammalian clans based upon phylogenetic analyses. Ferret immunoglobulin amino acid sequences were first aligned globally pairwise to generate distance matrix using

BLOSUM cost matrix with a gap open cost of 10 and gap extend cost of 0.1 and visualised using Geneious (10.1.3). Phylogenetic relationships were subsequently determined based on the Jukes-Cantor model. Consensus phylogenetic trees were built using the Neighbour-Joining method with no outgroups and resampled by bootstrapping (100x) using Geneious tree builder (10.1.3).

64 3.3.3 Flow cytometric sorting of single ferret B-cells

Cryopreserved ferret single-cell spleen preparations were thawed in RF-10 media.

Cells were subsequently stained with live/dead stain, and excess live/dead stain was quenched by the addition of FACS wash. This was followed by the addition of surface stains anti-CD11b-BV510 (Biolegend: clone M1/70), anti-CD8-BV450 (Thermofisher: clone OKT8) and anti-ferret IgA/IgM/IgG-FITC (Rockland Immunochemicals cat.618-

102-130). Cells were washed twice to remove excess staining antibodies. Stained cells were resuspended in OptiMEM (ThermoFisher) before single, live, surface

Immunoglobulin positive B cells were sorted into 96-well PCR plates and stored at -

20oC before RT-PCR amplification of immunoglobulin gene sequences.

3.3.4 RT-PCR recovery of immunoglobulin sequences from single sorted ferret B- cells

A B cell receptor (BCR) sequencing protocol was developed based upon the multiplex nested RT-PCR approaches previously described for humans [170] and mice [171].

Reverse transcription of total cellular RNA from single sorted ferret B cells was performed in 25 L reaction volumes in the sort plate using 450 ng random hexamers

(Bioline), 50 U Superscript III (Thermofisher), 1 X First Strand buffer (Thermofisher),

8 U RNAsin (Promega), 0.125 pmol (DTT) (Thermofisher), 0.8% v/v IGEPAL CA-630

(Sigma Aldrich) and 0.8mM deoxynucleotide triphosphate (dNTP) (Bioline). Cycling conditions for cDNA synthesis were: 42oC for 10 min, 25oC for 10mins, 50oC for 60 mins and 94oC for 5min.

3 L of unpurified cDNA was used as template in multiplex nested PCR reactions to amplify paired recombined ferret heavy and kappa and lambda light chain sequences.

65 Primary reactions were carried out in 50 L volumes using Hotstart Taq plus polymerase (Qiagen), 1 X reaction buffer, 2.0 mM MgCl2, 0.1 mM dNTP (Bioline) and five nanomol each of primary forward and reverse primer pools (Table 3-1). 4 L of the primary PCR product was used as the template in a secondary, nested PCR (50

μL volume) containing 1X reaction buffer, 1.5 mM MgCl2, 1 x solution Q and five nanomol secondary forward and reverse primer pools (Table 3-1). Secondary amplification products were resolved by 1% agarose gel electrophoresis and sequenced by standard Sanger sequencing using the IgM (Heavy), kappa and lambda chain reverse primers from the secondary amplification step.

66 Table 3- 1: Ferret immunoglobulin specific primers. (A) Ferret immunoglobulin heavy chain primer pools. (B) Ferret immunoglobulin Kappa chain primer pools. (C) Ferret immunoglobulin lambda chain primer pools.

FerretHeavy_clan_ III-Fwd_1.1 ATGGACTTTGTGCTTGGCTGGGTTTTC FerretHeavy_clan_ III-Fwd_1.2 ATGGAATTTGTACTTGGCTGGGTTTTCC FerretHeavy_clan_ III-Fwd_1.3 ATGGAATTTGTGCTGGGCTGGGTTTTCC FerretHeavy_clan_ III-Fwd_1.4 ATGGAATTTGTATTGGGCTCGGTTTTCC FerretHeavy_clan_ III-Fwd_1.5 ATGGAGTTTGCACTTGGCTGGGTATTCC Heavy Primary FerretHeavy_clan_ III-Fwd_1.6 ATGGGATTTGTGCTTGGCTGGGTTTTCC forward pool FerretHeavy_clan_ III-Fwd_1.7 ATGGAGCTTGTGCTTGGCTGGGTTTTCC FerretHeavy_clan_ III-Fwd_1.8 ATGAAGTTTGTGCTTGGCTGGGTTTTCC FerretHeavy_clan_ I-Fwd1.1 CTGGAGCTGGAGAATCCTCTTCTTG FerretHeavy_clan_ I-Fwd1.2 CTGGAGCTGGAGAATCCTCTTCCT FerretHeavy_clan_ II-Fwd1 ATGCAGCTGCTGTGGTCCCTCC

Ferret_IgA-Rev1 CACCAGGCAGGCGATGACCAC Heavy Primary Ferret_IgG-Rev1 GAATTCCAAGATACGGTTACAGGCTCG reverse pool Ferret_IgM-Rev1 CACTGTCATTCTTGTAGGTCCAGGAG Ferret_IgD-Rev1 GTCCTTGTGCCAAGCAGGCCAG

FerretHeavy_clan_ III-Fwd_2.1 GCTTGGCTGGGTTTTCCTTGTTGC FerretHeavy_clan_ III-Fwd_2.2 GCTTGGCTGGGTTTTCCTTGTTGTTC FerretHeavy_clan_ III-Fwd_2.3 ACTTGGCTGGGTTTTCCTTGTTGCTC FerretHeavy_clan_ III-Fwd_2.4 GCTCAGCTGGCTTTTCCTTGTTTCAG FerretHeavy_clan_ III-Fwd_2.5 CACTTGGCTGGGTATTCCTTGTTTCTC Heavy FerretHeavy_clan_ III-Fwd_2.6 ATTGGGCTCGGTTTTCCTTCTTGCTC Secondary FerretHeavy_clan_ III-Fwd_2.7 GCTTGGCTGGGTTTTCCTTCTTGC forward pool FerretHeavy_clan_ III--Fwd_2.8 GCTGGGCTGGGTTTTCCTTCTTGC FerretHeavy_clan_ I-Fwd_2.1 GGTGGCCCTGGCTACAGGTAAG FerretHeavy_clan_ I-Fwd_2.2 GGTGGTGTTGGCTACAGGTAGG FerretHeavy_clan_ I-Fwd_2.3 GGTTGCCCTGGCTACAGGTAAG FerretHeavy_clan_ II-Fwd_2 CTCCTCTGCCTTCTGGCAGCTC

Ferret_IgA-Rev2 CTTCGTCACAGCTGCAGAGGCTC Heavy Ferret_IgG-Rev2 GTAACCTGATACAAGGCATGCAAGGG Secondary Ferret_IgM-Rev2 GTCTCATCGGACTGGAAACTCTCAC reverse pool Ferret_IgD-Rev2 GCCTTGCTTCGGGACTTTACACTC

FerretKappa_ Fwd_1.1 ATGAGGTTCCCTTCTCAGCTCCTG FerretKappa_ Fwd_1.2 ATGAGGTTCCCTGCTCAGCTCCTG FerretKappa_ Fwd_1.3 ATGAGGTTCCCTTCTCAGCTCCTA FerretKappa_ Fwd_1.4 ATGAGGTTCCCTTTTCAGCTTCTG FerretKappa_ Fwd_1.5 ATGAGGTTTCCTGCTCAGCTCCTG FerretKappa_ Fwd_1.6 ATGAAGTTCCCTTCTCAGCTCCTG FerretKappa_ Fwd_1.7 ATGAGGTTCCCATCTCAGTTCCTG FerretKappa_ Fwd_1.8 ATGAGGTTCCCTGCTCAGCTCCTT FerretKappa_ Fwd_1.9 ATGAGGTTCCCTGCTCAGCTCTTT FerretKappa_ Fwd_1.10 ATGAGGTTCCCTGCTCAGCACTTT Kappa Primary forward pool FerretKappa_ Fwd_1.11 ATGAGGTTCCCTGTTCAGCTCCTT FerretKappa_ Fwd_1.12 ATGAGGTTGCCTTCTCAGTTCCT FerretKappa_ Fwd_1.13 ATGAGGTTCCCTGTTCAGCTCCTG FerretKappa_ Fwd_1.14 ATGACTTCCCCTGCTCAGCTCCTG FerretKappa_ Fwd_1.15 ATGGTGACTCCATCACAGCTTCTT FerretKappa_ Fwd_1.16 ATGGGAGTCCTGACCCAACTCCTC FerretKappa_ Fwd_1.17 ATGGGAGTCCCAACCCAACTCCTC FerretKappa_ Fwd_1.18 ATGGGAATCCTGACCCAACTCCTC FerretKappa_ Fwd_1.19 ATGGGGTCCGGGACTCCCCTGCTA FerretKappa_ Fwd_1.20 ATGGTGGGCAAACTCACTGTGGC

Kappa Primary Reverse primer FerretKappa_ Rev_1 CCATCAACCTTCCATTTGACATTGAC

FerretKappa_ Fwd_2.1 CTCCTGGGGCTGCTGATGCTC FerretKappa_ Fwd_2.2 CTCCTGGGGCTGCTAATGCTC FerretKappa_ Fwd_2.3 CTCCTGGGGGTGCTGATGCTC FerretKappa_ Fwd_2.4 CTCCTGGGGCTGATGATGCTC FerretKappa_ Fwd_2.5 CTCCTGGGGCTACTGATGCTT FerretKappa_ Fwd_2.6 CTTCTGGGGCTGCTGATGCTC FerretKappa_ Fwd_2.7 TTCCTGGGGCTGCTGATGCTC FerretKappa_ Fwd_2.8 CTCCTGGGGCTAATGATGCTC FerretKappa_ Fwd_2.9 TTCCTGGGGCTTCTGATGCTC FerretKappa_ Fwd_2.10 CTCCTTGGGCTGCTAATGCTT FerretKappa_ Fwd_2.11 CTCTTTGTACTGCTAATGCTTTGG Kappa Secondary forward pool FerretKappa_ Fwd_2.12 CACTTTGCACTGCTAATGGTTTGG FerretKappa_ Fwd_2.13 CTCCTTGGGCTCCTCATACTT FerretKappa_ Fwd_2.14 CTCCTGGGGTTCCTCATGCTC FerretKappa_ Fwd_2.15 CTCCTGGGGCTCTTCATGCTC FerretKappa_ Fwd_2.16 CTCCTGGGGCTCATGATGCTC FerretKappa_ Fwd_2.17 CTCCTGGGGCTGCTGATTCTC FerretKappa_ Fwd_2.18 CTTCTTGGCCTTCTGCTCCTC FerretKappa_ Fwd_2.19 CTCCTCTGCCTTCTGCTGGC FerretKappa_ Fwd_2.20 CTCCTCTGTGTTCTGCTGGCC FerretKappa_ Fwd_2.21 CTGCTATGGATCCTGCTGCTC FerretKappa_ Fwd_2.22 CTGTGGCTTCGATACTTTTGGTATC

Kappa Secondary reverse primer FerretKappa_ Rev_2 CAGAGGCACTGCCGGTATGTAAC

FerretLambda_ Fwd_1.1 ATGGCCTGGATTCCTGTCCTCC FerretLambda_ Fwd_1.2 ATGGCCTGGACTCTTGTTCTCC FerretLambda_ Fwd_1.3 ATGGCCTGGATTCCAGTCCTCT FerretLambda_ Fwd_1.4 ATGGCCTGGACCCCTCTTTCAC FerretLambda_ Fwd_1.5 ATGGCCTGGTCCCCTGTACTC FerretLambda_ Fwd_1.6 ATGGCCTGGAGCCCTGTCCTC FerretLambda_ Fwd_1.7 ATGGCCTGGGCCACGGTCCTC FerretLambda_ Fwd_1.8 ATGGCCTGGACTCCTCTCCTTC FerretLambda_ Fwd_1.9 ATGGCCTGGACTCCTCTCTCAC FerretLambda_ Fwd_1.10 ATGGCCTGGAGCCCTCTCTCAC FerretLambda_ Fwd_1.11 ATGGCCTGGAGCACTCTTTCAC Lambda primary Forward Primer Pool FerretLambda_ Fwd_1.12 ATGGCCTGGAACCCTCTCTCAC FerretLambda_ Fwd_1.13 ATGGCCTGGACCCCTCTTCTG FerretLambda_ Fwd_1.14 ATGGACTGGATCCCTCTCCTAC FerretLambda_ Fwd_1.15 ATGTACTGGATCCCTCTCCTAC FerretLambda_ Fwd_1.16 ATGGCCTGGGCCCCACTC FerretLambda_ Fwd_1.17 ATGGCCTGGTCCCCTCTTCTC FerretLambda_ Fwd_1.18 ATGGCCTGGACACCTCTCCTC FerretLambda_ Fwd_1.19 ATGGCCTCGACCCCTCTCCTC FerretLambda_ Fwd_1.20 ATGGCCTGGACGCCTCTCCT FerretLambda_ Fwd_1.21 ATGGTCTGGACCCCTCTCCTC FerretLambda_ Fwd_1.22 ATGGCCTTCATCATAGCTTTGTCCTC

Lambda Primary Reverse Primer FerretLambda_ Rev_1 GAAGTCACTGATGAGGCACACCAG

FerretLambda_ Fwd_2.1 CTTGGATTCCTGGCTCACTGC FerretLambda_ Fwd_2.2 CTTGGCCTCCTTGCTCACTGC FerretLambda_ Fwd_2.3 CTCCTTGTCCTCACTCTCTGCAC FerretLambda_ Fwd_2.4 CTCGGAATCCTGGCTCACTGC FerretLambda_ Fwd_2.5 CTCGGAGTCCTGGCTCACTG FerretLambda_ Fwd_2.6 CTTGGCCTCCTTGCTCATTGC FerretLambda_ Fwd_2.7 CTCAGCCTTCTTGCTCACTGC FerretLambda_ Fwd_2.8 CTCACCCTTCTGGCTCACTGTAC FerretLambda_ Fwd_2.9 CTCACCCTCCTCATTCAGTGC FerretLambda_ Fwd_2.10 CCTCTTGCTCCTCTGTCATTGC FerretLambda_ Fwd_2.11 CTCATGCTCCTCTGTCACTGC Lambda Secondary Forward Primer pool FerretLambda_ Fwd_2.12 CCTCATGTTCCTGTCTCACTGC FerretLambda_ Fwd_2.13 CTCATGCTCCTGTCTCACTGC FerretLambda_ Fwd_2.14 CCTCATGCTCCTGTCTCTCTG FerretLambda_ Fwd_2.15 CTCTTCATGCTCCTCTCCCAG FerretLambda_ Fwd_2.16 CACTTCTTGTCCTCACTCTCTGC FerretLambda_ Fwd_2.17 CTCCTCGTCCTCACTCTCTG FerretLambda_ Fwd_2.18 CTCCTCCTCATCACTCTCTGC FerretLambda_ Fwd_2.19 CACTCCTCTTCCTCACTTTCTGC FerretLambda_ Fwd_2.20 GCTCCCAGTCCTCACTCTTTG FerretLambda_ Fwd_2.21 CTCCCCGTCCTCACTCTATG FerretLambda_ Fwd_2.22 CTCCCCGTCCTCACTCTCTG FerretLambda_ Fwd_2.23 CCTTGTTTTCCTGGCTCACTGC

Lambda Secondary Reverse Primer FerretLambda_ Rev_2 CTTGCTGGCGGCGAGTTCCTC

69 3.3.5 Analysis of recovered immunoglobulin sequences

Full length recombined VH, V and V sequences recovered from single sorted ferret

B-cells were analysed using Geneious (10.1.3). A DNA database of previously annotated germline ferret variable gene segments was first created. Sequences coding for recovered ferret variable gene segments was subsequently identified by alignments to annotated ferret variable gene segments using protein BLAST. CDR3 sequences and lengths for both heavy and light chain loci were determined by counting amino acid residues immediately after FR3 starting from the conserved cysteine (C) residue and ending with a conserved tryptophan (W) or phenylalanine

(F).

3.3.6 RNA-seq validation of ferret immunoglobulin constant region sequences

RNA was extracted from five million cryopreserved PBMCs derived from a single immunologically naïve ferret spleen using RNeasy Plus Micro Kit (Qiagen) according to the manufacturers’ instructions. mRNA libraries were prepared using Illumina

Truseq Stranded mRNA kit, and 100 bp single-end reads were obtained using Illumina

HiSeq 3000. The analysis was performed using Galaxy (https://usegalaxy.org) [336].

Sequences were filtered (Q>30) and trimmed (Trim) to remove Illumina adapter sequences. Contigs were assembled de-novo with > 40 bp minimum read overlap for path extension using Trinity [337]. Contigs were aligned to ferret genomic sequences using MAGIC-BLAST [338] to identify putative ferret immunoglobulin constant genes.

70 3.3.7 Human CD16 / CD32 dimer ELISA assay to detect cross-reactive ferret IgG binding activity

96- well ELISA plates were coated with 50 ng per well of chimeric ferret/human antibody, purified polyclonal human IgG1 (Sigma-Aldrich), mouse IgG1

(Thermofisher), mouse IgG2a (Sigma-Aldrich) or Rat IgG2a (Biolegend) antibodies in

1 X PBS and incubated overnight at 4oC. Wells were blocked for 1 h with ELISA blocking buffer at 25oC and washed thrice in ELISA wash buffer. Biotinylated human

CD16 or CD32 dimers (starting at 0.1 g / mL) [124] that were previously serially diluted 3 fold in ELISA dilution buffer (starting at 1:50 dilution) were added to wells and incubated for 1 h at 25oC. Following the wash, CD16 / CD32 binding was detected by incubation for 1 h at 25oC with HRP- Streptavidin diluted at 1:10000 in ELISA blocking buffer. OD630 readings were obtained after addition of Sureblue TMB peroxidase substrate (Seracare) and TMB BlueSTOP Solution (Seracare).

71 3.4 Results

3.4.1 Annotation of ferret germline variable, diversity and joining genes

3.4.1.1 Ferret Immunoglobulin heavy chain locus

The initial publication of the ferret genome was reported in 2014 [312]. However, assembly and annotation of immunoglobulin loci are currently incomplete. Therefore, we identified and annotated genomic contigs containing potential heavy chain variable

(IGHV) genes. 18 IGHV genes retaining an open reading frame (ORF), downstream recombination signal sequence (RSS; heptamer and nonamer) and critical amino acid residues, for example, Cys 74 were considered potentially functional. Based on DNA sequence homology, three groupings of immunoglobulin genes, corresponding to the three vertebrate clans [339] could be delineated (Figure 3-2 A): clan I (3 genes), clan

II (1 gene) and clan III (16 genes). In line with reports from other Carnivora such as dogs [340] and cats [341], the majority of IGHV gene diversity in ferrets lies within Clan

III (human IGHV3 / canine IGHV3), with sequence diversity concentrated within the

CDR-H1 and CDR-H2 regions (Figure 3-2 A). Based upon the conserved arrangement of RSS sequences, we identified 7 putative D gene segments (numbered IGHD1-7)

(Figure 3-2 B), of which 3 appear orthologous to canine and human variants. Similarly,

5 putative germline J gene segments (IGHJ1-5) were identified (Figure 3-2 C) including 2 conserved orthologues.

72 A

Name Contig Homology Leader CDR-H1 CDR-H2 I-HV1 897453 MRAQHFTTDWSWRILFLVVLATGSVYS / QVQLVQSEAEVRKPGESVKVSCKASGYTFTSYAMNWVQQAPGKSLQYMGWIDTNTGKPTYAPGFSGRFVFSTDTSVSTAYLQMNSLNSEDTAVYYCAR hsVH7-4-1, cfVH1-30 I-HV2 897498 MTWSWRILFLVALATGKCVYA / QVHLLQSGAEVRNPGASVKVSCKASGYTFTDYYMHWVRQAPERGLEWMGRIDPEDGATNIAQKFQARVTLMADTSTSMAYMELRSLRSEDTALYYCAR hsVH1-2, cfVH1-30 I-HV3 897453 MTWSWRILFLVALATGKCVYA / QVHLLQSGAEVRNPGASVKVSCKASGYTFTNYYMHWVQQAPERGLEWMGQIDPEDGATNIAQKYQARVTLMADTSTNMAYMELRSLRPEDTALYYCAR hsVH1-69, cfVH1-30 CDR-H1 CDR-H2 II-HV1 897427 MQLLWSLLCLLAAPLGVLS / QLTLQESGPGLVKPSQTLSLTCVVSGGSVTSSYYWNWIRQRPGKALEWMGYWTGSTRYNPAFQGRISITADTSKNQFSLQLSSMTTEDTAVYYCAR hsVH4-61, cfVH4-1 CDR-H1 CDR-H2 III-Consensus EVQLVESGGDLVKPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLQWVAYISNDGSSTYYADSVKGRFTISRDNGKNTLYLQMNSLRAEDTAVYYCAR III-HV1 898421 MEFVLGWVFLVALLKGVQC / EVQLVESGGDLVKPGGSLRLSCTAS GFTF SSYSMQWV RQAP GKGL QWVA YIRY DGGSTSYA DSVK GRFT ISRDNGKNTLYL QMNS LRAE DTALYYCAR hsVH3-30, cfVH3-5 III-HV2 897360 MEFVLSWLFLVSVLKGAQC / EMQLVESGGDLVKPGGSLRLSCEAS GFTF SGYG MSWV RQAP GKRI ELVSNIDA GGGSTSYTDSVKGRFTISRDNAKNTLYL QMNS LRTEDTAMYFCAK hsVH3-23, cfVH3-23 III-HV3 897360 / EMKLVESGGDLVKPGGSLRLSCVAS GFTF SSYG MTWV CQDPGKGPQWVAGIWI DGSFTSYVDSVKGQFT ISRDNGKNTLYL QMNS LRSN DMDVYYCAR hsVH3-33, cfVH3-38 III-HV4 897360 / EMQLVESGGDLVKPEGS LRLS CAAS GFTF SSYG MSWV RQAP GNGL QWVA GISYDGSS TYYA DSVK GRFT ISRDNGKNTLYL QMNS LRAE DTAV YYCAT hsVH3-21, cfVH3-41 III-HV5 897556 MEFVLGWVFLVALLKGVLC / EVQLVESGGDLLKPGGSLRLSCAASGFTFSSYG MSWV HQAPGKGLQWVADISKGGSYTYYTDSVKGRFTISRDNGKNMLYLQMNSLRAEDTAVYYCAT hsVH3-21, cfVH3-43 III-HV6 897453 MELVLGWVFLVALLKGVQC / EVQLVESGGDLVKPGGSLRLSCAASGFIFSSYWMRWV RQAP GKGL KWVTSISNTGSNTYYADSVKGRFTISRDNGKNTLYLQMNSLRAEDTAVYYCAR hsVH3-21, cfVH3-26 III-HV7 897498 MDFVLGWVFLVVLLKGVEC / EVQLVESGGDLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLQWVTSISNTGSNTYYADSVKGRFTISRDNGKNMLYLQMNSLKAE DTAV YYCVK hsVH3-23, cfVH3-35 III-HV8 897795 MDFVLGWVFLVALLKGVQS / EVKLVESGGDLVKPGGSLRLSCAASGFTFSNYDMNWV RQAP GKGL QWVA YISSGGSSTYYADSVKGRFTISRDNDKDMLYLQMNSLRAEDTAMYYCAR hsVH3-69, cfVH3-35 III-HV9 897360 MGFVLGWVFLLALLKGVQC / EVQLVESGGDLVKPGGSLRLSCAASGFTISNNE MNWV RQAP GMGL QWVA YINS VGS-TYYADSVKGRFTISRDNSKNTLHLHMSN LNAE DTAV YYCA R hsVH3-66, cfVH3-35 III-HV10 897427 MKFVLGWVFLVALLKGVQC / EVQLVESGGDLVKPGGSLRLSCAASGFTFSNYGMSRVRQAPGKGLQWVAWISSRSSYTYYTDSVKGQFT ISRDNSKNTLYL QMSSLRAEDTAVYYCAR hsVH3-21, cfVH3-41 III-HV11 897498 / EVQLVESGGDLAKPGGSLRLSCAASGFTFSNYGMSWVRQTPGKGLQWVAYISTSSSYTYYADSVKGQFT VSRDNGKNTLYLQMSSLRAEDTAVYYCAR hsVH3-21, cfVH3-35 III-HV12 897498 MEFVLGWVFLLALLKGVQC / EVQLVESGGDLVKPEGS LRLS CAAS GFTF SNYYMYWV RQAP GKGL QWVA EISNTGSSTYYTDSVKGRFTISRDNGKNTLYLQMNSLRAEDTAMYYCVR hsVH3-69, cfVH3-41 III-HV13 897558 / VVQLVESGGDLVKPRGS LRLS CAAS GFTF SNYHMDWV HQAPGKGLQWVAEISNTGSSTNYA DSVK GQITISRDNGKNTLYLQMNSLRVEDTAVYYCAT hsVH3-69, cfVH3-9 III-HV14 897564 MEFVLGSVFLLALLKGVQC / EVQLVESGEDLVKPGGSLRLSCAASGFTFSNYYMDWV RQAP GKGL QWVA DISGGGSSTSYA DSVK GRFT ISRDNGKNMLYLQMNSLRAEDTTL YYCAT hsVH3-69, cfVH3-2 III-HV15 897453 MEFVLGWVFLLALLKGVQC / EVQLVESGGDLVKPGGSLRLSCAASGFTFSNYYMERVRQAPGKGLQWVAGISRDGSSTSYA DSVK GRFT ISRDNGKNTLYL QINS LRSEDTAMYYCAR hsVH3-66, cfVH3-67 III-HV16 897558 MEFVLGWIFLLALLKGVQC / EVQLVESGGDLVKPGGSLRLSCAASGFTFSNYYMYWV RQAP GKGL QWVA GISRDGSSTSYA DSVK GRFT ISRDNGKNTLYL QINS LRSEDTAVYYCAR hsVH3-69, cfVH3-67

nonomer heptamer heptamer nonomer homology B HD1 GTTTTTTGACAAGGAACTTCCCACTGTGTTACTACGGTAGCTACAGC -----CACAGTGACAGACCCCGGGGCAATAACC cfHD2 IGHD1 L L R * L Q Y Y G S Y S T T V A T HD2 GTTTTTTGACAAGGAACTTCCCACTGTGATAACTACGATAACTAC ------CACAGCGACAGACCTCAGGGCGAAAACC IGHD2 I T T I T * L R * L N Y D N Y HD3 CCATATTGTCTGAGCGTCTGTCACTGTGGGTACTGGAATAGTATCTCC ----CACAGTGACATGTCCTGTGCTCAAAACC IGHD3 G T G I V S V L E * Y L Y W N S I S HD4 GGTTTCTGACCCCGCCTGTGTCACTGTGGCTACAGTAGCAGCTGGATC ----CACGGTGACACTCTCCAGGCCACAAACC cfHD4 IGHD4 A T V A A G L Q * Q L D Y S S S W I IGHD5 HD5 TGGGAAAATTGGACAGCCACGTACAGTGGAATGAAACTGGACCATTCACTTA CACTGTACACCAAGATAAATTCAAAATG E * N W T I H L N E T G P F T M K L D H D L HD6 TGAGAAGTGGTTGAGGGCATGTACAGTGGAATGTATCAACCATGATCCAGG -CACGGTGCACCCTGAAATGCTTCTGAGC IGHD6 E C I N H D P N V S T M I Q M Y Q P * S R HD7 GGTTTTGGCTGAGCCAGGAACCACAGTGCTAACTGGGGC------CACAGTGATTCACAACCCTACAGAAACC cfHD6 IGHD7 L T G * L G N W G C nonomer heptamer homology IGHJ1HJ1 GGGTCTTTGTCTGGGAGGCGCAGCTGGGCAGA GACTGTG-CTTCTGGGATTTGGTTTACTGGGGCCAAGGCTCCCTGGTCACCGTCTCCTCAG F W D L V Y W G Q G S L V T V S S IGHJ2HJ2 GCTTTCTGTGTGCAGCGGCTGGGCAGCTGCAC CAGTGTGGCCATGACTACTTGGACGTCTGGGGCCAGGGCACCCTGGTGACCGTCTCCTCAG H D Y L D V W G Q G T L V T V S S IGHJ3HJ3 GGTTTTTGTACACCCCCTGACGGGGCCTTTGG CAATGTG------AGTACTTTGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCAG Y F D Y W G Q G T L V T V S S hsHJ4, cfHJ2 IGHJ4HJ4 CGACTTCTTGTCCAGATTTCCTGCACAATTGT CACATTG-TGACAACTGGCTTGACTACTGGGGCCAGGGGACCCTGGTCACCGTGTCCTCAG D N W L D Y W G Q G T L V T V S S HJ5 GGGTTTTGGTGGGGTGCGGATGGCGAATTCAC CACTGTGACTACTATGCTATGGGCTACTGGGGCCAGGGGACCTCGGTCACCGTGTCCTCAG IGHJ5 cfHJ3 Y Y A M G Y W G Q G T S V T V S S HJ(P) CGAGGTGGACGGTGCGGGCCAGGAGGGTCTCTGTTGGGGGAGAAGCAGGGACCGTTCCTGGGGCCAGGGGACCCTGGTCCCCATGTCCTCAG R S R D R S W G Q G T L V P M S S

73 Figure 3-2 Ferret immunoglobulin germline heavy variable, diversity and joining genes (A) Non- rooted phylogenetic tree of putative ferret heavy chain variable gene segments. Branch lengths are proportional to genetic distance as indicated. Multiple Sequence Alignments of annotated germline heavy variable genes. Amino acid sequences of IGHV genes were aligned. Residues that differ from the consensus sequence in redare higlighted. Closest human (Hs) and canine (cf) homologs are shown. (B) Coding and RSS sequences of ferret heavy chain diversity gene (IGHD) segments. (C) Coding and RSS sequences of ferret heavy joining gene (IGHJ) segments

74 3.4.1.2 Ferret immunoglobulin kappa chain locus

48 ferret kappa variable germline genes were identified and divided among the three mammalian IGKV clans with the majority (44) belonging to clan II (human IGKV2, 3 and 4/ canine IGKV 2,3,4) and 2 genes each in clans I and II (Figure 3-3 A). Similar to the heavy chain variable gene transcripts, the majority of the sequence diversity is concentrated in the CDR1 and CDR2 regions. As for joining genes, five putative genes

(IGKJ 1-5) were identified and orthologous to human and canine homologues, but only two (IGKJ1 & IGKJ3) have functional RSS sequences (Figure 3-3 B). The 0.7Mb kappa chain locus is contained within a single contig of the ferret genome (contig

GL896905.1) (Figure 3-3 C) enabling the determination of relative positions and orientations of the variable gene segments. Alignments of the identified ferret IGKV genes are available in appendix 3.1.

CDR1 CDR2

Homology B

76 Figure 3-3 Ferret immunoglobulin germline kappa variable and joining genes. (A) Non-rooted phylogenetic tree of kappa variable gene segments detailing the three potential clans identified. Branch lengths are proportional to genetic distance as indicated (B) Immunoglobulin kappa joining genes. Coding and 5’RSS sequences are shown as indicated. All sequences were extracted from contig GL896905.1. Closest human (Hs) and canine (cf) homologs are shown (C) Schematic of the kappa locus (contig GL896905.1) detailing the orientation of putative functional IGKV segments relative to the joining and constant genes

77 3.4.1.3 Ferret immunoglobulin lambda chain locus

Contigs bridging the lambda chain locus were extracted and analysed for lambda variable gene segments (IGLV) (Figure 3-4). 32 potentially functional IGLV gene segments were identified, spanning four probable clans, with 2 genes each in clans I and IV, 22 genes in clan II (human IGLV3/ canine IGLV3) and 6 genes in clan V (Figure

3-4 A). We did not identify any clan III sequences in the current draft copy of the genome, suggesting the presence of unknown lambda chain variable genes yet to be discovered in the ferret genome. Sequences are homologous to human/canine orthologs, and diversity is concentrated in the CDR1 and CDR2 regions. 4 lambda joining gene segments (IGLJ) were identified and appear orthologous to human and canine variants (Figure 3-4 B). Alignments of the annotate ferret lambda variable genes are available in appendix 3.2.

78 A

Homology

Figure 3-4 Ferret immunoglobulin germline lambda variable and joining genes. (A) Non-rooted phylogenetic tree of lambda variable gene segments detailing the four potential clans identified. Branch lengths are proportional to genetic distance as indicated (B) Immunoglobulin kappa joining genes. Coding and 5’-RSS sequences are shown as indicated.

79 3.4.2 Single-cell RT-PCR for recovery of ferret immunoglobulin sequences

Multiplex PCR primers (Table 3-1) targeting leader and constant regions were designed to investigate the expressed repertoire of germline immunoglobulin gene segments. Using an approach analogous to other mammals [170, 171], single ferret B cells were sorted from cryopreserved splenocyte preparations by flow cytometry using a simple antibody panel and gating scheme, which enabled the isolation of single immunoglobulin positive ferret B-cells (Figure 3-5-A). This facilitated the recovery of recombined heavy (IgM), kappa and lambda mRNA transcripts.

3.4.2.1 Recovery of ferret immunoglobulin heavy chain sequences cDNA was generated, and immunoglobulin sequences were recovered by nested multiplex PCR with ferret specific immunoglobulin primers. 121 functional recombined

IgM+ heavy chain sequences were recovered from 480 sorted ferret B cells from three genetically outbred ferrets (25% recovery). In line with the frequency of germline gene segments, the majority of immunoglobulins were derived from clan III genes, with only two sequences recovered from clan II and a single example of a sequence from clan

I (Figure 3-5 B).

An accurate assessment of germline IGHV gene utilisation is difficult due to the current inability to segregate naïve versus B cells that are somatically mutated (memory) during the sort, the limited genomic information surrounding the ferret IGHV locus and a poor understanding of any allotypic variation within these outbred animals.

Nevertheless, we recovered examples of 7 IGHV gene sequences that were highly conserved (>99% amino acid sequence homology) to predicted germlines from sequences annotated from a single ferret including examples from two of the three

80 clans (II-HV1, III-HV7, III-HV8, III-HV9, III-HV12, III-HV16). A single clan I sequence was recovered, albeit with more limited homology to the annotated germline gene

(97.6%). The 18 ferret IGHV genes found within genomic contigs is less than observed in felines (24 genes) and canines (38 genes) [342], suggesting additional variable germlines may remain undiscovered. Supporting this, we repeatedly recovered multiple heavy chain immunoglobulin sequences that shared identical IGHV gene sequences but recombined with different D and J genes, strongly suggestive of a common germline progenitor (III-HV17, III-HV18, III-HV19 and III-HV20, III-HV21). The existence and identity of these additional putative germlines (Appendix 3.3) will be clarified as genomic sequencing of ferrets continues. The utilisation of all five predicted

IGHJ gene segments and 6 of 7 IGHD gene segments (not IGHD6) was evident within recombined BCR sequences. CDR-H3 regions, often a critical determinant for antigen recognition, ranged from 5 to 25 amino acids (mean 13.2) (Figure 3-5 C) in length; broadly comparable to canines [343] and potentially shorter on average than observed in humans [344].

3.4.2.2 Recovery of ferret immunoglobulin kappa chain sequences

99/480 (20.6%) functional and recombined kappa immunoglobulins were recovered, all of which were derived from variable gene segments belonging to clan II and recombined with 4 of 5 predicted IGKJ gene segments (not IGKJ2). Germline variable gene segments II-KV13, II-KV18, II-KV19 II-KV20, II-KV21, II-KV22. II-KV23, II-KV29,

II-KV36, II-KV40, II-KV41, II-KV42 and II-KV43 were recovered and highly conserved

(>99% identity) to the annotated germline genes (Figure 3-5 B). CDR3 lengths of the recovered sequences ranged from 5 to 11 amino acids, with a modal AA length of 9

(Figure 3-5 C) Four additional potential novel germlines were evident within recovered

81 sequences (II-KV45-48) (table 3) and most likely represented allelic variants of clan II germlines (Listed in appendix 3.4).

3.4.2.3 Recovery of ferret immunoglobulin lambda chain sequences

144/384 (37.5%) productive and recombined lambda chain transcripts were recovered. Most sequences are homologous to annotated clan I and clan II germline sequences (II-LVI1, II-LV2, II-LV4, II-LV5, II-LV6, II-LV7, II-LV8, II-LV11, II-LV12, II-

LV14, II-LV15, II-LV16, II-LV17, II-LV18, II-LV20, I-LV1) (Figure 3-5 B) and recombined with 3 out of 4 identified lambda joining genes (not IGLJ4). Additional potential variable genes were also recovered (II-LV22, II-LV23) as were sequences putatively derived from clan V but with limited homology to the annotated genes. The lengths of the CDR-L3 ranged 8-13 for the lambda locus, with a modal length of 11 AA

(Figure 3-5 C). Nucleotide sequences of annotated and recovered IGLV genes are listed in appendix 3.5. Overall, this initial pilot allowed about 31/480 (6.5%) recovery efficiency of functional kappa chain coding mAb sequence pairs and 29/480 (6.0%) lambda chain coding mAb sequence pairs from single sorted ferret B cells. Further improvements to this multiplex PCR approach will no doubt be possible with continued refinements to primer design and flow cytometric panels, and as more genomic information becomes available.

A Lymphocytes Singlet DUMP Ferret B-cells

A -

SSC

SSC AQUA/CD11b

Ferret IgA/G/M Ferret

FSC-A SSC-H CD8

Heavy Kappa Lambda B

60 40 80 60

s 20

e e

c 20 c

n n

60 n

u u u 40

q 15

e e

e 20

b b

b 20

m m

m 5

n n n 0 0 0 8 9 0 1 2 3 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 5 6 7 8 9 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 1 1 CDR-H3 length CDR-L3 length (Kappa) CDR-L3 length (lambda)

Figure 3-5 Genetic features of recovered ferret germline immunoglobulin sequences. (A) Gating scheme for recovery of recombined ferret immunoglobulin sequences. Ferret cells were first gated on single, live lymphocytes. Dead cells, CD11b myeloid cells and CD8+ T cells were dumped before gating for IgA/IgG/IgM positive ferret B-cells (B) Distribution of variable germline genes recovered from productive, recombined heavy and light chain immunoglobulins are shown as pie charts. The width of each segment is proportional to the number of recovered sequences. Sequences corresponding to predicted germlines are shown in solid (less than 1% variable), while sequences with poor alignment to predicted germlines are hatched and indicated in red. (C) Distribution of CDR-H3 and CDR-L3 lengths among recovered immunoglobulin sequences.

83 3.4.3 Sequence validation of ferret immunoglobulin constant gene segments

Using a next-generation sequencing approach analogous to previous reports [345], we recovered cDNA sequences of ferret constant regions and investigated potential immunoglobulin subclasses. RNA was extracted from ferret splenocytes and subjected to RNA-Seq. Putative mRNA transcripts were assembled de novo and 5 heavy chain isotypes (IgM, IgG, IgE, IgD and IgA) and two light chain (IgK and IgL) constant genes identified. Minimal variation was observed to artificially spliced genomic sequences, except for mutations which resulted in single amino-acid substitutions (Figure 3-6) which could indicate allelic variation within outbred ferrets.

Notably, we were unable to identify any IgG subclass variants using this approach, which with four distinct subclasses identified in other carnivores [346], might reflect low transcript abundance within our samples precluding sufficient subclass cDNA recovery.

84 ferret_kappa_Transcript 1 RNDAQPSVFLFQPSPDQLHTGSASVVCMLNGFYPREVNVKWKVDGVTKNTGILESVTEQD 60 RNDAQPSVFLFQPSPDQLHTGSASVVCMLNGFYPREVNVKWKVDGVTKNTGILESVTEQD ferret_kappa_Genomic 1 RNDAQPSVFLFQPSPDQLHTGSASVVCMLNGFYPREVNVKWKVDGVTKNTGILESVTEQD 60 ferret_kappa_Transcript 61 SKDSTYSLSSTLTMPSTEYLSHEKYSCEVTHKSLSSPLVKSFQRSECQ 108 SKDSTYSLSSTLTMPSTEYLSHEKYSCEVTHKSLSSPLVKSFQRSECQ ferret_kappa_Genomic 61 SKDSTYSLSSTLTMPSTEYLSHEKYSCEVTHKSLSSPLVKSFQRSECQ 108

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ferret_lambda_Transcript 1 QPKSAPSVTLFPPSSEELAASKATLVCLISDFYPSGMTVAWKADGSPVTQGVETTKPSKQ 60 QP SAPSVTLFPPSSEELAASKATLVCLISDFYPSG+TVAWKADGSPVTQGVETTKPSKQ ferret_lambda_Genomic 1 QPTSAPSVTLFPPSSEELAASKATLVCLISDFYPSGVTVAWKADGSPVTQGVETTKPSKQ 60 ferret_lambda_Transcript 61 SNNKYAASSYLSLSPDKWQSHSSFSCLVTHEGNTVEKKVVPSQCS 105 SNNKYAASSYLSLSPDKWQSHSSFSCLVTHEGNTVEKKVVPSQCS ferret_lambda_Genomic 61 SNNKYAASSYLSLSPDKWQSHSSFSCLVTHEGNTVEKKVVPSQCS 105 ______

Ferret_IgM_Transcript 1 ENPSPPNLFPLITCESFQSDETLVAMGCLAGDFLPDTVTFSWTYKNDSEVSRQNVKYFPS 60 ENPSPPNLFPLITCESFQSDETLVAMGCLAGDFLPDTVTFSWTYKNDSEVSRQNVKYFPS Ferret_IgM_Genomic 1 ENPSPPNLFPLITCESFQSDETLVAMGCLAGDFLPDTVTFSWTYKNDSEVSRQNVKYFPS 60

Ferret_IgM_Transcript 61 VLREGKYVATSQVFLPSVDVLQGSEDYLTCKVKHTKGNKSVHVPLPAAVELPPNVTVFIP 120 VLREGKYVATSQVFLPSVDVLQGSEDYLTCKVKHTKGNKSVHVPLPAAVELPPNVTVFIP Ferret_IgM_Genomic 61 VLREGKYVATSQVFLPSVDVLQGSEDYLTCKVKHTKGNKSVHVPLPAAVELPPNVTVFIP 120

Ferret_IgM_Transcript 121 PRDAFSGNGQRTSQLLCQARGFSPKQISVSWFRDGKPLASGIDTGKVEADGKVSGTVTYR 180 PRDAFSGNGQRTSQLLCQARGFSPKQISVSWFRDGKPLASGIDTGKVEADGKVSGTVTYR Ferret_IgM_Genomic 121 PRDAFSGNGQRTSQLLCQARGFSPKQISVSWFRDGKPLASGIDTGKVEADGKVSGTVTYR 180

Ferret_IgM_Transcript 181 VLSTLTITESAWLSQSVFTCQVEHSGVTSERNVSSVCTSNPAVGIRVFTIPPSFANIFLT 240 VLSTLTITESAWLSQSVFTCQVEHSGVTSERNVSSVCTSNPAVGIRVFTIPPSFA+IFLT Ferret_IgM_Genomic 181 VLSTLTITESAWLSQSVFTCQVEHSGVTSERNVSSVCTSNPAVGIRVFTIPPSFASIFLT 240

Ferret_IgM_Transcript 241 KSAKLSCLVTDLGTYDSLTITWTRQNGEPLKTHTNISESHPNITFSAMGEATVCVEDWES 300 KSAKLSCLVTDLGTYDSLTITWTRQNGEPLKTHTNISESHPNITFSAMGEATVCVEDWES Ferret_IgM_Genomic 241 KSAKLSCLVTDLGTYDSLTITWTRQNGEPLKTHTNISESHPNITFSAMGEATVCVEDWES 300

Ferret_IgM_Transcript 301 GEQFTCTVTHTDLPSPLKKTISRPKGVPKHMPSVYVLPPSREQLSLRESASVTCLVTGFS 360 GEQFTCTVTHTDLPSPLKKTISRPKGVPKHMPSVYVLPPSREQLSLRESASVTCLVTGFS Ferret_IgM_Genomic 301 GEQFTCTVTHTDLPSPLKKTISRPKGVPKHMPSVYVLPPSREQLSLRESASVTCLVTGFS 360

Ferret_IgM_Transcript 361 PPDVFVQWLQKGQPVPPNTYVTSAPMPEPQAPGLYFVHSTLTVSEEDWSAGETYTCVVGH 420 PPDVFVQWLQKGQPVPPNTYVTSAPMPEPQAPGLYFVHSTLTVSEEDWSAGETYTCVVGH Ferret_IgM_Genomic 361 PPDVFVQWLQKGQPVPPNTYVTSAPMPEPQAPGLYFVHSTLTVSEEDWSAGETYTCVVGH 420

Ferret_IgM_Transcript 421 EVLPHMVTERSVDKSTGKPTLYNVSLVLSDTAGTCY 456 EVLPHMVTERSVDKSTGKPTLYNVSLVLSDTAGTCY Ferret_IgM_Genomic 421 EVLPHMVTERSVDKSTGKPTLYNVSLVLSDTAGTCY 456

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ferret_IgG_Transcript 1 ASTTAPSVFPLAPSCGATPGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSS 60 GSLTSGVHTFPSVLQSS Ferret_IgG_Genomic* 1 ------GSLTSGVHTFPSVLQSS 17 ferret_IgG_Transcript 61 GLYSLSSMVTVPSSRWPSDTFICTVAHPASNTKVDKRVTQGGPPHTDPCKKCPQPPACDM 120 GLYSLSSMVTVPSSRWPSDTFICTVAHPASNTKVDKRVTQ GP HTDPCK CPQPPACDM Ferret_IgG_Genomic 18 GLYSLSSMVTVPSSRWPSDTFICTVAHPASNTKVDKRVTQRGPTHTDPCKNCPQPPACDM 77 ferret_IgG_Transcript 121 LGGPSVFMFPPKPKDTLSISRTPEVTCMVVDLEDPEVQISWFVDNQEVHAAKTNSREQQF 180 LGGPSVFMFPPKPKDTLSISRTPEVTCMVVDLEDPEVQISWFVDNQEVHAAKTNSREQQF Ferret_IgG_Genomic 78 LGGPSVFMFPPKPKDTLSISRTPEVTCMVVDLEDPEVQISWFVDNQEVHAAKTNSREQQF 137 ferret_IgG_Transcript 181 NSTFRVVSVLPIQHQDWLKGKVFKCKVNNKALPSPIERTISKARGEPHQPSVYVLPPPRD 240 NSTFRVVSVLPIQHQDWLKGKVFKCKVNNKALPS IERTISKARGEPHQPSVYVLPPPRD Ferret_IgG_Genomic 138 NSTFRVVSVLPIQHQDWLKGKVFKCKVNNKALPSAIERTISKARGEPHQPSVYVLPPPRD 197 ferret_IgG_Transcript 241 EMSRTTISVTCLVKDFYPPDIDVEWQSNGRQLPEASVRTTPPQLDADGSYFLYSKLSVDK 300 EMSRTTISVTCLVKDFYPPDIDVEWQSNGRQLPEASVRTTPPQLDADGSYFLYSKLSVDK Ferret_IgG_Genomic 198 EMSRTTISVTCLVKDFYPPDIDVEWQSNGRQLPEASVRTTPPQLDADGSYFLYSKLSVDK 257 ferret_IgG_Transcript 301 AHWQRGDTFTCAVLHEALHNHHTQKSISQSPGK 333 AHWQRGDTFTCAVLHEALHNHHTQKSIS+SPGK Ferret_IgG_Genomic 258 AHWQRGDTFTCAVLHEALHNHHTQKSISKSPGK 290

Ferret_IgE_Transcript 1 ASSQGLSVFPLTPCCKGMAGATSVSLGCLVSGYLPMPVTVTWDTGSLNKSVATVPATFDQ 60 ASSQGLSVFPLTPCCKGMAGATSVSLGCLVSGYLPMPVTVTWDTGSLNKSVATVPATFDQ Ferret_IgE_Genomic 1 ASSQGLSVFPLTPCCKGMAGATSVSLGCLVSGYLPMPVTVTWDTGSLNKSVATVPATFDQ 60

Ferret_IgE_Transcript 61 TSGLHNAISQVTSWGEWAKHTFTCSVAHAASPAINKTFRACAMNFSPPSVKLFHSSCNPL 120 TSGLHNAISQVTSWGEWAKHTFTCSVAHAASPAINKTFRACAMNFSPPSVKLFHSSCNPL Ferret_IgE_Genomic 61 TSGLHNAISQVTSWGEWAKHTFTCSVAHAASPAINKTFRACAMNFSPPSVKLFHSSCNPL 120

Ferret_IgE_Transcript 121 GDTHTTIQLLCLISGYVPGDMEVIWLVDGQRVTDMFSYTAPGTQEGNVTSTHSELNITQG 180 GDTHTTIQLLCLISGYVPGDMEVIWLVDGQRVTDMFSYTAPGTQEGNVTSTHSELNITQG Ferret_IgE_Genomic 121 GDTHTTIQLLCLISGYVPGDMEVIWLVDGQRVTDMFSYTAPGTQEGNVTSTHSELNITQG 180

Ferret_IgE_Transcript 181 EWVSQKTYTCRVSYQGFHFEDHALKCTESDPRGVSSYLSPPSPLDLYVHKSPKITCLVVD 240 EWVSQKTYTCRVSYQGFHFEDHALKCTESDPRGVSSYLSPPSPLDLYVHKSPKITCLVVD Ferret_IgE_Genomic 181 EWVSQKTYTCRVSYQGFHFEDHALKCTESDPRGVSSYLSPPSPLDLYVHKSPKITCLVVD 240

85 Ferret_IgE_Transcript 241 LASTEGMSLTWSRESGEPVHPDGLKEETQFNGTVSVTSTLPVDTQDWVEGEAYHCTVNHP 300 LASTEGMSLTWSRESGEPVHPDGLKEETQFNGTVSVTSTLPVDTQDWVEGEAYHCTVNHP Ferret_IgE_Genomic 241 LASTEGMSLTWSRESGEPVHPDGLKEETQFNGTVSVTSTLPVDTQDWVEGEAYHCTVNHP 300

Ferret_IgE_Transcript 301 DLPKSLVRSIAKTPGQRAAPEVHVFLPPEEDESTTDRVTLTCLIQNFFPADISVQWLRND 360 DLPKSLVRSIAKTPGQRAAPEVHVFLPPEEDESTTDRVTLTCLIQNFFPADISVQWLRND Ferret_IgE_Genomic 301 DLPKSLVRSIAKTPGQRAAPEVHVFLPPEEDESTTDRVTLTCLIQNFFPADISVQWLRND 360

Ferret_IgE_Transcript 361 RPMQAGQQATTRPHKVPGARRAFFVFSRLEVSRRDWEEQNSFACQVVHEALPKSRVFKKT 420 RPMQAGQQATTRPHKVPGARRAFFVFSRLEVSRRDWEEQNSFACQVVHEALPKSRVFKKT Ferret_IgE_Genomic 361 RPMQAGQQATTRPHKVPGARRAFFVFSRLEVSRRDWEEQNSFACQVVHEALPKSRVFKKT 420

Ferret_IgE_Transcript 421 VSKTPGK 427 VSKTPGK Ferret_IgE_Genomic 421 VSKTPGK 427 ______

Ferret_IgD_Transcript 1 ETLHLFPLVSECKVPKQGDSLGLACLAQGPSVESLRVASSSSSGPQTTTMITLGSRERMQ 60 ETLHLFPLVSECKVPKQGDSLGLACLAQGPSVESLRV SSSSSGPQTTTMITLGSRERMQ Ferret_IgD_Genomic 1 ETLHLFPLVSECKVPKQGDSLGLACLAQGPSVESLRVVSSSSSGPQTTTMITLGSRERMQ 60

Ferret_IgD_Transcript 61 LSFLSVFWKPDPHFCRAVVSGRPQQKPIPWPESWERKTDPPAHGPSPRETSTPSAPVSST 120 LSFLSVFWKPDPHFCRAVVSGRPQQKPIPWPESWERKTDPPAHGPSP ETSTPSAPVSST Ferret_IgD_Genomic 61 LSFLSVFWKPDPHFCRAVVSGRPQQKPIPWPESWERKTDPPAHGPSPPETSTPSAPVSST 120

Ferret_IgD_Transcript 121 RHTRTQAAESGSPVDATLRDCRNHTHPPSLYLLQPPLRGPWLQGEATFTCLAVGDDLQEA 180 RHTRTQAAESGSPVDATLRDCRNHTHPPSLYLLQPPLRGPWLQGEATFTCLAVGDDLQEA Ferret_IgD_Genomic 121 RHTRTQAAESGSPVDATLRDCRNHTHPPSLYLLQPPLRGPWLQGEATFTCLAVGDDLQEA 180

Ferret_IgD_Transcript 181 RMSWAVARAPPSGAVEEELREEHTNGSQSLSSRLALPVSLWASGTSIACTLSLPNRPAQV 240 RMSWAVARAPPSGAVEEELREEHTNGSQSLSSRLALPVSLWASGTSIACTLSLPNRPAQV Ferret_IgD_Genomic 181 RMSWAVARAPPSGAVEEELREEHTNGSQSLSSRLALPVSLWASGTSIACTLSLPNRPAQV 240

Ferret_IgD_Transcript 241 ASVAPGQHAATAPSSLTVRVLTVRQAASWLLCEVSGFSPPDILLTWLKGRTEVDPGAFAT 300 ASVAPGQHAATAPSSLTVRVLTVRQAASWLLCEVSGFSPPDILLTWLKGR EVDPGAFAT Ferret_IgD_Genomic 241 ASVAPGQHAATAPSSLTVRVLTVRQAASWLLCEVSGFSPPDILLTWLKGRMEVDPGAFAT 300

Ferret_IgD_Transcript 301 ARPMVQPGNSTFWTWSVLRVLAAQSPGPATYTCVVRHDASRKLFNSSQSLDAGLATTPPP 360 ARPM QPGNSTFWTWSVLRVLAAQSPGPATYTCVVRHDASRKLFNSSQSLDAGLA TPPP Ferret_IgD_Genomic 301 ARPMAQPGNSTFWTWSVLRVLAAQSPGPATYTCVVRHDASRKLFNSSQSLDAGLAMTPPP 360

Ferret_IgD_Transcript 361 PQSHEESRGYATDLGDATDLEDTGGLWPTFAALFLLALLYSGFVTFIKVK 410 PQSHEESRGYATDLGDATDLEDTGGLWPTFAALFLLALLYSGFVTFIKV+ Ferret_IgD_Genomic 361 PQSHEESRGYATDLGDATDLEDTGGLWPTFAALFLLALLYSGFVTFIKVR 410

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Ferret_IgA_Transcript 1 EPKASPSVFPLSLCSCDEAGHVVIACLVQGFFPPEPVKVTWSPGKEGASVRSFPPVKATA 60 EPKASPSVFPLSLCSCDEAGHVVIACLVQGFFPPEPVKVTWSPGKEGASVRSFPPVKATA Ferret_IgA_Genomic 1 EPKASPSVFPLSLCSCDEAGHVVIACLVQGFFPPEPVKVTWSPGKEGASVRSFPPVKATA 60

Ferret_IgA_Transcript 61 GSLYTMSSQLTLPADQCPAGSSLQCHVQHLSDPSKAVSVPCQGRGLCPPHCQCPSCDQPR 120 GSLYTMSSQLTLPADQCPAGSSLQCHVQHLSDPSKAVSVPCQGRGLCPPHCQCPSCDQPR Ferret_IgA_Genomic 61 GSLYTMSSQLTLPADQCPAGSSLQCHVQHLSDPSKAVSVPCQGRGLCPPHCQCPSCDQPR 120

Ferret_IgA_Transcript 121 LSLHPPALEDLLVTSNGSLTCTLSGLKDPKGASFSWTPSGEKDAIQKAPKRDACGCYSVS 180 LSLHPPALEDLLVTSNGSLTCTLSGLKDPKGASFSWTPSGEKDAIQKAPKRDACGCYSVS Ferret_IgA_Genomic 121 LSLHPPALEDLLVTSNGSLTCTLSGLKDPKGASFSWTPSGEKDAIQKAPKRDACGCYSVS 180

Ferret_IgA_Transcript 181 SVLPGCAAPWNSGVTFSCTATHPESKSPITGNISKLLGNTFRPQVHLLPPPSEELALNEL 240 SVLPGCAAPWNSGVTFSCTATHPESKSPITGNISKLLGNTFRPQVHLLPPPSEELALNEL Ferret_IgA_Genomic 181 SVLPGCAAPWNSGVTFSCTATHPESKSPITGNISKLLGNTFRPQVHLLPPPSEELALNEL 240

Ferret_IgA_Transcript 241 VSLTCLVRGFSPKDVLVRWQQGTQELPPEKYTTWKSLKEPGRGSPTFAVTSVLRVDAEAW 300 VSLTCLVRGFSPKDVLVRWQQGTQELPPEKYTTWKSLKEPGRGSPTFAVTSVLRVDAEAW Ferret_IgA_Genomic 241 VSLTCLVRGFSPKDVLVRWQQGTQELPPEKYTTWKSLKEPGRGSPTFAVTSVLRVDAEAW 300

Ferret_IgA_Transcript 301 KQGDKFSCVVGHEALGPANFTEKTIDRLAGKPTHVNVSVVVAEADGVCY 349 KQGDKFSCVVGHEALGPANFTEKTIDRLAGKPTHVNVSVVVAEADGVCY Ferret_IgA_Genomic 301 KQGDKFSCVVGHEALGPANFTEKTIDRLAGKPTHVNVSVVVAEADGVCY 349

Figure 3-6. Alignments of transcript and artificially spliced ferret immunoglobulin constant regions. Amino acid substitutions are indicated in red. *5’- end of ferret IgG genomic sequences are missing due to incomplete sequencing of the ferret genome.

86 3.4.4 Recombinant Expression of chimeric human/ferret antibodies

We next developed the capacity to express and purify recombinant ferret IgG.

Constant genes for ferret IgG and lambda chains were synthesised and cloned into human expression vectors. Recombined human VDJ (heavy) and VJ (lambda) genes from influenza-specific human antibody CR9114 [326] were joined to ferret constant regions to create chimeric IgG coding expression plasmids. The recombinant expression of chimeric human/ferret kappa chain utilising antibodies was unsuccessful and requires further development.

Co-transfection of heavy and lambda chain plasmids into a eukaryotic expression system (Expi293) enabled the purification of chimeric ferret/human IgG using standard protein-A purification (Figure 3-7 A), with the resultant antibody retaining HA-specificity

(Figure 3-7 B). We next attempted to examine human CD16/32 dimer cross-reactivity with this ferret IgG subclass, which would enable the detection of antibodies involved in ADCC responses. No binding was observed, showing the lack of cross-reactivity with human CD16/32, similar to mouse IgG1 and rat IgG2A (Figure 3-7 C). This highlights the need for the development of reagents suitable for studying NK-cell mediated functions such as ADCC in ferrets.

Figure 3-7 Expression and recovery of chimeric ferret IgG/IgL antibody expressing human CR9114 variable regions. (A) Denaturing SDS-PAGE gel of recombinant chimeric ferret antibody. Heavy (50kDa) and Light chain (25kDa) bands are shown and indicated. (B) HA-specific ELISAs to validate chimeric ferret antibody. Ferret-Human CR9114 mAb and serum samples from immunologically naïve ferrets (naïve serum) or ferrets infected with 1000 TCID50 A/California/04/2009 (infected serum) (28 d.p.i) and serially diluted in PBS to detect A/California/04/2009 HA binding. 1x PBS, as well as serum samples derived from naïve or ferrets infected with A/California/04/2009, was included as a control. (C) CD16/32 dimer binding ELISAs. 96-well ELISA plates were first coated with IgGs from various species. CD16/CD32 binding was detected using biotinylated CD16/32 dimers. The binding was detected subsequently using SA-HRP and TMB, followed by OD630 measurements.

88 3.5 Discussion

In this chapter, we sought to improve the utility of ferrets as an immunological model by deriving tools for the analysis of B cell immunity. V, D, J and C segments were identified in the draft copy of the ferret genome using conserved human and canine immunoglobulin gene orthologues. This facilitated the design of novel ferret immunoglobulin specific nested multiplex PCR primers, which enabled the recovery of germline variable segment coding genes from single ferret B-cells.

A lack of available reagents currently limits the phenotypic potential of our sort panel, especially an inability to sort memory B cells or distinguish between different heavy chain subclasses. Nevertheless, a broad cross-section of predicted germline segments from both heavy and light chain loci was amplified, including several potentially novel germline genes currently absent from available genomic contigs. We noted highly biased V gene utilisation for both heavy and light chain immunoglobulin repertoires, with a majority of recovered sequences derived from the most numerous

V gene clans (which we termed HV-III, KV-II and LV-II respectively). This observation mirrors the gene distribution in other carnivores, where the majority of recovered heavy chain sequences are similarly biased [340, 341, 347, 348]. Light chain bias has been reported for mice (kappa) [349] and both dogs and felines (lambda) [350]. Humans display more balanced usage of both the kappa and lambda chains [348, 351], as did ferrets in the current study. The distribution of ferret CDR-H3 (mode 14 AA) and lambda and kappa CDR-L3 lengths (mode 11 AA and 9 AA respectively) were broadly similar to reports from cats [341], dogs [347] and humans [352].

89 The utility of BCR repertoire analysis to study antigen-specific B-cell responses in ferrets will increase as key knowledge gaps are bridged. Novel ferret-specific flow cytometry reagents are required to enable the resolution of different ferret B-cell populations, in particular, markers such as IgD will be required to resolve antigen- activated B cell populations to facilitate the study of rare antigen-specific B-cell populations.

As ferret genome sequencing is still a work-in-progress, as shown by the fragmented contigs containing heavy chain and lambda chain genes, more immunoglobulin coding genes such as IGLV clan III sequences remain undiscovered. Increased genomic information, particularly, the confirmation of ferret immunoglobulin germline genes and allelic variation among outbred ferrets will allow the development of comprehensive gene databases such as those maintained by International ImMunogeneTics (IMGT)

[315]. This is critical to enable further improvements to the currently presented primer sets to improve the recovery rates of ferret immunoglobulin transcripts. This information will also be useful to differentiate allelic variants and somatically induced mutations in clonally expanded B-cell subsets when analysing antigen-specific responses in ferrets. Such improvements will eventually facilitate in-depth immune repertoire analysis using next-generation sequencing approaches that are in wide use for humans and other model animal species[353].

While we employed a multiplex approach to amplify immunoglobulin genes, the use of template switching based methods such as 5’RACE has been applied to annotate immunoglobulin genes from animal models such as dogs [340]. This approach may

90 reduce the potential for primer bias driving the preferential recovery of specific immunoglobulin gene segments.

Alternatively, next-generation high throughput approaches and tools for the analysis of large RNA-seq data sets such as VDJPuzzle [354], BraCer [355], BALDR [356] and

BASIC [357] have also enabled the recovery and analysis of entire antibody repertoires for animal models of increasing interest such as felines [341] and rhesus macaques [358]. While the ferret kappa locus is contained within a single contig within the current draft of the ferret genome, the lambda and heavy chain loci were contained in unassembled contigs. The complete assembly of these loci using high throughput long sequence reads (1500-20000 bp) will be potentially useful as shown by the assembly of reference high coverage immunoglobulin gene loci in rhesus macaques

[358]. This has enabled in-depth analysis of antigen-specific immunoglobulin gene repertoire in rhesus macaques, such as the identification of antigenic epitopes with immunodominant features [359], which is critical for informing vaccine development.

Heuristic germline inference methods have also enabled the accurate identification of germline immunoglobulin genes which differ between outbred individuals. Methods such as IgDiscover [360] and TIgER [361] have been successfully used to identify germline V genes variants more accurately in humans, mice and rhesus macaques.

The application of these described tools for ferrets will enable the recovery of accurate and representative germline datasets for downstream analyses.

We successfully validated a single IgG isotype which enabled the expression of chimeric human-ferret CR9114 IgG mAb which retained HA binding. This will be useful for studies with less concern regarding ferret anti-human antibody responses,

91 as fully human mAb studies have shown unusually short half-lives when administered to ferrets [362]. Further work clarifying the range of ferret IgG subtypes and the engagement with cellular Fc-receptors (FcR) is also required. The presence of four different isotypes in closely related species such as minks [363], dogs [346] and three in felines [364] suggests these may exist in ferrets. Recent studies have proposed a critical role for antibody effector functions for protection against viral pathogens such as influenza [365, 366]. Further characterisation of ferret IgG and the respective FcRs to mediate ADCC and other Fc-dependent antibody responses is needed.

3.6 Conclusion

In this chapter, the capacity to recover and express ferret monoclonal antibodies was established. Annotation of ferret immunoglobulin V, D, J and C genes enabled recovery of recombined immunoglobulin gene sequences from single sorted ferret B- cells. Validation of constant chain coding genes facilitated the design of ferret IgG/IgL coding plasmids which enabled the expression of human-ferret chimeric CR9114 mAb retaining HA-specificity. With future improvements in ferret specific flow cytometric reagents and well-validated ferret immunoglobulin gene databases, the methodology described in this chapter will be useful for in-depth studies of ferret antigen-specific B- cell responses.

92 Chapter 4

Proof of concept for recovering HA-specific mAbs from

influenza-infected ferrets

4.1 Abstract

Ferrets are an important virologic model for human influenza viruses due to the capacity to be directly infected with human strains. Serum based assays such as HAI using ferret serum is critical for seasonal influenza vaccine strain selection, which is based on the elicitation of B-cell responses against viral surface HA proteins. Previous studies have shown species-specific differences in antibody recognition profiles of HA, but in-depth studies at the mAb level have not been feasible in ferrets due to the lack of reagents and tools. In this chapter, we aimed to use the method developed in the previous chapter of this thesis to examine influenza-specific B-cell and mAb responses. We recovered clonally expanded immunoglobulin transcript sequences from influenza-infected ferrets which enabled the reconstitution of HA-specific ferret mAbs. These mAbs were neutralizing and forced escape in the Sa region of the HA head, a site also commonly recognised by human HA-specific neutralizing antibody responses. This work shows proof of concept of studying ferret B-cell responses at the mAb level and provides additional tools to advance the ferret model for influenza and other infectious diseases. Future improvements in flow cytometric panels will enable a larger panel of mAbs to be recovered for detailed HA epitope mapping studies.

93 4.2 Introduction

B-cell responses are a critical arm of adaptive humoral immunity. Antibodies generated by influenza-specific B cells can provide protection against influenza via neutralisation, ADCC or complement activation. Currently, evaluation of B-cell responses in ferrets is dependent on polyclonal serum-based assays such as ELISA and HAI, or cellular responses by ELISpot or transcriptomics. Such studies have been informative, but do not necessarily correlate with antigen-specific B cell responses.

The ability to recover antigen-specific mAbs in humans and other animal models such as mice have enabled detailed examination of B-cell responses at the mAb level that is lacking in ferrets to date. The use of B-cell antigen probes in humans [173] and other animal species have been useful for the dissection and interrogation of B-cell responses for influenza and other diseases such as HIV. Ferret polyclonal sera are commonly used in influenza studies [268], but the extent of which ferret antibody responses recapitulate corresponding human responses remains unknown.

In chapter 3 of this thesis, a ferret specific single-cell RT-PCR methodology was established, which enabled the recovery of ferret immunoglobulin transcripts from single B-cells facilitating recombinant mAb expression. Recombinant HA probes were utilised to capture, recover and analyse immunoglobulin transcript sequences from

HA-specific B-cells (Figure 4-1). Clonally expanded sequences from HA-specific B- cells were isolated from two genetically outbred ferrets infected with 2009pdm

(A/California/04/2009) H1N1 virus. Two antibodies belonging to the same heavy chain clonal family were identified and exhibited HA binding and influenza virus neutralisation activity in-vitro. Subsequent viral escape analysis enabled the

94 identification of neutralisation epitopes, providing proof of concept for recovering ferret mAbs for HA epitope mapping studies.

Figure 4-1 Single-cell recovery of HA-specific mAbs from influenza-infected ferrets. (A) Two genetically outbred ferrets were infected with 1000 TCID50 2009 pdm H1N1 (A/California/04/2009), and parapharyngeal lymph node cells were cryopreserved. (B) Biotinylated HA probes labelled with streptavidin-PE and streptavidin-APC were used to stain HA-specific B-cells to excluded non-specific B-cell populations such as fluorophore specific B-cells. (C) HA-specific ferret B-cells were single-cell sorted for the recovery of cognate immunoglobulin heavy and light chain pairs. (D) Variable coding sequences of candidate mAb sequences were subsequently cloned into ferret immunoglobulin expression vectors and expressed for screening. (E) mAbs were screened for recombinant HA (ELISA) and native HA binding (HAI/microneutralisation assays). Neutralisation epitopes were subsequently validated via microneutralisation and HA viral escape assays.

97 4.3 Materials and Methods

4.3.1 Single-cell sorting of HA-specific class-switched B-cells from infected ferrets

Two genetically outbred ferrets were infected with 1000 TCID50 units of egg-derived

2009 pdm H1N1 (A/California/04/2009) virus. Ferrets were euthanised 28 d.p.i. and single-cell suspensions of parapharyngeal lymph nodes were prepared by passing the organs through a 0.22 m filter (Miltenyi Biotec). Cells were washed with RF-10 media twice and cryopreserved in liquid nitrogen prior to staining. Cryopreserved cells were thawed, washed twice in 1 X PBS and stained with live/dead stain for 3 min at room temperature. Excess live dead stain was quenched with FACS wash and surface stains were subsequently performed using anti-CD11b-BV510 (Biolegend: clone

M1/70), anti-ferret IgA/IgM/IgG-FITC (Rockland Immunochemicals cat.618-102-130) and anti-CD8 eFluor450 (eBioscience Clone OKT8) for 30 min at 4oC. Biotinylated recombinant full-length hemagglutinin (HA) probes derived from A/California/04/2009

[367] labelled in excess with streptavidin-PE or streptavidin-APC (Invitrogen) were used to capture single HA-specific B cells into 96-well PCR plates and stored at -20oC prior to RT-PCR amplification. All ferret experiments were approved by the University of Melbourne animal ethics committee (approval no. (#CT-FER-17-05).

4.3.2 Recovery of immunoglobulin gene transcript sequences from single sorted ferret B-cells.

Amplification of ferret heavy and light chain immunoglobulin gene transcripts was performed as described in chapter 3 (section 3.3.4) except for heavy chain gene transcript sequences. Reverse heavy chain primer pools were replaced with a single reverse primer targeting IGHJ region in both primary and secondary amplification steps. Due to the proximity of the IGHJ primer to CDR3 region, primers targeting FR2

98 regions (5’- ATTGGAACTGGATCCGCC/5’- TGGGTCCGCCAGGC) were applied to recover CDR3 sequences to enable reconstitution of full-length (FR1-FR3) ferret heavy chain immunoglobulin transcripts (Figure 4-2)

Figure 4-2 Recovery of full-length class-switched heavy chain ferret immunoglobulin transcripts. A single IGHJ reverse primer was used to amplify and recover FR1-FR3 heavy chain sequences from IHA-specific B-cells. FR2 specific primers were used to facilitate the recovery of CDR3 sequences. Full-length heavy chain immunoglobulin sequences (FR1-CDR3) were subsequently reconstituted and analysed.

99 4.3.3 Analysis of HA-specific sequences recovered from ferret B-cells

Immunoglobulin transcript sequences (FR1 – CDR3) recovered from single sorted ferret B-cells were analysed using Geneious 10.1.13. Recovered V gene segments

(FR1-FR2) were aligned to previously recovered germline immunoglobulin variable genes (Chapter 3) to identify germline ferret variable gene segments with the highest amino acid sequence identities. CDR3 sequences were determined by counting amino acid residues immediately after framework region 3, starting from the conserved cysteine (C) residue and ending with a conserved tryptophan (W) or phenylalanine

(F). Clonal families were defined as sequences sharing the same germline V-gene with identical CDR3 lengths, with sequence identities of at least 80% to account for

AID-dependent somatic hypermutation.

4.3.4 Mardin-Darby Canine Kidney (MDCK) cell culture handling and maintenance

1 x 107 cryopreserved MDCK cells were thawed and washed in MDCK maintenance media. Washed cells were seeded in T75 flasks and grown in 8% CO2 incubator at

37oC. Cells are sub-cultured once confluency reaches 80 - 90% every 3 – 4 days. To subculture MDCK cells, cells were first washed with 1 x PBS to remove serum. The monolayer was then detached by incubation with trypsin-EDTA solution at 37oC, 8%

CO2 for 10 min. MDCK maintenance media is then added to deactivate trypsin. Cells were centrifuged at 400g for 5 min at 4oC and resuspended in fresh MDCK maintenance media before splitting at a ratio of 1:4. Passage number was maintained below P20 for MDCK-cell-based assays.

100 4.3.5 Influenza virus neutralisation assay

MDCK cells were seeded at 1.5 x 105 cells/well in 96 well flat-bottomed tissue culture

o plates and grown to 80-90% confluency for 24h at 37 C, 5%CO2 using MDCK media.

Ferret mAbs found to bind to HA in ELISA were diluted serially 5-fold starting at 0.01 mg/mL in quadruplicates using influenza media. 100 L serial dilutions of antibodies

o were incubated with 100 TCID50 (100 L) of H1N1 A/California/04/2009 for 1 h at 37 C,

5% CO2. MDCK cells were washed with 1 x PBS thrice and incubated with antibody-

o virus mix for 1 h at 37 C, 5% CO2. Virus antibody mix was subsequently removed, and cells were washed thrice with 1 x PBS. Influenza virus growth media was subsequently

o added to wells for incubation (72 h, 37 C in 5% CO2). Cells were subsequently observed microscopically under an inverted microscope for cytopathic effects (CPE), which is the loss of cell monolayer. Endpoint neutralising titres were defined as the lowest concentration of mAb which prevented CPE in at least 3 out of 4 wells.

4.3.6 Haemagglutination Inhibition (HAI) assay

50L of 1 x PBS was added to each well of a 96-well V bottom plate (Greiner). 50L candidate mAbs were added to each well in duplicates and diluted 2 folds. 1% turkey erythrocytes were subsequently added to each well. Plates were tilted for 30-45 minutes at room temperature until erythrocytes had settled to the bottom of the plate, which is defined as the absence of haemagglutination. Endpoint titres were defined as the reciprocal of the lowest concentration of mAbs which prevented hemagglutination in both replicates. Endpoint mAb concentrations were subsequently determined by dividing the concentration of antibodies by the endpoint titres.

101 4.3.7 Influenza viral escape assay

2.5 x 105 MDCK cells in 2 mL MDCK maintenance media were seeded and cultured in 24 well plates till 80-90% confluency in 8% CO2 incubator at 37oC. 400 PFU

A/California/04/2009 was incubated in the presence of four different amounts of HA- specific antibodies (20 g, 2 g, 0.2 g, 0.02 g) in a total volume of 100 L in influenza virus growth media for 1 h at 37oC. in duplicates. Cells were washed with 1 x PBS to remove MDCK maintenance media. Virus- antibody mix was added to MDCK cells and topped up with 1 mL influenza media. Cultures were observed 3 – 4 days post- incubation for CPE. Supernatants from CPE positive wells were collected and stored at -20oC. This was repeated for at least 5 passages until HA mutations were detected by RT-PCR as below in the presence of media only controls.

4.3.8 RT-PCR amplification and sequencing of HA genes

RNA was extracted from CPE positive wells using viral RNA extraction kit (Qiagen).

Extracted RNA was used as the template for one-step RT-PCR (Invitrogen). Full- length HA gene was amplified in 2.5mM MgCl2, 1 nanomol each of HA-specific forward

(5’-ATGAAGGCAATACTAGTAG-3’) and reverse primers (5’-CTCATGCTTCTGA-3’),

5 L extracted RNA, 1 x Reaction mix, 1 L Superscript III/Platinum taq Mix in a total reaction volume of 50 L. Cycling conditions are as follows: 1 cycle of 42 oC for 1 h,

94oC for 2 min; 5 cycles of 94oC for 20 s, 50oC for 30 s, 68oC for 2 min; 40 cycles of

94oC for 20 s, 58oC for 30 s 68oC for 2 min. Final extension was performed for 10 min at 68oC. PCR amplicons were resolved by agarose gel electrophoresis and gel purified according to the manufacturers’ instructions (Qiagen) before standard Sanger sequencing with internal primer targeting HA1 (5’- CCGTACCATCCATCTACCATC).

102 4.4 Results

4.4.1 Single-cell sorting of HA-specific ferret B-cells

Single-cell BCR sequencing has been employed in mice [171], guinea pigs [172], macaques [331] and humans [170] to study antigen-specific B-cell responses and recover mAbs for characterisation. It is currently difficult to perform similar studies in ferrets due to the lack of both immunological reagents and sequencing protocols to study different ferret B-cell populations. HA reactive B-cells from two outbred ferrets infected with 2009pdm H1N1 (A/California/04/2009) influenza virus were therefore sorted using a panel of currently available ferret B-cell reagents (Figure 4-3 A) to facilitate the recovery of immunoglobulin transcript sequences using the primers developed in chapter 3 of this thesis.

HA is the main target of influenza-specific antibody responses and is widely studied due to antigenic drift which ablates protective immunity. Previous studies using human and mouse mAbs have enabled the generation of HA recognition maps leading to the identification of important epitopes for vaccine development. For example, the binding epitope of broadly neutralising antibody 32D6 against influenza A viruses were recovered recently from human memory B-cells [368], which is an important step for the development of universal influenza vaccines. To isolate HA-specific ferret B-cells, we utilised previously reported HA probes labelled with two spectrally distinct fluorophores, PE and APC. Comparisons of flow cytometric staining profiles of immunologically naïve and influenza-infected ferrets revealed the presence of HA- probe positive B cells in infected ferrets, suggesting that these populations express

HA-specific BCRs/antibodies (Figure 4-3 B).

103 A B

Figure 4-3 Gating scheme to recover clonally expanded HA transcript sequences from infected ferrets. Single-cell suspensions were prepared from parapharyngeal lymph nodes from either naïve or ferrets infected with 2009 pdmH1N1 (A/California/04/2009). Live single Ig+ lymphocytes were selected, and dead cells were excluded. HA-specific cells were isolated using biotinylated HA probes and detected using SA conjugated to PE or APC. (A) Gating Scheme (B) HA probe binding for immunologically naïve and infected ferrets.

104 4.4.2 Genetic features of sequences recovered from HA-specific ferret B-cell populations

HA probe positive B cells were subsequently single-cell sorted into 96-well plates for

RT-PCR amplification of immunoglobulin gene transcripts using the ferret primers developed in chapter 3 of this thesis. As we were unable to recover any heavy chain gene sequences from the single sorted ferret B-cells using constant chain gene- specific primers, we designed a single primer targeting IGHJ region. The use of this primer enabled the recovery of ferret heavy chain transcripts for analysis.

The analysis revealed preferential usage of germline variable gene segments across both heavy and light chain loci (Figure 4-4 A), consistent with the selection and expansion specific B-cell clones in response to influenza infection in ferrets. 288 heavy chain transcripts were recovered from 1024 (28.1%) sorted HA-specific B-cells sorted from two genetically outbred ferrets. Majority of the heavy chain variable sequences belong to clan III. Preferential usage of germline variable gene segments

III-HV8, III-17c, III-19b and III-HV20 and III-HV21 (Figure 4-4 A) was observed and a single transcript belonging to clan II (II-HV1) was recovered. CDR-H3 lengths were correspondingly skewed with modal AA length of 12 (Figure 4-4 B) as compared to bulk ferret B-cell populations (14 AA) as determined in chapter 3 (Figure 3-5 C).

As for the light chain loci, 84 (8.2%) lambda chain and 26 (2.5%) kappa chain transcripts were recovered from 1024 sorted HA-specific B-cells. Analysis of immunoglobulin gene usage similarly revealed a preference for light chain gene variable segments. Majority of recovered kappa chain sequences utilised II-KV46, whereas for lambda chain, most belonged to II-LV4 and V-LV3 (Figure 4-4 A). The

105 distribution of modal CDR L-3 lengths was also skewed, with a preference of 7 AA for kappa chain and 10 AA for lambda chain (Figure 4-4 B) as compared to 9 AA and 11

AA respectively from bulk ferret B-cell populations as determined in chapter 3 (Figure

3-5 C).

106 A Heavy Kappa Lambda

B 25 100 40

20 80 30

c n

n 60

n 15

e e

u 20

u u

e e

10 40 r

F F F 10 5 20

0 0 0 7 8 9 0 1 2 3 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 7 8 9 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 1 CDR-H3 length CDR-L3 length CDR-L3 length

Figure 4-4 Genetic features of ferret HA-specific immunoglobulin Heavy and Light chain sequences. (A) Recovered heavy, kappa and lambda chain sequences were converted to fasta format and analysed using geneious to identify the closest ferret variable germline gene. Segments are coloured according to genetic clans the recovered sequences belong to. The width of each segment is proportional to the number of variable gene sequences recovered (B) Distribution of CDR3 (AA) lengths across heavy, kappa and lambda loci of recovered sequences.

107 4.4.3 Clonal expansion of HA specific ferret B-cells

Clonotype analysis revealed the recovery of 84 heavy chain clonal families, 6 kappa chain clonal families and 17 lambda chain clonal families (Figure 4-5 A). We also noted amino acid substitutions in the recovered sequences, with a mean pairwise identity of

94.0% for heavy chain transcripts 96.2% for kappa chain transcripts and 96.0% for lambda chain transcripts as compared to germline V-gene sequences showing AID- mediated affinity maturation (Figure 4-5 B). Supporting this, the examination of transcript sequences within clonal families revealed the presence of amino acid substitutions at different positions of the recovered transcripts (Appendix 4.1, 4.2, 4.3).

Overall, this study enabled the recovery of 1.5% (15/1024) heavy-lambda chain paired antibody sequences and 0.8% (8/1024) heavy-kappa paired chain antibody sequences from HA-specific B-cells.

108

A Heavy Kappa Lambda

288 26 84

3B03/4A06

e e

e 100 100 100

e e

l m 90 m 90

m 90

t t

i 80

e e

A A

70 A

A 70 70

% % %

Figure 4-5. Clonal expansion of HA-specific B-cell transcripts. (A) Heavy, kappa and lambda chain clonotypes were found and grouped in the same family. Each segment represents a clonally expanded immunoglobulin family and the size of each segment is proportional to the number of sequences. The total number of sequences analysed is indicated in the centre of the pie chart. (B) Percent amino acid sequence identities of recovered heavy, kappa and lambda as compared to germline V-genes recovered in chapter 3.

Heavy Kappa Lambda

109

4.4.4 Recovering and validating ferret HA-specific mAbs from infected ferrets

Next, we derived fully ferret HA-specific mAbs from the recovered sequences for validation. As we are currently only able to express lambda chain ferret mAbs, cognate heavy and lambda chain sequence pairs derived from HA- specific B-cells were selected for expression (Table 4-1). Out of 15 heavy-lambda chain pairs, we identified a single clonally expanded antibody family containing 2 members (4A06/3B03). Three other mAbs (1A04/1B02/1C08) representing families containing only a single member was also selected for expression as negative controls, likely derived from B-cells that bound non-specifically to the HA probes. These candidates were successfully expressed using the plasmids developed in chapter 3.

mAbs were examined for full-length HA or stem-HA binding by ELISA to determine HA binding capacity. Representative mAbs derived from the single clonally expanded family (3B03/4A06) (Figure 4-6 B), were found to bind full-length HA but not stem-HA, suggesting specificity for HA1, which is the main target for HA neutralising antibodies

(Figure 4-6 A). We subsequently determined if mAbs 3B03 and 4A06 were neutralising. HAI and microneutralization assays revealed the ability of both mAbs to block HA- sialic acid interactions (4A06- 8 x 10-5μg/μL; 3B03- 4 x 10-4 μg/μL) and prevent viral-induced MDCK cell pathology (4A06 – 1.9 x 10-4 μg/μL; 3B03 – 3.9 x 10-

4 μg/μL) (Figure 4-6 C).

110 Table 4-1: Amino acid sequences of expressed candidate HA-specific mAbs. Closest ferret germline V-gene and amino acid sequence identities are indicated. mAb candidates from clonally expanded families are highlighted in bold.

Closest

Clone Germline ferret

V-gene

Heavy III-HV17a EVQLVESGGDLVKPGGSLRLSCAASGFTFSSYDMYWVRQAPGKGLQCVAWINTGGSSTSYADSVKGRFTISRDNGKNTLYLQMNSLRAEDTAMYYCAKITQSQYCDSYGYCNLHDYLDVWGQGTLVTVSSASTS 1A04 Lambda V-LV3 PVLTQPPSLSASPGTTARLTCTLSRDISVGSKYMHWYQQKPGTPPRYLLYYYSDSSTQLGPGIPSRFSGSKDTSANAGILLISGLQPEDEADYYCAVWHSGAIVFGGGTQLTVLGQPTSAPSVTLFPPSS

Heavy III-HV19b EVQLVESGGDLVKPGGSLRLSCAASGFTFSNDYMIWVRQAPGKGLQWVAWIAPGGSRTHYADSVKGRFTISRDNGKNTLYLQMTSLRAEDTAVYYCVTTVNWNLQTHWGQGTLVTVSSASTS 1B02 Lambda V-LV-3 PVLTQPPSLSASPGTTARLTCTLSRDISVDSKYMHWYQQKPGTPPRYLLYHYSDSTTQLGPGIPSRFSGSEDTSGNAGILLISGLQPEDEADYYCALWHSGVYVFGGGTQLTVLGQPTSAPSVTLFPPSS

Heavy III-HV-8 EVKLVESGGDLVKPGGSLRLSCAASGFTFSNYDINWVRQAPGKGLQWVAYISSGGSSTYYADSVKGRFTISRDNDKDMLFLQMNSLRAEDTAMYYCARAGSTEATAYYAMDYWGQGTLVTVSSASTS 1C08 Lambda II-LV18 FVLTQPPSMSVNLGQTVRMTCGGNNIGRKSVPWYQQKPGLAPVMIIYGDSSRPSGIPDRFSGTNSGNTATLTISGVRAEDEADYYCQVWDNSADAWVFGGGTQLTVLGQPTSAPSVTLFPPSS

Heavy III-HV19b EVQLVESGGDLVKPGGSLRLSCAASGFTFNNYYMSWVRQAPGKGLQWVAWINTGGSSTFYADSVKGRFTISRDNAKNTLYLQMNGLRAEDTAMYYCVRAGGSSWYNWLDYWGQGTLVTVSSASTS 3B03 Lambda V-LV3 PVLTQPPSLSASPGTSARLTCTLSRDISVGSDYMHWYQQKPGTPPRYLLYYYYSDSTTQLGPGIPSRFSGSKDTSANAGLLLISGLQPEDEADYYCAVWHSNTYVFGGGTQLTVLGQPTSAPSVTLFPPSS

Heavy III-HV19b EVQLVESGSDLVRPGGSLRLSCAASGFTFTNYYMSWVRQAPGKGLQWVAWINTGGSNTYYADSVKGRFTISRDNGKNTLYLQMNSLSAEDTAIYYCARAGGSSWYNWLDFWGQGTLVTVSSASTS 4A06 Lambda V-LV3 PVLTQPPSLSASPGTTARLTCTLSRDIIVGSKYMHWYQQKPGTPPRYLLYYYSDSTTQLGPGIPSRFSGSEDTSANAGILLISGLQPEDEADYYCAVWHSRLYVFGGGTQLTVLGQPTSAPSVTLFPPSS

111

Figure 4-6 Validation of recovered HA-specific mAbs. (A) 96 well ELISA plates were coated with recombinant full-length HA (HA-FL) protein or stem only mini HA protein (HA-SS). Candidate ferret mAbs were serially diluted 5 folds and added to wells for binding. Binding was detected using donkey anti-ferret IgG, anti-donkey IgG conjugated to HRP and subsequent addition of TMB. Readings were measured at 630nm using a 96 well plate spectrophotometer. (B) Denaturing SDS-PAGE gels 3B03 and 4A06 mAbs. Heavy (50kDa) and light chains (25kDa) are indicated. Lane 1: Molecular weight marker; Lane 2: 3B03; Lane 3: 4A06. (C) Endpoint mAb concentrations for HA-specific mAbs (HAI/microneutralization assays) Endpoints of HAI assays are defined as the lowest concentration of mAb which prevents agglutination of turkey red blood cells in duplicates. Endpoints of microneutralization assays are defined as the lowest concentration of mAb which prevented CPE in at least 3 out of 4 quadruplicates.

112 4.4.5 Viral escape mapping validation of 4A06 mAb

To further confirm these findings, the neutralisation epitopes were determined via previously described influenza viral escape assays. These assays have been used to generate HA epitope recognition maps of humans/mice and useful for dissecting influenza-specific B-cell responses at the mAb level. 2009pdm (A/California/04/2009)

H1N1 virus was incubated with limiting dilutions of either 3B03 or 4A06 mAbs to generate influenza escape mutants for at least five passages. Neutralisation resistant viruses displayed a K163E mutation (Appendix 4.4) by passage 3, previously associated in human populations with escape from pandemic H1N1 serum neutralising activity [369, 370]. Visualising the mutation on the 3D structure of HA revealed localisation to the Sa site surrounding the RBD (Figure 4-7).

113

Figure 4-7 Viral escape mapping of 4A06 mAb. A/California/04/2009 virus was incubated with limiting dilutions of 4A06 mAb and sequenced for at least five passages before sequencing for HA mutations. K163E mutation is indicated in red.

114 4.5 Discussion

Single-cell BCR sequencing has been established in mice and humans and used to study B-cell responses and recover mAbs. Pathogen-specific B-cell responses are often characterised by the preferential use of specific V-regions and CDR3 amino acid sequences. We recovered clonally expanded sequences from ferret HA-specific populations, suggesting that the isolated ferret B-cell populations were derived from populations which proliferated and diversified BCR repertoires in response to influenza infection.

The use of a single reverse IGHJ reverse primer meant that it is currently not possible to delineate the recovered sequences based on heavy chain subclasses. This approach also required an additional sequencing step for the recovery of CDR3 portions of the heavy chain transcript sequences, decreasing the efficiency of the presented method. The optimisation of amplification protocols using ferret constant chain targeting primers or exploring other 5’RACE based amplification approaches is important and will enable the future recovery of a larger panel of HA specific mAbs for examination.

The ability to differentiate mAbs based on heavy chain subclass is critical, as different heavy chain subclasses have different antibody-mediated effector functions. While most serum immunoglobulins have been reported to be IgG+, the identification of IgA mAbs, for example, will increase our understanding of mucosal defences against influenza in ferrets. Nasal IgA has been shown in humans to be a critical barrier against influenza infections in subjects with low HAI titres [371]. In-depth ferret mAb

115 studies will complement the examination of influenza mucosal IgA responses in ferrets

[372].

Poor recovery of light chain sequences (2.5%-kappa, 8.2%- lambda) was noted, which decreased the overall efficiency of ferret HA-specific mAb sequence recovery in this chapter (2.3%). This could be accounted for by FcR mediated background binding of the flow cytometric mAbs and the absence of FcR blocking reagents as previously reported in the literature [258]. Another factor that could have contributed to this is the incomplete state of the ferret genome, which could have precluded the recovery of undiscovered immunoglobulin light chain coding sequences (chapter 3), such as clan

III lGLV variable genes.

Improvements to the flow cytometric panel are required to improve the recovery of HA transcript sequences from infected ferrets. This could be improved by the inclusion of a monocyte dump gate for example, and other effector B-cell markers such as CD138 for antibody-secreting B-cells or IgD for immunologically naïve B-cells. The development of FcR blocking reagents, ferret flow cytometric reagents as described

(chapter 5) and improvements to gene annotations (chapter 3) is essential, as it will facilitate the increased resolution of antigen-specific ferret B-cells on flow cytometry and enhance the recovery of immunoglobulin transcripts.

The current sort panel also did not have the capacity to differentiate geminal centre, plasma, and memory B-cell effector subsets due to the lack of immunological reagents targeting these markers. The delineation of ferret immune responses into these B-cell subsets using validated reagents will enable the in-depth analysis of B-cell dynamics

116 and kinetics which are critical for informing vaccine development as shown in mice and human B-cell studies [362].

We noted a disproportionately higher recovery of lambda light chain transcripts in this study as compared to kappa light chains. These results reflect the preferential expansion of lambda chain expressing rather than kappa chain expressing B-cells in influenza-infected ferrets, consistent with previous ferret studies showing lambda chain bias by examination of serum light chain responses [268]. Similar studies in mice revealed a preference for kappa light chain usage for HA-specific B-cells [373], showing species-specific differences in immune responses against influenza.

While amino acid substitutions which indicated AID mediated affinity maturation of influenza-infected ferrets was present, delineating these mutations into germline allelic variants and somatically mutated residues was not possible due to the lack of validated databases of germline ferret immunoglobulin genes, such as those maintained by

International ImMunogeneTics (IMGT) [315]. Nevertheless, HA-specific ferret mAbs were isolated from HA-specific ferret B-cell populations. These fully ferret antibodies showed HA binding specificity and influenza neutralisation activity, validating the use of the tools for generating HA-specific ferret mAbs.

A much wider panel of mAbs is required to assess if ferrets recognise canonical HA head and stem sites that humans do as this is critical for vaccine selection each year.

This requires higher efficiencies in ferret BCR transcript recovery, which can be improved by the inclusion of mAbs to resolve different ferret immune cell populations

(Chapter 5).

117 4.6 Conclusion

In this chapter, we utilised the tools developed in chapter 3 of this thesis to recover

HA-specific transcript sequences and HA-specific mAbs. Further improvements to the tools developed in this thesis will enable more efficient recovery of immunoglobulin transcript sequences from ferrets for HA epitope mapping studies.

118 Chapter 5

Isolating recombinant murine anti-ferret antibodies using single

cell-RT PCR

5.1 Abstract

Ferrets are an excellent virologic model to study human respiratory diseases, but the ongoing lack of reagents has limited the scope of immunological studies for serological or transcriptional analysis. To address this technological gap in the ferret field, this chapter aimed to establish a methodology to recover murine anti-ferret mAbs. We aimed to develop key B (CD19, CD138 & IgD) and NK cell reagents (NKp46 & LAMP-

1) to enable a wider range of studies to be conducted in ferrets. To achieve this aim, candidate recombinant ferret antigens were first identified, characterised, and expressed as human Fc-fusion constructs to facilitate purification using Protein A.

Immunisation of C57BL/6 mice with these recombinant ferret antigens enabled the recovery of probe-specific B cells for these ferret markers. Cognate immunoglobulin sequence pairs from single sorted ferret CD19 and IgD specific murine memory B- cells were subsequently identified. Reconstitution of these murine anti-ferret mAbs enabled recovery of recombinant ferret CD19 and IgD protein binding mAbs from immunised mice. Although the mAbs recognised cognate recombinant ferret antigen, they lacked the capacity to resolve ferret cell-populations by flow cytometry. This general methodology will improve the ferret model for influenza and other relevant diseases by enabling the recovery of ferret antigen specific mAbs to enable in-depth studies of pathogen specific immune responses.

119 5.2 Introduction mAbs are an essential tool in basic and applied immunological research. mAbs are broadly used in key techniques such as flow cytometry, confocal microscopy and

ELISA, and together have provided detailed insights of immune responses in different contexts such as genetic diseases and infections such as influenza [374]. Most influenza-related immunological studies are conducted in either humans or mice due to the wide availability of immunological reagents which allows in depth interrogation of multiple aspects of immune responses, including antigen-specific B and T cell responses [375]. Ferrets are an excellent virologic model for studying influenza and other human respiratory viruses due to anatomical similarities in the respiratory tract conferring the ability to be infected directly with human viral strains and the capacity for transmission. Immune responses in the ferret model are however, understudied due to limited ferret specific immunological reagents. Thus, there is a significant global effort aimed at improving immunologic reagents to study ferret immunity [259].

Currently, most ferret flow cytometric analyses are based on a very limited number of effective markers targeting key immune cell populations such as CD4+ T cells, CD8+

T cells, CD11b+ myeloid cells and CD79α/CD20+/immunoglobulin (Ig+) B-cells [253,

258]. As compared to human and mice, it is currently not possible to resolve important subpopulations of cells such as memory or activated B/T lymphocytes which play important roles in protection against viral infections. Currently available cross-reactive mAbs targeting pan-B-cell markers CD20, CD79 and CD79 for ferrets unfortunately target intracellular epitopes. Intracellular staining requires fixation and therefore severely limits downstream molecular analyses due to the deleterious effects of fixatives on DNA/RNA [376]. A pan-surface marker of ferret B cells such as CD19 and

120 other markers defining B-cell subsets such as IgD (activated B-cells) and CD138

(plasma cells) would aid in the study of humoral immunity by enabling studies of B- cell effector functions and improve the recovery of HA-specific mAbs (Chapter 4). The development of NK-cell markers NKp46 and LAMP-1 will also enable other ADCC studies to be carried out in ferrets, expanding the scope of functional antibody studies.

These studies have revealed the importance of ADCC as a functional mechanism underlying universal protection against influenza [377].

Herein, this chapter describes the development of a methodology suitable for the recovery of murine anti-ferret mAbs to improve flow cytometric/confocal microscopy panels available for ferrets. We developed this method from recent murine work [378] and our laboratory’s expertise in using recombinant protein probes to identify antigen- specific B-cells [379] (Figure 5-1). Clonally expanded murine memory B-cells likely responding to recombinant ferret CD19 and IgD protein were identified which enabled the recovery of mAb coding transcript sequences. Candidate mAbs were subsequently expressed and screened by ELISA and flow cytometry. Reagents developed using this methodology will ultimately be useful for future ferret B-cell studies.

121

122

Figure 5-1. Single cell recovery and validation of ferret antigen specific mAbs. Key steps using this methodology are highlighted in this figure. Each group of three mice were vaccinated with CD19, IgD, CD138, NKp46 or LAMP-1 (A) Candidate ferret antigens are identified, cloned, expressed and purified (B) Immunisation of C57BL/6 mice to induce murine anti-ferret responses. (C) Isolation of ferret antigen specific murine B-cells. Recombinant ferret antigens conjugated to two spectrally distinct fluorophores were used to capture ferret antigen specific murine B-cells to exclude non-specific B-cell populations such as single positive fluorophore specific populations. (D) Antigen specific murine B-cells were subsequently single-cell sorted into 96-well PCR plates, which facilitated the RT-PCR recovery of cognate heavy and light chain immunoglobulin gene transcripts for analysis. (E) Recovered heavy and light chain immunoglobulin genes were cloned into eukaryotic expression vectors which enabled the recombinant expression of candidate murine anti-ferret antibodies for screening. (F) Screening of mAbs for recombinant antigen binding (ELISA) and native cell surface antigen binding (Flow cytometry)

123 5.3 Materials and methods

5.3.1 Identification and bioinformatics validation of ferret proteins

Full length amino acid coding sequences of predicted ferret CD19 ECD (Uniprot identifier M3YN68) were extracted as FASTA files from Uniprot. Mammalian homologues of CD19 were downloaded from the following Genbank accession nos.

(mouse –AAA37390.1; human –AAA69966.1; cat – XP 019676303.1; dog –

XP_022275622, rat- NP_001013255.2; macaque- EHH31541.1.) CH1 domain of ferret IgD was identified, annotated and validated by RNA-seq in chapter 3.

Mammalian homologues of IgD (CH1) were extracted from IMGT accession nos.

(human – X57331; mouse – V00786; rat – RatNor_6_chr6; crab eating macaque –

Macfas_2_Chr7; dog – IMGT000001). Full length CD138 coding sequences from ferrets and other related mammals were extracted from Uniprot as FASTA files with the following uniprot identifiers (mouse- P18828; rat – P26260; human – P18827; macaque – A0A2K5WT52; cat – M3W3X8; dog – E2RT70; ferret – M3YQM0). LAMP-

1 protein sequences were extracted from Uniprot using the following identifiers

(human – P11279; mouse – P11438; rat – P14562; macaque – A0A2K5UV67; dog –

F1Q260; cat – M3WKD0; ferret – M3YEW6) as FASTA files. NKp46 proteins sequences were extracted from Uniprot using the following identifiers (human –

O76036; mouse – Q8C567; rat- Q9Z0H5; macaque – Q95JB9; bovine – Q863H2; chimpanzee – Q08I02 ferret – M3XT06) as FASTA files.

Extracted protein sequences were subsequently multiple aligned against ferret homologues using Clustal Omega tool [380]. Phylogenetic trees were constructed based on the Juke-Cantor genetic model. The Neighbour-Joining method was used, with no outgroups and resampled by bootstrapping (100x) using Geneious tree builder

124 (10.1.3). Protein structures of validated ferret protein sequences were subsequently predicted in silico by comparison with crystal structures using SWISS-MODEL tool

[381].

5.3.2 Immunisation of mice with recombinant ferret proteins

18-week-old female C57BL/6 mice were immunised subcutaneously with 20 g each ferret protein in 100 L TitremaxGOLD adjuvant on day 0 in triplicates. On day 28, mice were given booster shots of 100 g in 100 L PBS and blood was collected by cheek bleeds on Day 42 for ELISA validation of serological responses in mice. Mice were restrained by scruff and a 4.0mm lancet was used to collect 100 L blood from the submandibular vein. Mice were subsequently monitored for at least seven days for signs of wound infection after immunisation and cheek bleeds. All mouse experiments were approved by the University of Melbourne animal ethics committee (approval no.

1714193).

5.3.3 ELISA validation of ferret immunogen specific serological responses

96-well ELISA plates were each coated with 50 ng per well of recombinant ferret antigen in 1 X PBS at 4OC overnight respectively. Wells were then blocked with ELISA blocking buffer for 1 h at room temperature and washed with ELISA wash buffer five times. Ten-fold serial dilutions of serum (collected on day 42 post immunisation; starting at 1:10 dilution) were prepared in ELISA dilution buffer from cheek bleeds and prepared by spinning at 10000g for 15 min at 4oC. Serum samples were incubated on coated plates at room temperature for 1 h, followed by washing with ELISA wash buffer. Detection was subsequently performed by staining with goat anti-mouse IgG

(H+L) at 1:10000 in ELISA blocking buffer for 30 min. OD630 readings were obtained

125 after addition of Sureblue TMB peroxidase substrate (Seracare) and TMB BlueSTOP

Solution (Seracare).

5.3.4 Single cell sorts for ferret antigen specific murine B-cells

Immunised mice were sacrificed 42 days post immunisation after validation of serum immune responses against ferret antigens by CO2 asphyxiation (Flow rate of 3 litres per minute) for 10 minutes. Single cell preparations were prepared by passing spleens through 0.22 m filters in RF10 media (RPMI + 10% FCS + 1 X PSG). To remove red blood cells, single cell suspensions were treated with 4 mL 1 X Pharmlyse (BD) diluted in distilled water for 7 min and washed by centrifugation at 500 g for 5 min at 4oC. To remove remaining red blood cells, supernatant was removed, and cells were treated with 1 mL 1 X Pharmlyse subsequently for another 3 min. Cells were washed with 3 mL 1 X PBS to remove remaining. Pharmlyse solution and subsequently stained with live/dead stain for 3 min at room temperature. Excess stain was quenched using FACS wash buffer. Following blocking with anti-mouse CD16/32, surface stains anti- CD3

BV786 (Biolegend: Clone 145-2C11), anti-F4/80 BV786 (Biolegend: Clone BM8), anti-

CD45 APC-Cy7 (Biolegend: Clone 30-F11), anti-CD38 PE-Cy7 (Biolegend: Clone 90), anti-IgD PercpCy5.5 (Biolegend: Clone 11-26c-2a) and anti-B220 BV650 (Biolegend:

Clone RA3-6B2) were added for 30 min at 4oC. Human IgG tag specific cells were excluded using purified human IgG preparations (Rockland no. 009-0102) conjugated to AF488 (Abcam). Cells were washed twice and resuspended in OptiMEM media

(Thermofisher). Ferret antigen specific memory B-cells (CD45+B220+IgD-CD38+) were subsequently gated with PE or APC conjugated (Abcam) ferret antigen probes respectively and single cell sorted into 96-well PCR plates. Plates were stored at -

20oC prior to RT-PCR amplification.

126 5.3.5 RT- PCR recovery of immunoglobulin sequences from single-sorted murine B- cells

RT-PCR procedures were based on previously reported protocols [171, 378]. Reverse transcription of total cellular RNA from single sorted murine B cells was performed in

14 L reaction volumes in the sort plate using 450 ng random hexamers (Bioline), 50

U Superscript III (Thermofisher), 1X First Strand buffer (Thermofisher), 14 U RNAsin

(Promega), 0.1 pmol (DTT) (Thermofisher), 0.7 % v/v IGEPAL CA-630 (Sigma Aldrich) and 2.4mM mM deoxynucleotide triphosphate (dNTP) (Bioline). Cycling conditions for cDNA synthesis were: 42oC for 5 min, 25oC for 10 min, 50oC for 60 mins and 94oC for

5min. 3L of unpurified cDNA was used as template in multiplex nested PCR reactions to amplify paired recombined murine heavy and light chain (IgG, IgK) sequences.

For Heavy chain (IgG) amplifications, primary reactions were carried out in 40 L volumes using 2.0 U Hotstart Taq plus polymerase (Qiagen), 1X reaction buffer, 2.0 mM MgCl2, 0.3mM dNTP (Bioline) and 10 picomol each of primary forward and reverse primer pools (Table 4-1 A) using 4L of cDNA as template. 4 L of primary PCR product was used as template in a secondary, nested PCR (40 l volume) containing

2.0 U Hotstart Taq plus polymerase (Qiagen), 1X reaction buffer, 2.0 mM MgCl2, 0.3 mM dNTP (Bioline) and 10 picomol each of secondary forward and reverse primer pools (Table 4-1 A).

As for kappa chain, primary reactions were carried out in 40 L volumes using 1.25 U

Hotstart Taq plus polymerase (Qiagen), 1X reaction buffer, 1.5 mM MgCl2, 0.3mM dNTP (Bioline) and 8 picomol each of primary forward and reverse primer pools (Table

5-1 B) using 4L of cDNA. 4 L of primary PCR product was used as template in a

127 secondary, nested PCR (40 l volume) containing 1.25 U Hotstart Taq plus polymerase (Qiagen), 1X reaction buffer, 1.5 mM MgCl2, 0.3 mM dNTP (Bioline) and

8 picomol each of secondary forward and reverse primer pools (Table 5-1B).

Secondary amplification products were sequenced by standard sanger sequencing using the IgG (2mRG) and kappa chain (5’mVkappa) forward primers from the secondary amplification step.

128 Table 5-1: Mouse immunoglobulin specific primers. (A) Mouse immunoglobulin heavy chain primer pools. (B) Mouse immunoglobulin Kappa chain primer pools.

1mFH_I AGGAACTGCAGGTGTCC A 1mFH_II CAGCTACAGGTGTCCACTCC 1mFH_III TGGCAGCARCAGCTACAGG 1mFH_IV CTGCCTGGTGACATTCCCA 1mFH_V CCAAGCTGTGTCCTGTC Mouse Heavy Primary 1mFH_VI TTTTAAAAGGTGTCCAGKGT Forward Primer pool 1mFH_VII CCTGTCAGTAACTRCAGGTGTCC 1mFH_VIII TTTTAAAAGGGGTCCAGTGT 1mFH_IX CGTTCCTGGTATCCTGTCT 1mFH_X ATGAAGTTGTGGYTRAACTGG 1mFH_XI TGTTGGGGCTKAAGTGGG

Mouse Heavy Primary 1mRG* AGAAGGTGTGCACACCGCTGGAC Reverse Primer (Gamma)

Mouse Heavy Secondary Forward 2mFG* GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG Primer

Mouse Heavy 2mRG* Secondary Reverse GCTCAGGGAARTAGCCCTTGAC /Gamma) Primer

B 5′ L-Vκ_3 TGCTGCTGCTCTGGGTTCCAG 5′ L-Vκ_4 ATTWTCAGCTTCCTGCTAATC 5′ L-Vκ_5 TTTTGCTTTTCTGGATTYCAG Mouse Kappa 5′ L-Vκ_6 TCGTGTTKCTSTGGTTGTCTG Primary Forward Pool 5′ L-Vκ_6,8,9 ATGGAATCACAGRCYCWGGT 5′ L-Vκ_14 TCTTGTTGCTCTGGTTYCCAG 5′ L-Vκ_19 CAGTTCCTGGGGCTCTTGTTGTTC 5′ L-Vκ_20 CTCACTAGCTCTTCTCCTC Mouse Primary Reverse Primer mCκ GATGGTGGGAAGATGGATACAGTT

Mouse Kappa Secondary Forward Primer 5′ mVkappa GAYATTGTGMTSACMCARWCTMCA mJK01 TTTGATTTCCAGCTTGGTG Mouse Secondary mJK02 TTTTATTTCCAGCTTGGTC Reverse Primer Pool mJK03 TTTTATTTCCAACTTTGTC mJK04 TTTCAGCTCCAGCTTGGTC

129 5.3.6 Analysis of recovered immunoglobulin sequences

Full length recombined VH, V and V nucleotide sequences recovered from single sorted mouse B-cells were analysed using IMGT high-V quest to identify murine germline V, D and J members with the highest sequence identity. CDR-3 sequences and lengths for both heavy and light chain loci were determined by counting amino acid residues immediately after framework region 3 starting from the conserved cysteine (C) residue and ending with a conserved tryptophan (W) or phenylalanine

(F). Clonal families were defined as sequences with identical CDR3 amino acid lengths with identical V-gene usage and at least 80% sequence identities to account for AID- dependent somatic hypermutation.

5.3.7 ELISA validation of recombinant murine antibodies

96-well ELISA plates were each coated with 50 ng per well of recombinant ferret

CD19, ferret IgD or polyclonal human IgG1 in 1 X PBS at 4OC overnight respectively.

Wells were then blocked with ELISA blocking buffer for 1 h at room temperature and washed with ELISA wash buffer five times. Ten-fold serial dilutions of recombinantly expressed murine antibodies from immunised C57BL/6 mice in ELISA dilution buffer

(starting at 1:10 dilution) were added onto coated plates at room temperature for 1 h, followed by washing with thrice. Detection was subsequently performed by staining with goat anti-mouse IgG at 1:10000 in ELISA blocking buffer for 30 min. OD630 readings were obtained after addition of Sureblue TMB peroxidase substrate

(Seracare) and TMB BlueSTOP Solution (Seracare).

130 5.3.8 Flow cytometric staining profiles of recombinant ferret specific murine antibodies

Cryopreserved parapharyngeal lymph node cell preparations from immunologically naïve ferrets were thawed and washed twice in RF10 media. To establish a flow cytometric panel to validate other murine ferret mAbs recovered using the technique established in this chapter, ferret cells were first preincubated in 1% mouse serum and

1% ferret serum to reduce Fc mediated antibody binding for 30 mins at room temperature. Staining was subsequently performed using anti-CD11b-BV510

(Biolegend: clone M1/70), anti-CD8 eFluor450 (eBioscience Clone OKT8) and anti- ferret CD4 [257]. Cells were subsequently washed and fixed with fixing solution. Cell permeabilization was performed with 100 L 1 X Permeabilising Solution 2 diluted in distilled water (BD) for 7 min. Cells were washed twice to remove residual

Permeabilising Solution, followed by staining with anti-human CD79 PE-Cy7 (Clone

HM79) for 1 h at room temperature before data. All flow cytometry data was acquired with BD FACS Fortessa, with at least 106 events acquired and analysed using

Flowjo10 (BD). All ferret experiments were approved by the University of Melbourne animal ethics committee (approval no. #CT-FER-17-05).

Cells were subsequently stained with Live/Dead stain and preincubated in 1% mouse serum and 1% ferret serum to reduce Fc mediated antibody binding for 30 mins at room temperature. Surface stains were performed with anti-CD11b-BV510

(Biolegend: clone M1/70) murine anti-ferret CD19 or anti-ferret IgD mAbs and incubated for 30 mins at room temperature. Excess staining mAb was removed by washing twice, and native antigen binding was detected using anti-mouse IgG-BV605

(BD Biosciences). Cells were subsequently permeabilised and stained with anti- human CD79 PE-Cy7 (Clone HM79) as described above before data acquisition.

131 5.4 Results

This chapter began a process of developing ferret specific antibodies to assist our work on B cell and NK cell immunity in the ferret model. We chose to work towards developing murine anti-ferret antibodies against CD19, IgD, CD138, NKp46 and

LAMP-1 (CD107a). The overall development pathway is shown above in Figure 5-1.

The stage to which each set of reagents was developed to is summarised below in

Table 5-2.

Table 5-2: Summary of mAb development for ferret B-cell and NK-cell antigens

CD19 IgD CD138 NKp46 LAMP-1 Cloning and √ √* √* √ √ expression Mouse √ √ √ √ √ Immunisations Isolation of √ √ √ √ √ antigen specific memory B-cell Single-cell √ √ X** X** X** RT-PCR recovery of antibody coding genes Expression of √ √ mAbs Antigen √ √ specificity (ELISA) Antigen X*** X*** specificity (Flow cytometry)

*Anomalous SDS-PAGE migration of proteins **Failed to recover IgG+ transcripts ***Failed to recognise native epitopes on ferret cells

132 5.4.1 Identification and expression of ferret antigens for mAb development

5.4.1.1 CD19

To increase the utility of the ferret model to study B-cell responses, the pan B-cell marker CD19 was chosen as there is currently no mAbs targeting cell surface pan-B- cell antigens. While cross-reactive antibodies have been reported to bind B-cells such as CD79 and CD20, the currently available antibodies target intracellular epitopes which require fixation, severely limiting the downstream molecular analyses due to

DNA/RNA damage [376]. The use of surface CD19 targeting stain will enable sorting of live cells to preserve the integrity of genetic material for subsequent RT-PCR amplification. Based on the draft copy of the ferret genome, the predicted ferret CD19 amino acid sequence is available (Uniprot M3YN68) and was extracted for further analyses. To increase the likelihood that the mAbs target cell surface epitopes, sequences coding for the extracellular domain (ECD) was identified based on previous topological descriptions of curated human CD19 sequences (Uniprot P15391)

(residues 20-291). Sequence comparisons with mammalian CD19 ECD domain orthologues (Figure 5-2 A) show high sequence conservation (55.68% - 84.98%), in agreement with important housekeeping functions of CD19 such B-cell development and signal transduction [382]. For example, CD19 associates with CD21 and enhances B-cell signalling in response to T cell-dependent complement tagged antigens [383]. Comparisons with other mammalian orthologues also enabled the correct prediction of phylogenetic relationships between species. Ferret CD19 is predicted to be most closely related to dogs, followed by cats, macaques, humans, rat and mouse (Figure 5-2 B), consistent with the conservation of other proteins [384].

133 We next confirmed the structure of the extracellular domain of CD19 by comparisons with known crystal structures of homologous proteins (homology modelling) [381]

(Figure 5-2 C). Previously elucidated structure of the extracellular domain of human

CD19 (PDB ID 6al5.1.A; with a sequence identity of 58.50%) was identified and used as the template. To evaluate the quality of the predicted model, the GMQE and

QMEAN scores were examined. GMQE combines properties from the target-template alignment and reflects the expected accuracy of a model built with a particular template and ranges from 0 to 1 with scores closer to 1 reflecting higher accuracies.

QMEAN provides quality estimates of a single model globally and locally and scores above -4.0 suggests good quality models. The GMQE score (0-1) of 0.73 and QMEAN score of -2.91 suggests good accuracy of the predicted model. Following validation of ferret CD19, coding sequences of the described proteins were cloned into eukaryotic expression vectors with a 5’- cleavable IL-2 secretion signal (predicted MW: 2.2kDa) upstream of the protein coding sequence (predicted MW; CD19 - 29.7kDa) to enable secretion of protein into cell transfection supernatants. A 3’ human IgG tag (25.5kDa) was included downstream of the ferret protein coding sequences to facilitate protein A purification. Transfection into eukaryotic Expi293 cells and subsequent protein A purification resulted in the expression of recombinant ferret CD19 (57.4 kDa) (Figure

5-2 D)

134 A

135

Figure 5-2 Characterization and recombinant expression of Extracellular domain (ECD) of ferret CD19. (A) Multiple Sequence Alignment of ferret CD19 ECD against mouse (AAA37390.1), rat (NP_001013255.2), cat (XP019676303.1), dog (XP_022275622), macaque (EHH31541.1) and human (AAA69966.1). Fully conserved residues are indicated by an Asterix (*), conservation of strongly similar properties are indicated by a colon (:) and a period (.) indicates conservation of weakly similar properties. Pairwise sequence identity of the various mammalian homologs with respect to ferret CD19 sequence is indicated in the lower panel. (B) Consensus phylogenetic tree. The tree was constructed using Geneious tree builder using the Jukes-Cantor genetic distance model. The tree was built using the neighbor-joining methods with no outgroups and resampled by bootstrapping (100 x). (C) Structural homology of ECD of ferret CD19 as compared with crystal structure of human CD19 using 6al5.1.A. (D) SDS PAGE gel of recombinantly expressed ECD of ferret CD19.

5.4.1.2 IgD

Currently, there is also a lack of flow cytometry markers to delineate different B-cell populations, such as immunologically naïve mature B-cells and activated B-cells.

Ferret IgD was chosen as the second marker for mAb development as a marker of class switched B-cells. Upon encountering antigen in secondary lymphoid organs, mature naïve B-cells downregulate IgD expression transcriptionally, before

136 undergoing AID dependent somatic hypermutation and class switch recombination to diversify the antibody repertoire [385]. The membrane distal CH1 domain, as previously validated in chapter 3, was selected instead of the membrane proximal domains (CH2; CH3) to avoid occluded epitopes as BCRs are organised in microclusters with other co-receptors such as CD19 and CD21 [386]. Sequence comparisons with mammalian homologues (Figure 5-3 A) revealed lower sequence conservation (22.50-45.98%), but phylogenetic analyses correctly predicted evolutionary relationships between different mammalian species (Figure 5-3 B).

Confirmation of structure by homologous modelling (Figure 5-3 C) revealed similar structure to CH1 domain of immunoglobulin gamma chain (4lm.1.A), with a lower

GMQE score of 0.54 and a QMEAN score of -6.61 suggesting lower quality of the predicted model compared to CD19 above. This can be attributed to the lack of x-ray structures for IgD as templates for modelling, and the intrinsically disordered properties of antibody CH1 domains [387]. Expression of the recombinant IgD

(predicted MW: 37 kDa) protein resulted in two distinct bands with apparent MW of

25kDa and 50kD (Figure 5-3 D). To further validate this anomalous pattern of protein migration protein mass spectrometry should be utilised to determine the MW or amino acid sequences de-novo [388].

137

138

Figure 5-3 Characterization and recombinant expression of CH1 domain of ferret IgD. (A) Multiple Sequence Alignment of ferret IgD (CH1) against mouse (V00786), rat (RatNor_6_Chr6), dog (IMGT00001), macaque (Macfas_2_Chr7) and human (X57331). Fully conserved residues are indicated by an Asterix (*), conservation of strongly similar properties are indicated by a colon (:) and a period (.) indicates conservation of weakly similar properties. Pairwise sequence identity of the various mammalian homologs with resepect to ferret IgD sequence is indicated in the lower panel. (B) Consensus phylogenetic tree. The tree was constructed using Geneious tree builder using the Jukes- Cantor genetic distance model. The tree was built using the neighbor-joining methods with no outgroups and resampled by bootstrapping (100 x). (C) Structural homology of CH1 domain of ferret IgD as compared with crystal structure of CH1 domain of human IgG using 4lm.1.A. (D) SDS PAGE gel of recombinantly expressed CH1 domain of ferret IgD.

5.4.1.3 CD138

Effector CD138+ plasma cells play an important role of antibody secretion to provide protection against influenza infections. Studies in humans have revealed the importance of long-lived plasma cell responses which is important for long term protection against influenza; plasma cells specific for pathogens encountered more than 40 years prior were identified in CD138+ B-cells [389]. As there are currently no available antibodies to define this subset of antibody-secreting cells in ferrets, we identified this antigen for murine anti-ferret mAb development. Amino acid sequence alignments revealed high sequence conservation (65.0%-85.1%) (Figure 5-4 A) and enabled the correct prediction of phylogenetic relationships between closely related mammalian species (Figure 5-4 B). No suitable template was available for homology modelling of ferret CD138; CD138 has been previously reported to be an intrinsically disordered integral membrane protein [390]. Expression and purification of the

139 recombinant construct (predicted MW: 52.7 kDa) revealed a broad band with apparent molecular weights ranging from 75 kDa to 100 kDa (Figure 5-4 C). This is consistent with the glycosylation of CD138 [391], which has shown to alter electrophoretic mobilities of proteins [392].

140 A

141 B

142

Figure 5-4 Characterization and recombinant expression of ferret CD138. (A) Multiple Sequence Alignment of ferret CD138 against mouse- P18828; rat – P26260; human – P18827; macaque – A0A2K5WT52; cat – M3W3X8 and dog – E2RT70 homologues. Fully conserved residues are indicated by an Asterix (*), conservation of strongly similar properties are indicated by a colon (:) and a period (.) indicates conservation of weakly similar properties. Pairwise sequence identity of the various mammalian homologs with respect to ferret CD19 sequence is indicated in the lower panel. (B) Consensus phylogenetic tree. The tree was constructed using Geneious tree builder using the Jukes- Cantor genetic distance model. The tree was built using the neighbor-joining methods with no outgroups and resampled by bootstrapping (100 x). (C) SDS PAGE gel of recombinant ferret CD138.

5.4.1.4 NKp46 and LAMP-1

Currently, there are mAb reagents defining ferret CD8+ T-cells and the cytokine IFN-

γ which enables the evaluation of cytotoxic T-cell responses. NK-cell responses have shown to be important for heterotypic protection in influenza [274] but at present there are no mAb reagents that can be used to identify ferret NK cells. A common NK cell marker used across several species is NKp46 and defining NK-cells through NKp46 expression would be useful for ferret immunological studies. Amino acid sequence alignments revealed high sequence conservation (Figures 5-5 A, B) of NKp46 across mammalian species with a sequence identity of 54.4%-60.3%. Subsequent homologous modelling of the ferret NKp46 using human homologue (PDB ID:

1o11.1A) revealed high structural homology with a GMQE score of 0.70 and QMEAN

143 score of -1.08 (Figure 5-5C). Expression and purification resulted in the recovery of recombinant NKp46 with a MW of 49.0kDa (Figure 5-5 D).

To measure NK-cell function, we also targeted LAMP-1 for murine mAb development.

LAMP-1 is commonly used as a degranulation marker for NK-cells and is useful for measuring NK-cell activation and cytotoxic potential [393]. It would also be a useful reagent to define the cytotoxic potential of antigen specific CD8+ T cells. Amino acid sequence alignments of LAMP-1 showed high sequence conservation (Figure 5-6 A,

B) across mammalian species for both LAMP-1 (64.2% - 87.1%). Subsequent homologous modelling using human homologue (PDB ID 5gv0.1A) revealed significant structural homology with GMQE and QMEAN scores of 0.37 and -1.14 respectively (Figure 5-6 C). Expression and purification resulted in the recovery of recombinant LAMP-1 protein with a MW of 66.9 kDa (Figure 5-6 D).

144

145

D C MW Nkp46

75kDa

50kDa

Figure 5-5 Characterization and recombinant expression of ferret NKp46. (A) Multiple Sequence Alignment of ferret NKp46 against human – O76036; mouse – Q8C567; rat- Q9Z0H5; macaque – Q95JB9; bovine – Q863H2; chimpanzee – Q08I02 homologues. Fully conserved residues are indicated by an Asterix (*), conservation of strongly similar properties are indicated by a colon (:) and a period (.) indicates conservation of weakly similar properties. Pairwise sequence identity of the various mammalian homologs with respect to ferret CD19 sequence is indicated in the lower panel. (B) Consensus phylogenetic tree. The tree was constructed using Geneious tree builder using the Jukes- Cantor genetic distance model. The tree was built using the neighbor-joining methods with no outgroups and resampled by bootstrapping (100 x). (C) Homology modelling of predicted NKp46 protein as compared to human NKp46 (PDB ID: 5gv0.1A) (D) SDS PAGE gel of recombinantly expressed NKp46.

146 A

147 B

C D

MW LAMP

75kDa 50kDa

Figure 5-6 Characterization and recombinant expression of ferret LAMP-1. (A) Multiple Sequence Alignment of ferret LAMP-1 against human – P11279; mouse – P11438; rat – P14562; macaque – A0A2K5UV67; dog – F1Q260; cat – M3WKD0 homologues. Fully conserved residues are indicated by an Asterix (*), conservation of strongly similar properties are indicated by a colon (:) and a period (.) indicates conservation of weakly similar properties. Pairwise sequence identity of the various mammalian homologs with respect to ferret CD19 sequence is indicated in the lower panel. (B) Consensus phylogenetic tree. The tree was constructed using Geneious tree builder using the Jukes-

148 Cantor genetic distance model. The tree was built using the neighbor-joining methods with no outgroups and resampled by bootstrapping (100 x). (C) Homology modelling of predicted LAMP-1 protein against human LAMP-1 homologue (PDB ID: 1o11.1A) (D) SDS PAGE gel of recombinantly expressed LAMP-

5.4.2 Immunisation of C57BL/6 mice with recombinant ferret antigens

C57BL/6 mice were subsequently subcutaneously immunised in triplicates with 20

g of recombinant ferret antigens in 100 L TitremaxGOLD adjuvant (Figure 5-7 A).

TitremaxGOLD adjuvant was included in the initial dose to boost and maximise antibody responses [394] as the purified recombinant protein antigens lack PAMPs, which may lead to weak innate stimulation and subsequently diminished B-cell responses. To elicit memory B-cell responses, which are independent of innate stimulation, mice were boosted with 100 g protein in PBS 28 days later. Two weeks later, immunised mice were sacrificed and ferret antigen specific ELISAs were performed to validate serological responses against ferret antigens. Polyclonal serological responses against each ferret antigen were observed in all immunised mice, showing the elicitation of antibody responses against the recombinant ferret antigens (Figure 5-7 B). We next single sorted ferret antigen specific B-cells from these immunised mice to enable the recovery of antibody coding sequences of potential mAb candidates.

149 A

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Figure 5-7 Immunisation and serological responses against ferret antigens in C57BL/6 mice (A) Immunisation regime of C57BL/6 mice. Each ferret antigen was administered to separate groups of mice in triplicates. 20 μg each of recombinant ferret proteins were resuspended in 100 μL Titremax and administered subcutaneously on Day 0. Each antigen was administered in separate groups of mice in triplicates. A single booster shot comprising 100 μg of protein in 100 μL PBS was administered on day 28. Cheek bleeds were collected on Day 42, and ferret antigen specific ELISAs were subsequently performed to confirm polyclonal antibody responses against recombinant ferret proteins. Spleens were harvested for processing for subsequent flow cytometric sorting steps. (B) Ferret antigen ELISAs to confirm polyclonal serum responses in immunised mice. Serum from immunised and naïve mice were diluted 4 folds starting from 1:10 and added to plates coated with recombinant ferret antigens. Ferret antigen serum polyclonal antibody binding was detected using anti-mouse-IgG (H+L) (HRP), followed by the addition of TMB substrate and measurement of OD630.

150

5.4.3 IgD-CD38+ memory B-cell responses to ferret antigens

Memory B-cell responses were subsequently examined in immunised mice to facilitate the recovery of high affinity murine anti-ferret mAbs (Figure 5-8 A) on day 42. Single spleen cell preparations recovered from immunised mice were first gated for single, live lymphocytes. CD45+ leukocytes were selected while human IgG1 tag specific B- cells, CD3+ T cells and F4/80+ macrophages were excluded. Mouse IgD-

CD38+B220+ memory B-cells were gated and antigen specific populations were isolated using recombinant ferret probes conjugated to spectrally distinct fluorophores

(either PE or APC) to increase the probability of sorting B-cells that are specific for the ferret antigens. Comparisons of ferret antigen probe staining in naïve and immunised mice showed the presence of well-defined double positive populations in immunised mice (Figure 5-8 B), showing the presence of B220+IgD-CD38+ memory B-cell responses likely responding to immunising antigens by 42 days post vaccination. We next recovered paired heavy and light chain immunoglobulin sequences from these populations to enable the selection and reconstitution of ferret antigen binding mAbs.

5.4.4 RT-PCR recovery of immunoglobulin sequences from ferret CD19/IgD specific murine B-cells

IgD-CD38+ murine memory B-cells were classically thought to be class switched

(IgG+), but studies have shown the presence of non-switched memory (IgM+) populations with a different functional role [395, 396]. IgM+ memory B-cells have lower frequencies of mutations as compared to IgG+ memory populations and thought to be the low affinity reservoir of central memory B-cells which is important for the maintenance of memory B-cell numbers. IgG+ memory B-cell populations, thought to

151 be the effector pool, have higher BCR affinities [395, 396] and differentiate into plasma cells upon activation by antigen.

Using the gating scheme shown in figure 5-8, ferret antigen specific memory B-cells were single sorted into 96-well PCR plates to enable the isolation of high affinity IgG+ mAbs. We first attempted to isolate IgG+ antibody transcript sequences from the probe positive memory B-cells. We are currently unable to recover any IgG+ sequences from

CD138, NKp46 and LAMP-1 specific memory-B cells though there were strong serum

IgG titres in the immunised mice. We amplified IgK+ transcript sequences successfully from these populations, suggesting that the isolated memory B-cell populations were not class-switched (IgG+) (Appendix 5.1). There is a need for further optimisation of antigens [397], vaccination strategies [398] or flow cytometric gating strategies to target antibody secreting plasma cells, rather than memory B-cells [399] as discussed in further detail below (section 5.5) to recover IgG+ sequences from these mice.

We recovered both IgG+ and IgK+ sequences from both CD19 and IgD specific memory-B cells to enable reconstitution of IgG+ antibody coding sequence pairs.

Consistent with the selection and proliferation of B-cells specific for ferret CD19 and ferret IgD, clonal expanded immunoglobulin transcript sequences were recovered. A total of 288 CD19 and IgD binding memory B-cells were sorted from three C57BL/6 mice each, and genetic features of recovered sequences were characterised to identify mAb candidates for expression.

152

Figure 5-8 Memory B-cell responses in ferret antigen immunised mice. Pharmlyse treated cells derived from splenocytes of immunised mice were first gated for single, live lymphocytes. CD45 was included as a pan-leukocyte marker. CD3 and F4/80 was used to exclude non-T-cells and macrophages. Memory B-cells (B220+, IgD-, CD38+) were gated subsequently. Human IgG specific B- cells were excluded and antigen specific B-cells were isolated using probes labelled with either PE or APC. Antigen specific memory B-cells were single sorted into 96-well PCR plates for recovery of recombined transcripts coding for murine heavy/light immunoglobulin chains. (A) Gating scheme (B) Comparison of ferret antigen probe binding in naïve and immunized mice.

153 5.4.5 Analysis of recovered murine immunoglobulin transcript sequences

5.4.5.1 Genetic features of ferret CD19 specific murine memory B-cells

A total of 99/288 (33.3%) functional class switched recombined heavy chain (IgG) transcripts and 50/288 (17.4%) functional recombined kappa chain transcripts were recovered from ferret CD19 specific murine memory B-cells. In line with previous reports, there is a preferential usage of the IGHV1 family of V-gene segments [400] in ferret CD19 positive transcripts, with 81% sequences belonging to IGHV1. The remaining (19%) sequences belonged to IGHV4 and IGHV6 family of variable chain genes.

While variable genes from 13 IGHV families and 21 IGKV families were recovered, preferential usage of variable gene segments (Figure 5-9 A) and CDR3 lengths (Figure

5-9 B) were noted in both heavy and kappa chain loci. For heavy chain sequences, these major families are IGHV1-12 (39.4%), IGHV-1-55 (28.3%) and IGHV6-3

(15.1%). As for kappa chain variable genes, IGKV1-135 (42%) was noted to be a major gene family recovered, consistent with the selection of B-cell populations in response to ferret CD19 immunisation. Supporting this, biased usage of CDR-H3 lengths of 9 and 14 AA were noted, and deviates from previously reported average CDR-H3 lengths of 11 AA in mice (Figure 5-9 B). The majority of the light chain transcripts showed amino acid lengths of 9 AA, reported to be the most frequent length in naïve mice [400].

154

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r F

F 10

0 0 9 0 1 2 3 4 5 6 7 8 5 6 7 8 9 0 1 2 3 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CDR-L3 length CDR-H3 length 155 Figure 5-9 Genetic features of ferret CD19 specific murine immunoglobulin sequences. Recovered heavy chain sequences were converted to fasta format and analysed using IMGT highV-QUEST. Distribution of Germline Variable (V) and CDR-H3 lengths (AA) of recovered sequences are shown. (A) Characteristics of recovered murine immunoglobulin Heavy and Kappa chain variable genes. Segments are coloured according to murine immunoglobulin gene families (B) CDR3 amino acid length distribution. Segments are coloured according to V-gene family.

156 5.4.5.2 Clonal expansion of ferret CD19 specific murine memory B-cells

The transcript sequences were subsequently grouped into clonal families (Figure 5-

10 A). A total of 23 heavy chain and 25 kappa chain clonal families were recovered from CD19 immune mice. Comparisons of recovered transcript V-gene sequences to germline V-genes revealed presence amino acid substitutions in recovered heavy and light chains, with a mean percent amino acid sequence identity of 93.1% and 96.01% respectively (Figure 5-10 B). Supporting this, sequences within clonal families showed amino acid substitutions at different positions, suggesting AID-mediated affinity maturation in these B-cell populations (Heavy – Appendix 5.2; Kappa- Appendix 5.3).

This method enabled the recovery of 13/288 (4.86%) potential anti-CD19 antibody sequence pairs for further examination.

157 Heavy (IgG) Kappa A

99 50

e n

B n e e 100

100 g

n n

i 95 i

95 l

g g

90 90

i t

n 85

A A

A 80

% %

Figure 5-10 Clonal expansion of CD19 specific immunoglobulin transcripts. (A) Clonal analysis of recovered heavy and kappa chain sequences. Heavy and light chain clonotypes were found and grouped in the same family. Each segment represents a clonally expanded immunoglobulin family and the size of each segment is proportional to the number of sequences. The total number of productive, recombined sequences analysed is indicated in the centre of the pie chart. (B) Amino acid sequence identity of recovered V-gene segments as compared to germline murine sequences.

158 5.4.5.3 Genetic features of ferret IgD specific murine memory B-cells

From three C57BL/6 mice, 54/288 (18.75%) functional recombined heavy chain (IgG) transcripts and 67/288 (23.3 %) functional recombined kappa chain transcripts were recovered from IgD specific murine memory B-cells. Similar to CD19 immune mice, preference for IGHV1 gene family (85.1%) was noted with remaining sequences belonging to IGHV5 and IGHV6.

We recovered sequences from 20 different IGHV families and 30 different IGKV families. (Figure 5-11 A). Bias for heavy variable genes IGHV1-12 (13%), IGHV1-74

(13%), IGHV1-55 (9.3%), IGHV1-80 (13%) and kappa chain genes IGKV3-2 (10.4%),

IGKV10-96 (10.4%), IGKV14-111 (17.9%) were noted. The diversity of CDR-3 lengths recovered are shown in figure 5-11 B, with a preference for CDR-H3 lengths of 6 /14

AA and CDR-L3 length of 9 AA.

159 A Heavy (IgG) Kappa

25 100

20 80

c c n 15

n 60

q q

e 10 e

r 40

F F 5 20

0 0 5 6 7 8 9 0 1 2 3 4 5 6 7 8 8 9 0 1 1 1 1 1 1 1 1 1 1 1 1 CDR-H3 length CDR-L3 length 160 Figure 5-11 Genetic features of ferret IgD specific murine immunoglobulin sequences. Recovered heavy chain sequences were converted to fasta format and analysed using IMGT highV-QUEST. Distribution of Germline Variable (V) and CDR-H3 lengths (AA) of recovered sequences are shown. (A) Characteristics of recovered murine immunoglobulin Heavy and Kappa chain variable genes. Segments are coloured according to V gene families (B) CDR3 amino acid length distribution.

161 5.4.5.4 Clonal expansion of ferret IgD specific murine memory B-cells

Clonal analysis of the amino acid sequences revealed 31 distinct heavy and 35 kappa chain clonal families (Figure 5-12 A). Similar to the transcripts recovered from CD19 specific populations, AID-somatic hypermutation was also evident, with a mean AA sequence identity of 92.55% for heavy chains and 96.45% for light chains (Figure 5-

12 B) consistent with amino acid substitutions at different positions within clonal families (Heavy – Appendix 5.4; Kappa – Appendix 5.5) This method enabled the recovery of 6/288 (2.08%) potential anti-IgD mAb pairs for subsequent expression and screening.

162 Kappa Heavy (IgG)

A IGK

54 67

n e

e 100

g -

e 100

n i

e 95 l

i 95

g e 90

90 t

i t

n 85 n

85 e

A 80

% %

Figure 5-12 Clonal expansion of ferret IgD-specific murine immunoglobulin sequences (A) Clonal analysis of recovered heavy and kappa chain sequences. Heavy and light chain clonotypes were found and grouped in the same family. Each segment represents a clonally expanded immunoglobulin family and the size of each segment is proportional to the number of sequences. The total number of productive, recombined sequences analysed is indicated in the centre of the pie chart. (B)Amino acid sequence identity of recovered V-gene segments as compared to germline murine sequences.

163 5.4.6 ELISA validation of murine anti-ferret CD19/IgD IgG+ mAbs

Candidate mAbs were derived from clonally expanded sequences as described previously. Noting different light chains associated with the same heavy chain sequence families, Representative CD19 antibodies from 8 heavy chain clonal families were expressed in previously reported murine IgG/IgK expression plasmids [171]

(Table 5-3). 8 out of 13 CD19 antibodies (clones 2,3,5,7,8,12 & 13) and all 6 out of 6

IgD antibodies were successfully expressed. To examine the binding specificities of the isolated murine mAbs, human IgG1 and ferret CD19/IgD ELISAs was performed to differentiate ferret antigen specific and human IgG purification tag specific mAbs.

Two out of 8 (Figure 5-13 A) expressed anti-CD19 clones (clones 5 & 12) were found to be specific for the recombinant ferret CD19 protein and not to the human IgG1 purification tag (Figure 5-13 B). Out of 6 IgD specific clones, 5 clones (IgD clones 2,

3, 4, 5 & 6) exhibited ferret IgD binding activity by ELISA. While IgD clones 4 and 5 bound with higher affinities than the other clones, human IgG1 binding was present, thus likely indicating binding specificities for both antigen and purification tag of the recombinant proteins. The remaining 3 IgD clones (2, 3 and 6) appeared specific for recombinant ferret IgD by ELISA (Figure 5-13 B). These findings confirm the capacity of the methodology we developed to derive antigen specific mAbs from immunised mice.

164 Table 5-3: Amino acid sequences of expressed murine anti-ferret CD19 and IgD mAbs. Successfully expressed antibodies are indicated in bold

CD19 Closest Amino Amino

Germline acid

V-gene sequence

identity

Clone 1 Heavy IGHV1-55 89.8% QVQLQQPGAEVVKPGASVKMSCETSGYTFTSYWITWVNQRPGQGLEWIGELYPGSGRINYNEKFKSKATLTVDTSSSTGYMQLSSLTAEDSAVYYCARGNGNSQSYYFDYWGQGTTLTVSSASTS

Kappa IGKV1-135 98% DIVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIK

Clone 2 Heavy IGHV1-55 89.8% QVQLQQPGAEVVKPGASVKMSCETSGYTFTSYWITWVNQRPGQGLEWIGELYPGSGRINYNEKFKSKATLTVDTSSSTGYMQLSSLTAEDSAVYYCARGNGNSQSYYFDYWGQGTTLTVSSASTS

Kappa IGKV8-30 98% DIVVTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIK

Clone 3 Heavy IGHV1-74 94.8% QVQLQQPGAELVKPGASVKVSCKASGYIFISYWMHWVKQRPGQGLEWIGRIHPSDSDTDYNQNFKGKATLTVDKSSSTAYMQLSSLTSDDSAVYYCTTRAGRAWFAYWGQGTLVTVSAASTS

Kappa IGKV6-15 92.6% DIVVTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQRPGQSPKALIYSASYRNSGVPDRFTGSGSGTDFTLTISNVQSEDSAEYFCQQYSTYPLTFGAGTKLELK

Clone 4 Heavy IGHV1-12 94.9% QAYLQQFGAELVRPGASVNMSCKASGNTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKNKATLTVDKSSITAYMQLSSLTSEDSAVYFCARIGSGFAYWGQGTLVTVSAASTS

Kappa IGKV2-109 96% DIVETQSPFSNPVTLGTSASISCRSSKSLLHSNGITFLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPFTFGSGTKLEIK

Clone 5 Heavy IGHV1-12 92.9% QAYLQQSGAELVRPGASVKMSCKASDYTFTSYNIHWVKQTLRQGLEWIGAIYPGNGASSYNQNFKGKATLTIDKSSSTAYMQLSSLTSEDSAVYFCARLTTVVATNYWGQGTTLTVSSASTS

Kappa IGKV8-19 95% DIVMTQSPSSLTVTAGEKVTLSCKSSQSLLHSGTQKNYLTWHQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDRAVYYCQNDYSYPLTFGAGTKLEIK

Clone 6 Heavy IGHV6-3 92.9% EVKLEESGGGLVRPGGSMKLSCIASGFTFSKYWMNWVRQSPEKGLDWVTQIRLKSENYATHYAESVKGRFTISRDDSKSSVYLEMNNLRAEDTGIYYCTDYDVWGTGTTVTVSSASTS

Kappa IGKV1-133 97% DIVVTQTTLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFLGTFGGGTKLEIK

Clone 7 Heavy IGHV1-12 92.9% QAYLQQSGAELVRPGASVKMSCKASDYTFTSYNIHWVKQTLRQGLEWIGAIYPGNGASSYNQNFKGKATLTIDKSSSTAYMQLSSLTSEDSAVYFCARLTTVVATNYWGQGTTLTVSSASTS

Kappa IGKV1-135 99% DVVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIK

Clone 8 Heavy IGHV1-12 98% QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQVSSLTSEDSAVYFCARSGDRLGYPFAYWGQGTLVTVSAASTS

165 Kappa IGKV8-28 98% DIGATQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLEIK

Clone 10 Heavy IGHV1-53 99% QVQLQQPGTELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVHYCARGSLRQYFDVWGTGTTATVSSASTS

Kappa IGKV6-17 87.2% DIVMTQSYKFMSTSVRDRVSITCKASLDTSTAVAWYQQKPGQSPKIMIYSASTRFTGVPHRFTGSGSGTDFTFTISSVQAEDLAVYYCRATYSTLRTFGGGTKLEIK

Clone 12 Heavy IGHV6-3 92.9% EVKLEESGGGLVRPGGSMKLSCIASGFTFSEYWMNWVRQSPEKGLDWVTQIRLKSENYATHYAESVKGRFTISRDDSKSSVYLEMNNLRAEDTGIYYCTDYDVWGTGTTVTVSSASTS

Kappa IGKV6-23 90.5% DIVMTQFHKFMSTSVGDGVSITCKASQDVGTAVAWYRQKPGQSPKLLIFWSSARHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNTYPLTFGTGTKLEIK

Clone 13 Heavy IGHV6-3 94% EVKLEESGGGLVQPGGSMKLSCVASGLTFSNYWMNWVRQSPEKGLEWVAQIRLKSDNYATHYAESVKGQFIISRDDSKNSVYLQMNNLRTEDTGIYYCTGGRDVEDYWGQGTTLTVSSASTS

Kappa IGKV3-4 100% DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPLTFGAGTKLEIK

IgD Closest Amino

Germline acid

V-gene sequence

identity

Clone 1 Heavy IGHV1-55 89.8% QVQLQQPGAEVVKPGASVKMSCETSGYTFTSYWITWVNQRPGQGLEWIGELYPGSGRINYNEKFKSKATLTVDTSSSTGYMQLSSLTAEDSAVYYCARGNGNSQSYYFDYWGQGTTLTVSSASTS

Kappa IGKV14- 98.9% EIQMTQSPSSISASLGDRITITCQATQDIVKNLNWYQQKPGKPPSFLIYYATELAEGVPSRFSGSGSGSDYSLTISNLESEDFADYYCLQFYEFPRTFGGGTKLEIK

130

Clone 2 Heavy IGHV1-80 88.8% QVQVQQSGAELVKPGASVKIPCKASGYAFSSYWMNWVKQRPGKGLEWIGQIYPGDGETRYNGQFKDRVTLTVDKSSSTAYMQFTSLTSEDSAVYFCARGTGYYFDYWGLGTTLTVSS

Kappa IGKV4-50 92.4% DIVLTQSPALMSASLGEKVTMSCRASSSVNYMYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGISYSLTISSLETEDTATYYCQQFTNFPYTFGGGTKLEIK

Clone 3 Heavy IGHV1-80 91.8% QVQVQQSGAELVKPGASVKIPCKASGYAFSSYWMNWVKQRPGKGLEWIGQIYPGDGETRYNGKFKDKATLTVDRSSSTAYMQFSSLTSEDSAVYFCARGTGYYFDYWGQGTTLTVSS

Kappa IGKV4-50 96.8% ENVLTQSPAIMSASLGEKVTLSCRASSSVNYMYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGISYSLTISSMEGEDAATYYCQQFTSFPYTFGGGTKLEIK

Clone 4 Heavy IGHV1-81 90.8% QVQLQQSGAELARPGASVKLSCKASGYTFRNYGITWMRERTGQGLEWIGEIYPTSDNTYYNEKFKGKATLTADKSSSTAYMEFRSLTSEDSAVYFCARSTYGSNYDDALDHWGQGTSVTVSSASTS

Kappa IGKV12-46 95.8% DIQMTQSPASLSVSVGETVTITCRASENIFSNLAWYQQKQGKFFQLLVYAATKLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPRTFGGGTKLEIK

166 Clone 5 Heavy IGHV1-81 94.9% QAQLQQSGAELARPGASVKLSCKTSGYTFTSFGIHWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEASAVYFCARSSYGSYYDDAVDYWGQGTSVTVSSASTS

Kappa IGKV12-46 100% DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPRTFGGGTKLEIK

Clone 6 Heavy IGHV1-74 92.9% QVQLQQPGAELVKPGASVKVSCKASGYIFVSFWMHWVKQRPGQGLEWIGRIHPSDSETYYNQKFKGRATLTVDTSSSTAYMQLSSLTSEDSAVYYCAIGFDYWGQGTTLTVSS

Kappa IGKV10-96 98.9% DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDTATYFCQQGNTLPLTFGAGTKLELK

167

Figure 5-13 ELISA validation of CD19/IgD specific mAbs. figure (A) Denaturing SDS-PAGE profiles of recombinant ferret CD19 and IgD binding mAbs. Heavy (50kDa) and light (25kDa) chains are shown. Lane 1/4 : MW marker. Lane 2: CD19 clone 5. Lane 3: CD19 clone 12. Lane 5:IgD clone 2. Lane 6: IgD clone 3. Lane 7: IgD clone 6 (B) 96 well ELISA plates were coated with recombinant ferret proteins or Human IgG1 protein. Candidate murine mAbs were serially diluted starting at 0.01mg/mL and added to wells for binding. Binding was detected using anti-mouse IgG conjugated to HRP and subsequent addition of TMB. Readings were measured at 630nm using a 96 well plate spectrophotometer

168 5.4.7 Flow cytometric staining profiles of CD19/IgD mAbs

We next established a flow cytometric panel to enable the validation of ferret mAbs isolated using the method presented in this thesis. A panel comprising previously reported reagents to stain ferret T and B lymphocytes was established using anti-

CD79 (HM47), anti-human CD8 (OKT8) specific antibodies and a novel anti-ferret

CD4 specific mAb [257] (Figure 5-14 A). This panel enabled the resolution of CD4+

T-cells, CD8+ T-cells and CD79α+ B-cells as distinct populations.

The candidate ferret specific IgG+ mAbs (2 for CD19 and 3 for IgD) were subsequently analysed for binding to natively expressed antigens by flow cytometry on lymphocytes derived from single cell preparations of parapharyngeal lymph nodes of immunologically naïve ferrets. Our isolated clones failed co-stain CD79α+ B-cell populations above background staining observed by a mouse secondary antibody alone (Figure 5-14 B), suggesting that the mAbs lacked specificities for native ferret

B-cell proteins. We speculate this could be due to the occlusion of epitopes that were targeted by the isolated mAbs, as CD19 is expressed on B-cells as a complex with other proteins such as CD21, CD82, CD225 [401] and cell surface immunoglobulins

[402]. These findings could also be attributed to structural or conformational differences between recombinant and natively expressed proteins [403] which abrogated mAb binding of cell surface proteins.

169 A

Anti-mouse IgG only CD19-5 CD19-12

CD79a

IgD-2 IgD-3 IgD-6

Anti-mouse IgG

Figure 5-14 Flow cytometric staining profiles of murine anti ferret mAbs. (A) Single ferret lymphocytes derived from parapharyngeal lymph nodes were first gated on live, CD11b- myeloid cells. Co-staining profiles of CD4+ and CD8+ populations are shown and CD79α expression of each of the indicated populations are shown. (B) Staining profiles of putative ferret CD19 and IgD mAb clones. Live, CD11b single lymphocytes were first gated and CD19/IgD antigen binding was examined using a secondary anti-mouse antibody conjugated to BV605.

170 5.5 Discussion

In this chapter, we present a general method to recover and express murine IgG+ mAbs specific for ferret antigens based on previously reported single cell murine RT-

PCR based protocols[171, 378]. The use of a flow cytometric sort panel targeting antigen specific murine memory B-cells enabled the recovery of immunoglobulin sequences and derivation of candidate mAbs from immunised mice. Here, we highlight key steps of this process that can be optimised to increase the probabilities of recovering the mAbs of interest using this approach.

Based on coding sequences derived from the ferret genome, ferret B-cell markers

CD19, IgD and CD138 and NK cell markers NKp46 and LAMP-1 were characterised using available bioinformatics tools and expressed to develop surface antigen specific mAbs. It is noted that coding sequences for recombinant proteins were based on the draft copy of the ferret genome from a single ferret, and genetic variants of these proteins are not yet characterised [404, 405]. Future characterisation of polymorphic variants across multiple outbred ferrets and breeding colonies would potentially enable the selection of potentially more suitable invariant epitopes as targets for immunisation.

We noted anomalous migration of ferret IgD and ferret CD138 proteins on SDS-PAGE gels. This highlights the limitations of SDS-PAGE, where proteins may display altered electrophoretic properties due to SDS binding characteristics [406] or post- translational modifications (PTMs) [392]. Further validation of such antigens by mass spectrometry will enable definitive determination of the identity of the expressed

171 recombinant proteins. This step is critical, as the specificities of the mAbs generated is dependent upon the immunising antigen.

The panel used in this chapter enabled the recovery of potential antibody coding sequences from B220+CD38+IgD- memory B-cells in the mice immunised with ferret

CD19 and IgD. Rare mouse memory B cell populations binding to ferret antigens were detected by antigen baiting. CD38 is reported to be expressed on all naïve B-cells but is downregulated in GC B-cells and subsequently restored on transition to long term memory B-cells [407]. The inclusion of this marker enabled the selection of affinity matured mouse CD38+IgD- memory B-cells derived from GC B-cells [156].

Clonally expanded sequences with somatic amino acid substitutions were recovered from both IgG+ and kappa chain loci from CD19 and IgD immune mice, indicating that the mice were generating effective switched (IgG) memory B cell responses to these ferret antigens using our current immunisation scheme. Memory, rather than GC populations were targeted to minimise the isolation of polyreactive populations that are selected against over time [408], as SHM has previously been reported to lead to antibody polyreactivity [327]. This marker is also important for the exclusion of polyreactive splenic T-cell independent marginal zone B cells which have the highest expression of CD38 among B-cells and involved in low affinity primary antibody responses [409].

We noted poor recovery of paired IgG+ antibody sequences from CD19 (4.86%) and

IgD specific memory B-cells (2.08%). This could likely be due to the exclusion of adjuvants in the boost vaccination step, which led to poor affinity matured B-cell

172 responses in the vaccinated mice. Recovery of IgG+ transcripts could be potentially improved by the inclusion of adjuvants or increasing the number of immunisations.

Bioinformatical tools to computationally predict MHC-II, TCR and BCR epitopes are also available and have been successfully used for the development of highly effective mAbs [410]. Since the generation of high affinity, class switched B-cell responses is contingent upon cognate T-cell help, such bioinformatical tools could be employed to identify antigenic T/B cell ferret peptides or epitopes to boost T-dependent B-cell responses and improve IgG+ responses. IgG binding antigenic epitopes have been evaluated using microarray peptide-immunoassays [398] and will be useful for the delineation of predicted ferret antigen epitopes likely to elicit strong IgG+ responses in immunised mice. Optimisation of vaccination regimes could also enhance IgG+ memory B-cell responses. We utilised a homologous prime-boost strategy to elicit antibody responses using only recombinant ferret protein. Heterologous prime-boost regimens using a combination of recombinant proteins and expression vectors have been reported to further enhance antibody responses [398].

We also noted our current inability to recover IgG+ transcripts from NKp46, LAMP-1 and CD138 memory B-cells though IgG serum responses were detected by ELISA in these groups of mice. Previous correlational studies showed weak associations between serum titres of antigen specific IgG and the frequencies of antigen-specific

IgG+ memory B-cells in mice [411]. The long-term persistence of circulating antibodies is not provided by circulating memory B-cells per se, but rather short lived plasma cells derived from antigen activated memory B-cells [412] or long-lived plasma cells [413] found in the bone marrow [414]. The isolation of these populations by the inclusion of

173 plasma cell marker CD138 [399] in the sort panel could lead to the recovery of IgG+ transcripts from these mice for the reconstitution of antigen specific mAbs.

Other enhancements to the current sort panel, such as the addition of mouse mAbs targeting surface IgM or surface IgG would also enable the exclusion of IgM+ or the selection of IgG+ populations to improve the efficiency of IgG+ transcript sequence recovery [330]. The relatively poor recovery of the transcript sequences could also be due to variable efficiencies of the multiplex primers used as reported previously [378,

415], or nucleotide substitutions due to AID-dependent SHM. Such mutations may have contributed to less efficient primer binding due to sequence mismatches with the murine immunoglobulin specific primers.

Using this method developed in the current chapter, candidate ferret CD19 and IgD specific mAbs recognising cognate recombinant antigen by ELISA were identified, validating this method to recover antigen binding murine mAbs. While our ferret mAbs subsequently did not possess the capacity to resolve ferret B-cells by flow cytometry, future improvements to the methodology as described in this chapter will increase the probabilities of recovering ferret specific murine mAbs. Figure 5-15 illustrates the recommended workflow as described in this chapter for generating antigen specific murine mAbs using this approach.

5.6 Conclusion

This chapter presents a general methodology to recover ferret antigen specific murine mAbs using modern single cell recombinant techniques. Further optimisation of antigens, vaccination regimes and flow cytometric panels will enable high affinity

174 mAbs to be isolated. This will enable the detailed study of ferret immune responses and improve the efficiency of ferret HA-specific mAb recovery in the future.

175

Figure 5-15 Workflow for generating ferret specific mAbs using murine single cell-RT-PCR. Key steps in this process is highlighted. Possible experiments or procedures for each key step is listed.

176 Chapter 6

General Discussion

6.1 Overview

Influenza is an important respiratory disease that causes annual widespread global epidemics and sporadic pandemic outbreaks. The mutability of the segmented viral

RNA genome and variable vaccine efficacies has led to the need for universal influenza vaccines. There are currently no licensed vaccines that provide broad protection across different influenza strains. More effective vaccines are required urgently to increase preparedness for future pandemic outbreaks.

Mice are widely used to characterise influenza-specific immune responses due to the wide availability of immunological reagents and readily available genetic sequence information. Detailed studies of pathogen-specific immune responses in mice have increased the understanding of host-pathogen interactions, which in turn informs influenza vaccine design. However, influenza studies in mice are limited to a number of mouse-adapted influenza strains such as PR8, and clinically relevant strains that are circulating among humans cannot be used to directly infect mice for in vivo studies.

Wild-type mice are also unsuitable for studying emerging human respiratory viruses such as coronaviruses [416] which are involved in the ongoing coronavirus (COVID-

19) pandemic. Mouse adapted coronavirus strains or transgenic hACE2 mice models, have been established and proven useful for characterising such viruses, but there is an increasing number of studies involving larger mammalian models such as NHP and ferrets. These animal models are more representative of human immune responses

177 due to similarities in respiratory structures and physiology. While much progress has been made NHP in terms of genomic sequencing and annotation [417] which has enabled the development of tools to interrogate immune responses [331], ferret genomic sequencing and annotation is still considered to be in its infancy, with a draft copy of the ferret genome [312] and annotation of the TCRB locus [311].

In this PhD thesis, we sought to fill knowledge gaps regarding ferret immunoglobulins and derive tools which will enable the interrogation of B-cell responses in ferrets, similar to those developed for humans and other animal models. We first established the capacity to recovery ferret mAbs (chapter 3). These tools were utilised (chapter 4) to analyse HA-specific B-cell repertoires and derive influenza neutralising HA-specific ferret mAbs for mapping HA epitopes. As we noted the poor recovery of ferret HA specific mAbs due to the lack of flow cytometric panels, we subsequently established a methodology and examined key steps in the process which will enable the recovery of ferret specific mAbs suitable for techniques such as flow cytometry and confocal microscopy (Chapter 5). These tools will improve the ferret model by enabling the in- depth interrogation of B-cell responses.

6.2 In-depth ferret B-cell studies are hindered by the lack of immunological reagents and immunogenetic information

Detailed immunological studies of host immune responses against human respiratory viruses in the ferret model are scarce. While there are beginning to be studies addressing immune responses in ferrets, most studies are based on molecular analyses or reagents targeting pan-immune T and B cell markers which does not enable the examination of different effector functions such as memory. Antibody

178 responses have been shown to be important for protective immunity in NHP models.

Curated genomic sequences for NHP are available, facilitating the development of tools and reagents for in-depth interrogation of NHP B-cell responses. For example, macaque mAbs targeting novel epitopes of emerging pathogens such as Ebola viruses

[418] were identified, which is important for informing vaccine design for emerging human pathogens. Detailed analysis of such responses is currently not feasible in ferrets due to the lack of reagents to resolve rare antigen-specific B-cell populations by flow cytometry, and the absence of well-established methodologies and tools to isolate antigen-specific ferret mAbs.

6.3 Development of techniques and tools to recover and express ferret mAbs

To address these knowledge and technological gaps in the ferret field, this thesis aimed to develop novel tools and reagents (Figure 6-1) which will enable the detailed study of pathogen-specific B-cell responses in ferrets.

As the annotation of the ferret genome is not complete, we first identified and annotated immunoglobulin coding genes (Chapter 3). While the ferret genome is currently incomplete, we identified a broad cross section of V, D and J genes across both heavy and light chain loci spanning the various immunoglobulin clans. This information allowed us to design novel multiplex ferret immunoglobulin specific primers which amplified paired heavy and light chain antibody coding sequences from single sorted ferret B-cells. While we did not identify any IgG subclasses, the validation of immunoglobulin chain constant regions enabled the construction of ferret IgG coding plasmids, which enabled the recovery and purification of a chimeric CR9114 ferret/human mAb retaining HA binding activity.

179 The development of these tools subsequently enabled the recovery immunoglobulin transcript sequences from influenza infected ferrets, enabling analysis of HA specific antibody repertoires in ferrets (Chapter 4). We subsequently identified and expressed a single clonally expanded family of mAbs which retained HA binding activity and neutralised influenza virus in-vitro, showing proof of concept of recovering HA specific mAbs of HA epitope mapping studies. We noted the poor recovery of ferret immunoglobulin transcript sequences, which could be attributed to the lack of flow cytometric panels available for ferrets.

We therefore next established a single-cell RT-PCR based methodology (Chapter 5) to recover ferret mAbs specific for B-cells and NK-cells in an attempt to broaden the scope of immunological analyses that can be carried out in ferrets. While we were unable to identify mAbs which were able to resolve any ferret cell population by flow cytometry, we studied key steps in the process which will inform future application of this approach to increase the probability of isolating the ferret antigen specific mAbs of interest, which will be discussed in further detail below.

180

Figure 6-1 Thesis overview. In this thesis, knowledge and technological gaps in ferret immunology were addressed which enabled the recovery of HA-specific ferret mAbs. These tools were developed based on genomic sequence information encoded within the ferret genome. Immunoglobulin coding and B-cell antigen sequences were extracted from the genome, which enabled the development of tools to isolate ferret antigen specific mAbs. To improve panels available for flow cytometry and confocal microscopy, we also developed a methodology to recover murine antigen specific mAbs. Together, these tools will advance the ferret model by providing novel methods and tools for the field.

181 6.4 Unidentified germline immunoglobulin ferret genes in the draft copy of the ferret genome

The knowledge of immunoglobulin coding genes in humans and mice have led to the design of PCR based techniques to examine B-cell immune repertoires against influenza. For aim 1 of this thesis, we attempted to bridge this knowledge gap by identifying ferret immunoglobulin genes in the draft copy of the ferret genome. The evolution of B-cells from jawed fish to mammals has led to the fine tuning of immune responses against a wide variety of pathogens [419]. As immunoglobulin genes are highly conserved across mammals, we inferred homology by sequence similarities using human and canine orthologs. This enabled the annotation and identification of ferret immunoglobulin genes on the ferret genome which facilitated the recovery and reconstitution of antibodies from single ferret B-cells.

A key limitation of this thesis is the incomplete draft copy of the ferret genome based on a single outbred ferret. As reported, only 2.28 Gb of the ferret genome out of 2.41Gb has been assembled, with 1.3 x 108 bp gaps between contigs. While putative germline immunoglobulin genes have been identified, with a genetic map constructed for the kappa chain locus (chapter 3), both heavy and lambda chain loci were noted contained in separate contigs. This suggests the presence of more unidentified immunoglobulin coding genes in the gap sequences, consistent with the current inability to identify genomic clan III IGLV sequences on the ferret genome, which may have accounted for the inefficient recovery of the transcript sequences.

This highlights the need for validated databases of germline ferret immunoglobulin genes, such as those maintained by IMGT to facilitate improvements in the primer sets

182 that were developed in this thesis to enable the amplification of a broader cross- section of immunoglobulin genes. Next-generation sequencing approaches have been applied in NHPs successfully to identify germline immunoglobulin genes. Using long sequence reads, reference Ig loci for rhesus macaques were successfully assembled and identified. Tools for analysis of large RNAseq sets such as VDJPuzzle [354],

BraCer [355], BALDR [356] and BASIC [357] have been deployed for analysis of antibody repertoires from cats [420] and rhesus macaques, which will facilitate future identification of ferret immunoglobulin genes. The ferret genome project conducted at the Broad Institute is underway, and a more complete copy of the ferret genome detailing genetic variations will be available in the near future. This approach will allow the eventual discovery of more immunoglobulin genes and allelic variants to facilitate future improvements to this methodology.

6.5 Recovery of antigen-specific B-cell transcripts and mAbs from ferrets

The development of the ferret single-cell RT-PCR technique as described in this thesis enabled the recovery and analysis of clonally expanded HA-specific immunoglobulin transcript repertoires from influenza-infected ferrets using HA probes. From the recovered sequences, two fully ferret mAbs belonging to the same clonal family were identified and showed HA binding by ELISA. Subsequent HAI/microneutralisation assay/viral escape assays confirmed the neutralisation capacity of these mAbs, providing proof of concept for recovering HA-specific ferret mAbs using tools developed in this thesis.

While we were successful in recovering these ferret mAb reagents, the transcript recovery rates of ferret mAb sequence pairs (2.3%) were noted to be much lower than

183 reported in humans (30-60%). This could be attributed to missing information in the draft copy of the ferret genome as mentioned previously and the inability to resolve important ferret B-cell populations such as IgD- activated or CD138+ plasma cell populations. Recovery of the immunoglobulin sequences could also be impacted by allelic variations of outbred ferrets and SHM, as in the case of amplifying sequences from somatically mutated ferret transcripts which may have lowered primer binding efficiencies.

To enable the isolation of effective anti-ferret murine mAbs the following should be explored. Antigen design could be improved (described above) to identify IgG+ and accessible epitopes to generate effective more effective mAbs for flow cytometry and confocal microscopy. Optimising mouse immunisation regimes such as the inclusion of adjuvants in boost steps could improve the recovery of more mAbs to screen.

6.6 Studying mAb recognition profiles of influenza HA

We now have the proof of concept for recovering ferret mAbs for mapping HA epitopes to compare species specific differences in the antibody responses against HA at the mAb level. Further improvements in the efficiency of recovery will enable a larger panel of ferret mAbs to be recovered for such studies. This will complement mapping studies which have been performed using polyclonal serum samples. For example, a study of influenza A (IAV) HA antigenic sites using engineered viruses with mutations in each of the antigenic sites surrounding the RBD revealed species-specific differences in antibody recognition [319]. HI activities against H1N1 (A/Michigan/45/2015) mutants as described revealed differences in antigenic epitopes recognised by mice, guinea pigs, ferrets and adult humans using serum samples. Neutralising antibodies in adult

184 humans recognised mostly Sb and Sa, while responses in ferrets were mostly directed to Sa [319]. Another similar study characterising humoral responses against influenza

B (IBV) using reverse engineered viral mutants showed that the proportion of human antibodies targeting non-canonical antigenic sites of IBV HA are comparable to canonical antigenic sites (120 loop, 150 loop, 160 loop and 190 helix) [421]. This contrasts with IBV specific responses in mice and ferrets, where most serum antibody is directed against canonical antigenic sites only. These results suggest inter-species differences in humoral recognition of the influenza HA. Other studies lend support, with a head to head comparison between humans and ferrets immunised or infected with various seasonal H3 vaccine strains showing key differences in serological recognition of viral HA [422] based on molecular modelling and antigenic cartography.

Similarly, cross-reactive H1N1 antibody responses in ferrets are not predictive of cross-reactive responses in humans [423], confirming fundamental differences in B- cell responses between humans and ferrets.

To improve the recovery of ferret anti-HA antibodies for such studies, the annotation of immunoglobulin genes should first be completed. The annotation performed, although allowing us to identify ferret immunoglobulin genes, was limited in that the genomic sequencing was incomplete, resulting in incomplete annotation. An improved annotation could be achieved by first completing genomic and transcriptomic sequencing of the ferret. Such improvements would enable the improved design of ferret-specific primers to recover broader families of immunoglobulin gene transcripts and subsequently anti-influenza mAbs.

185 An additional important step to improve the recovery of anti-influenza mAbs will be to optimise the PCR protocols. Rather than the multiplex approach we applied, a 5’-

RACE approach could be developed to reduce preferential bias of certain gene families to enable a clearer picture of the BCR repertoires analysed. This approach enables the addition of adaptor sequences, which can be targeted during subsequently amplification steps together with immunoglobulin constant regions to potentially improve amplification efficiencies allowing more candidate mAbs to be recovered for screening An alternative approach would be to use NGS to recover a larger repertoire of mAbs for sequencing (discussed further in section 6.9)

The flow cytometric panels available limited the capacity to isolate highly purified memory B cells to recover high affinity ferret anti-HA mAbs, an issue approached in

Chapter 5. Potential improvements in the efficiency of recovery ferret-specific mAbs is discussed in section 5.5.

6.7 FcR and IgG subclasses in ferrets

Currently, only one IgG subclass is identified in this thesis and there is also currently a lack of knowledge of FcR diversity in ferrets. Different IgG and Fc receptor subclasses in humans and mice have shown to be important for different antibody mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC).

Human Fc gamma receptor IIIa (FcγRIIIa; FcγRIV in mice) is the main receptor involved in ADCC and human IgG3 (mouse IgG2a) followed closely by human IgG1

(mouse IgG2b) display the highest affinities for this receptor. Advanced assays for

ADCC and other Fc-mediated responses to influenza have been developed in recent years in both NHP [365] and humans [124, 424]. Such non-neutralising mechanisms

186 have been shown to be important for broadly protective responses against antigenically distinct strains of influenza [365, 425, 426], and development of universal influenza vaccines.

Many of the current ADCC and related assays rely on the detection of Ab-mediated activation of NK cells to express cytokines such as IFN- or degranulation markers such as CD107a [366], but there are currently no ferret reagents to differentiate NK cells from other cytotoxic lymphocyte populations such as CD8+ T-cells [260]. While a ferret specific T cell IFN--expression assays [427] to measure CD8 T lymphocyte activation have been developed and validated, the future elucidation of corresponding ferret NK-cell reagents and ferret IgG/Fc receptor orthologues will enable ferrets to be used to evaluate antibody mediated ADCC responses.

6.8 Development of ferret reagents

A key technological gap that hinders detailed studies of ferret immune system by flow cytometric based assays is the lack of immunological reagents. Successful efforts have been made to identify mAbs to markers for T cells such as CD4 and CD8 and for

B cells such CD79α. mAbs against different ferret heavy chain subclasses (IgA, IgM,

IgE, IgG) are available for ferret B-cells, but currently do not have the capacity to resolve these populations on flow cytometry [263]. This is likely due to the concomitant ability of these reagents to bind and recognise immunoglobulin light chains. mAbs targeting pan B-cell markers available for ferrets such as CD20/CD79/CD79 only target intracellular epitopes to date, severely limiting downstream molecular analyses due to the requirement for cellular fixation which is deleterious for DNA/RNA. To address the lack of reagents, we next attempted to recover murine mAbs specific for

187 B-cell markers CD19, IgD and CD138, as well as NK-cell markers NKp46 and LAMP-

1. While our mAbs bound to cognate ferret antigens by ELISA, we noted the inability of our mAbs to resolve the intended ferret cell populations on flow cytometry. We identified key steps in the workflow presented in the thesis that are critical for developing high affinity ferret specific mAbs.

As mAb specificities is contingent upon the immunising antigen, careful evaluation of candidates is critical. An important consideration is the selection of solvent exposed

(hydrophilic) regions such as hydrophilic peptides for immunisation as most antigens are co-expressed with accessory proteins Tools to quantify hydrophilicity/hydrophobicity such as ProtScale [428] (ExPASy) are available and enables the selection of epitopes that are unlikely to be occluded as targets for immunisation. This can also be examined directly using validated 3D crystal [410] or predicted structures [429] of target antigens. Using serum from mice immunised with whole ferret antigens, important IgG+ epitopes can be identified using an array of peptides derived from the same antigen [430]. The identified ferret peptide antigens can be subsequently used to generate ferret antigen specific mAbs to increase the probabilities to recover mAbs of interest.

A wide variety of expression vectors (Viral, DNA Bacterial) have been developed, and studies have shown that boosting with a different vector carrying identical antigens

[431] or a combination of vectors and proteins enhances antibody responses [432].

There are effective flow cytometric panels and ELISPOT assays to evaluate class switched B-cell responses in mice [433, 434]. These tools can be applied to compare and optimise different vaccination strategies and regimes to optimise IgG+ B-cell

188 responses which may aid in the improvement of the recovery of IgG+ transcripts.

Improvements to the sort panel, such as the inclusion of CD138 to target plasma cells will also enable recovery of antibody sequences from these populations for screening and identification of potential candidates.

6.9 Cell display and NGS technologies to isolate antigen specific mAbs

We noted in this thesis is the relatively low recovery of mAb sequences using the single-cell RT-PCR approach. While we have addressed key limitations of such an approach, alternative methods should be considered. Cell display technology has enabled large libraries of potential mAbs and for rapid screening and identification.

The key advantage of this technique is the ability for biopanning [435]. By optimising selection and screening conditions, this technology has enabled the examination of large mAb libraries specific for conformations and epitopes. This approach has been successfully used to identify antigen specific mAbs [435] and will be useful for identifying anti-ferret mAbs suitable for flow cytometry and immunohistochemistry.

NGS technologies have enabled unprecedented depth and coverage of DNA sequencing and is routinely applied for a wide variety of applications such as genome assembly [436] and gene expression analysis [437]. While traditional display libraries are analysed by the isolation of potential clones in the order of 102 – 103 using Sanger sequencing [438], the application of NGS technologies in combination with cell display has increased the potential number of mAb sequences that can be screened up to 107 to increase the probabilities of isolating the mAbs of interest [439]. As this approach results in the loss of heavy and light chain sequence pairing information, the sheer

189 number of non-native heavy and light chain antibody pairs that are generated may decrease the efficiency of the screening process and cost effectiveness.

More recently, a novel NGS based approach retaining heavy and light chain pairing sequence information (LIBRA-Seq) was developed and shown to be useful for isolating antigen specific mAbs [440]. This method uses DNA-barcoded antigen baits for the sorting of antigen specific B-cells, enabling indexing using oligonucleotide labelled beads. This allows high throughput sequencing and mapping of both antigen barcodes and BCR sequences simultaneously at the single cell level to eliminate non- native sequence pairing for subsequent screens. Such technologies, when adapted will enable the efficient isolation of mAbs for the interrogation of ferret immunology in the future.

6.10 Concluding remarks

This thesis presents the proof of concept for recovering ferret HA-specific mAbs for

HA epitope mapping studies. Based on the draft copy of the ferret genome, ferret immunoglobulin coding genes were identified which facilitated the development of a novel single cell-RT-PCR assay to recover HA-specific mAb sequences from ferret B- cells. As the recovery of ferret antibody transcripts were poor, we subsequently developed a methodology to enable the recovery and screening of potential murine anti-ferret mAbs. Future improvements in genetic annotation and reagents, as well as the adaptation of more efficient technologies for mAb recovery will increase the utility of the ferret model for in-depth interrogation of pathogen specific humoral responses.

190 References

1. Ferkol, T. and D. Schraufnagel, The global burden of respiratory disease. Ann Am Thorac Soc, 2014. 11(3): p. 404-6. 2. Herold, S., et al., Influenza virus-induced lung injury: pathogenesis and implications for treatment. European Respiratory Journal, 2015. 45(5): p. 1463-1478. 3. Chen, Y., et al., Clinical Features of Severe Influenza A (H1N1) Virus Infection. The Indian Journal of Pediatrics, 2012. 80(2): p. 97-101. 4. Kao, K.C., et al., Predictors of survival in patients with influenza pneumonia-related severe acute respiratory distress syndrome treated with prone positioning. Ann Intensive Care, 2018. 8(1): p. 94. 5. Prevention, C.f.D.C.a., Mortality in the United States, 2017. November 2018. 6. Poon, A.F.Y. and J.O. Wertheim, The Re-Emergence of H1N1 Influenza Virus in 1977: A Cautionary Tale for Estimating Divergence Times Using Biologically Unrealistic Sampling Dates. PLoS ONE, 2010. 5(6). 7. Rozo, M., G.K. Gronvall, and M.R. Denison, The Reemergent 1977 H1N1 Strain and the Gain-of-Function Debate. mBio, 2015. 6(4). 8. Taubenberger, J.K. and D.M. Morens, Influenza: the once and future pandemic. Public Health Rep, 2010. 125 Suppl 3: p. 16-26. 9. Coleman, B.L., et al., Risk factors for serious outcomes associated with influenza illness in high- versus low- and middle-income countries: Systematic literature review and meta-analysis. Influenza Other Respir Viruses, 2018. 12(1): p. 22-29. 10. Putri, W.C.W.S., et al., Economic burden of seasonal influenza in the United States. Vaccine, 2018. 36(27): p. 3960-3966. 11. Gerhard, W., et al., Antigenic structure of influenza virus haemagglutinin defined by hybridoma antibodies. Nature, 1981. 290(5808): p. 713-7. 12. Choi, K.H., Viral polymerases. Adv Exp Med Biol, 2012. 726: p. 267-304. 13. Widjaja, L., et al., Molecular changes associated with adaptation of human influenza A virus in embryonated chicken eggs. Virology, 2006. 350(1): p. 137-45. 14. Skowronski, D.M., et al., Low 2012-13 influenza vaccine effectiveness associated with mutation in the egg-adapted H3N2 vaccine strain not antigenic drift in circulating viruses. PLoS One, 2014. 9(3): p. e92153. 15. Zost, S.J., et al., Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains. Proc Natl Acad Sci U S A, 2017. 114(47): p. 12578-12583. 16. Lewnard, J.A. and S. Cobey, Immune History and Influenza Vaccine Effectiveness. Vaccines (Basel), 2018. 6(2). 17. Bühler, S. and M. Ramharter, Flucelvax Tetra: a surface antigen, inactivated, influenza vaccine prepared in cell cultures. ESMO Open, 2019. 4(1). 18. Nakamura, K., et al., Systematic evaluation of suspension MDCK cells, adherent MDCK cells, and LLC-MK2 cells for preparing influenza vaccine seed virus. Vaccine, 2019. 37(43): p. 6526-6534. 19. Group, W.H.O.W., et al., Improving influenza vaccine virus selection: report of a WHO informal consultation held at WHO headquarters, Geneva, Switzerland, 14-16 June 2010. Influenza Other Respir Viruses, 2012. 6(2): p. 142-52, e1-5. 20. Lin, Y., et al., The characteristics and antigenic properties of recently emerged subclade 3C.3a and 3C.2a human influenza A(H3N2) viruses passaged in MDCK cells. Influenza Other Respir Viruses, 2017. 11(3): p. 263-274.

191 21. Tewawong, N., et al., Assessing Antigenic Drift of Seasonal Influenza A(H3N2) and A(H1N1)pdm09 Viruses. PLoS One, 2015. 10(10): p. e0139958. 22. Sutton, T.C., et al., Protective efficacy of influenza group 2 hemagglutinin stem- fragment immunogen vaccines. NPJ Vaccines, 2017. 2: p. 35. 23. Altman, M.O., D. Angeletti, and J.W. Yewdell, Antibody Immunodominance: The Key to Understanding Influenza Virus Antigenic Drift. Viral Immunol, 2018. 31(2): p. 142-149. 24. Groves, H.T., et al., Mouse Models of Influenza Infection with Circulating Strains to Test Seasonal Vaccine Efficacy. Front Immunol, 2018. 9: p. 126. 25. Bouvier, N.M. and A.C. Lowen, Animal Models for Influenza Virus Pathogenesis and Transmission. Viruses, 2010. 2(8): p. 1530-1563. 26. Tobita, K., [Classification and nomenclature of influenza viruses]. Nihon Rinsho, 1997. 55(10): p. 2512-4. 27. Bouvier, N.M. and P. Palese, The biology of influenza viruses. Vaccine, 2008. 26 Suppl 4: p. D49-53. 28. Zebedee, S.L. and R.A. Lamb, Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol, 1988. 62(8): p. 2762-72. 29. Nakatsu, S., et al., Complete and Incomplete Genome Packaging of Influenza A and B Viruses. mBio, 2016. 7(5). 30. Nakatsu, S., et al., Influenza C and D Viruses Package Eight Organized Ribonucleoprotein Complexes. Journal of Virology, 2018. 92(6). 31. Chen, W., et al., A novel influenza A virus mitochondrial protein that induces cell death. Nat Med, 2001. 7(12): p. 1306-12. 32. Hatta, M. and Y. Kawaoka, The NB protein of influenza B virus is not necessary for virus replication in vitro. J Virol, 2003. 77(10): p. 6050-4. 33. Lamb, R.A., C.J. Lai, and P.W. Choppin, Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins. Proc Natl Acad Sci U S A, 1981. 78(7): p. 4170-4. 34. Briedis, D.J., R.A. Lamb, and P.W. Choppin, Sequence of RNA segment 7 of the influenza B virus genome: partial amino acid homology between the membrane proteins (M1) of influenza A and B viruses and conservation of a second open reading frame. Virology, 1982. 116(2): p. 581-8. 35. Horvath, C.M., M.A. Williams, and R.A. Lamb, Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypeptide. EMBO J, 1990. 9(8): p. 2639-47. 36. Briedis, D.J. and R.A. Lamb, Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1 and NS2 proteins. J Virol, 1982. 42(1): p. 186-93. 37. Lamb, R.A., et al., Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza virus genome. Proc Natl Acad Sci U S A, 1980. 77(4): p. 1857-61. 38. Parrish, C.R., et al., Influenza Virus Reservoirs and Intermediate Hosts: Dogs, Horses, and New Possibilities for Influenza Virus Exposure of Humans. Journal of Virology, 2015. 89(6): p. 2990-2994. 39. Webster, R.G., et al., Evolution and ecology of influenza A viruses. Microbiol Rev, 1992. 56(1): p. 152-79. 40. Belser, J.A., J.M. Katz, and T.M. Tumpey, The ferret as a model organism to study influenza A virus infection. Dis Model Mech, 2011. 4(5): p. 575-9.

192 41. Webster, R.G. and E.A. Govorkova, Continuing challenges in influenza. Annals of the New York Academy of Sciences, 2014. 1323(1): p. 115-139. 42. Reperant, L.A., T. Kuiken, and A.D.M.E. Osterhaus, Influenza viruses. Human Vaccines & Immunotherapeutics, 2014. 8(1): p. 7-16. 43. Wu, Y., et al., Bat-derived influenza-like viruses H17N10 and H18N11. Trends Microbiol, 2014. 22(4): p. 183-91. 44. Tong, S., et al., New world bats harbor diverse influenza A viruses. PLoS Pathog, 2013. 9(10): p. e1003657. 45. Tong, S., et al., A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci U S A, 2012. 109(11): p. 4269-74. 46. Palese, P., Influenza: old and new threats. Nat Med, 2004. 10(12 Suppl): p. S82-7. 47. Bodewes, R., et al., Recurring influenza B virus infections in seals. Emerg Infect Dis, 2013. 19(3): p. 511-2. 48. Biere, B., B. Bauer, and B. Schweiger, Differentiation of Influenza B Virus Lineages Yamagata and Victoria by Real-Time PCR. Journal of Clinical Microbiology, 2010. 48(4): p. 1425-1427. 49. Thompson, W.W., et al., Influenza-associated hospitalizations in the United States. JAMA, 2004. 292(11): p. 1333-40. 50. Spoto, S., et al., Influenza B virus infection complicated by life-threatening pericarditis: a unique case-report and literature review. BMC Infect Dis, 2019. 19(1): p. 40. 51. Nobusawa, E. and K. Sato, Comparison of the Mutation Rates of Human Influenza A and B Viruses. Journal of Virology, 2006. 80(7): p. 3675-3678. 52. Buonagurio, D.A., et al., Noncumulative sequence changes in the hemagglutinin genes of influenza C virus isolates. Virology, 1985. 146(2): p. 221-32. 53. Matsuzaki, Y., et al., Clinical Features of Influenza C Virus Infection in Children. The Journal of Infectious Diseases, 2006. 193(9): p. 1229-1235. 54. Leung, H.S.Y., et al., Entry of Influenza A Virus with a 2,6-Linked Sialic Acid Binding Preference Requires Host Fibronectin. Journal of Virology, 2012. 86(19): p. 10704-10713. 55. Bottcher-Friebertshauser, E., et al., Cleavage of Influenza Virus Hemagglutinin by Airway Proteases TMPRSS2 and HAT Differs in Subcellular Localization and Susceptibility to Protease Inhibitors. Journal of Virology, 2010. 84(11): p. 5605-5614. 56. Gagneux, P., et al., Human-specific Regulation of α2–6-linked Sialic Acids. Journal of Biological Chemistry, 2003. 278(48): p. 48245-48250. 57. Ibricevic, A., et al., Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells. J Virol, 2006. 80(15): p. 7469-80. 58. Ma, W., R.E. Kahn, and J.A. Richt, The pig as a mixing vessel for influenza viruses: Human and veterinary implications. J Mol Genet Med, 2008. 3(1): p. 158-66. 59. Worch, R., Structural biology of the influenza virus fusion peptide. Acta Biochim Pol, 2014. 61(3): p. 421-6. 60. Pielak, R.M. and J.J. Chou, Influenza M2 proton channels. Biochim Biophys Acta, 2011. 1808(2): p. 522-9. 61. Cady, S.D., et al., Structure and function of the influenza A M2 proton channel. Biochemistry, 2009. 48(31): p. 7356-64. 62. Li, J., et al., Nucleocytoplasmic shuttling of influenza A virus proteins. Viruses, 2015. 7(5): p. 2668-82. 63. Boulo, S., et al., Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes. Virus Res, 2007. 124(1-2): p. 12-21.

193 64. Crow, M., et al., Mutational Analysis of the Influenza Virus cRNA Promoter and Identification of Nucleotides Critical for Replication. Journal of Virology, 2004. 78(12): p. 6263-6270. 65. De Vlugt, C., D. Sikora, and M. Pelchat, Insight into Influenza: A Virus Cap- Snatching. Viruses, 2018. 10(11). 66. Poon, L.L.M., E. Fodor, and G.G. Brownlee, Polyuridylated mRNA Synthesized by a Recombinant Influenza Virus Is Defective in Nuclear Export. Journal of Virology, 2000. 74(1): p. 418-427. 67. Hutchinson, E.C., et al., Genome packaging in influenza A virus. J Gen Virol, 2010. 91(Pt 2): p. 313-28. 68. McAuley, J.L., et al., Influenza Virus Neuraminidase Structure and Functions. Front Microbiol, 2019. 10: p. 39. 69. Kim, H., R.G. Webster, and R.J. Webby, Influenza Virus: Dealing with a Drifting and Shifting Pathogen. Viral Immunol, 2018. 31(2): p. 174-183. 70. Carrat, F. and A. Flahault, Influenza vaccine: the challenge of antigenic drift. Vaccine, 2007. 25(39-40): p. 6852-62. 71. Opanda, S., et al., Assessing antigenic drift and phylogeny of influenza A (H1N1) pdm09 virus in Kenya using HA1 sub-unit of the hemagglutinin gene. PLoS One, 2020. 15(2): p. e0228029. 72. Shao, W., et al., Evolution of Influenza A Virus by Mutation and Re-Assortment. Int J Mol Sci, 2017. 18(8). 73. Saunders-Hastings, P.R. and D. Krewski, Reviewing the History of Pandemic Influenza: Understanding Patterns of Emergence and Transmission. Pathogens, 2016. 5(4). 74. Taubenberger, J.K. and D.M. Morens, 1918 Influenza: the mother of all pandemics. Emerg Infect Dis, 2006. 12(1): p. 15-22. 75. Nachbagauer, R. and P. Palese, Is a Universal Influenza Virus Vaccine Possible? Annu Rev Med, 2020. 71: p. 315-327. 76. Stiver, G., The treatment of influenza with antiviral drugs. CMAJ, 2003. 168(1): p. 49-56. 77. Schnell, J.R. and J.J. Chou, Structure and mechanism of the M2 proton channel of influenza A virus. Nature, 2008. 451(7178): p. 591-5. 78. Hayden, F.G., H.E. Hoffman, and D.A. Spyker, Differences in side effects of amantadine hydrochloride and rimantadine hydrochloride relate to differences in pharmacokinetics. Antimicrob Agents Chemother, 1983. 23(3): p. 458-64. 79. Suzuki, H., et al., Emergence of amantadine-resistant influenza A viruses: epidemiological study. J Infect Chemother, 2003. 9(3): p. 195-200. 80. Thomaston, J.L. and W.F. DeGrado, Crystal structure of the drug-resistant S31N influenza M2 proton channel. Protein Sci, 2016. 25(8): p. 1551-4. 81. Jefferson, T., et al., Oseltamivir for influenza in adults and children: systematic review of clinical study reports and summary of regulatory comments. Bmj, 2014. 348(apr09 2): p. g2545-g2545. 82. Heneghan, C.J., et al., Zanamivir for influenza in adults and children: systematic review of clinical study reports and summary of regulatory comments. Bmj, 2014. 348(apr09 2): p. g2547-g2547. 83. Scott, L.J., Peramivir: A Review in Uncomplicated Influenza. Drugs, 2018. 78(13): p. 1363-1370. 84. Choi, W.-Y., et al., The Emergence of Oseltamivir-Resistant Seasonal Influenza A (H1N1) Virus in Korea During the 2008–2009 Season. Osong Public Health and Research Perspectives, 2011. 2(3): p. 178-185.

194 85. Trebbien, R., et al., Development of oseltamivir and zanamivir resistance in influenza A(H1N1)pdm09 virus, Denmark, 2014. Euro Surveill, 2017. 22(3). 86. Hurt, A.C., et al., Zanamivir-Resistant Influenza Viruses with a Novel Neuraminidase Mutation. Journal of Virology, 2009. 83(20): p. 10366-10373. 87. Alame, M.M., E. Massaad, and H. Zaraket, Peramivir: A Novel Intravenous Neuraminidase Inhibitor for Treatment of Acute Influenza Infections. Front Microbiol, 2016. 7: p. 450. 88. Lehnert, R., et al., Antiviral Medications in Seasonal and Pandemic Influenza. Dtsch Arztebl Int, 2016. 113(47): p. 799-807. 89. Pebody, R., J. McMenamin, and H. Nohynek, Live attenuated influenza vaccine (LAIV): recent effectiveness results from the USA and implications for LAIV programmes elsewhere. Arch Dis Child, 2018. 103(1): p. 101-105. 90. Yang, J., et al., Effectiveness, immunogenicity, and safety of influenza vaccines with MF59 adjuvant in healthy people of different age groups: A systematic review and meta-analysis. Medicine (Baltimore), 2020. 99(7): p. e19095. 91. Cox, M.M., et al., Safety, efficacy, and immunogenicity of Flublok in the prevention of seasonal influenza in adults. Ther Adv Vaccines, 2015. 3(4): p. 97-108. 92. Ekiert, D.C., et al., A Highly Conserved Neutralizing Epitope on Group 2 Influenza A Viruses. Science, 2011. 333(6044): p. 843-850. 93. Thomson, C.A., et al., Pandemic H1N1 Influenza Infection and Vaccination in Humans Induces Cross-Protective Antibodies that Target the Hemagglutinin Stem. Front Immunol, 2012. 3: p. 87. 94. Krammer, F., et al., Chimeric Hemagglutinin Influenza Virus Vaccine Constructs Elicit Broadly Protective Stalk-Specific Antibodies. Journal of Virology, 2013. 87(12): p. 6542-6550. 95. Liu, W.C., et al., Sequential Immunization With Live-Attenuated Chimeric Hemagglutinin-Based Vaccines Confers Heterosubtypic Immunity Against Influenza A Viruses in a Preclinical Ferret Model. Front Immunol, 2019. 10: p. 756. 96. Nachbagauer, R., et al., A universal influenza virus vaccine candidate confers protection against pandemic H1N1 infection in preclinical ferret studies. NPJ Vaccines, 2017. 2: p. 26. 97. Valkenburg, S.A., et al., Stalking influenza by vaccination with pre-fusion headless HA mini-stem. Scientific Reports, 2016. 6(1). 98. Prachanronarong, K.L., et al., Mutations in Influenza A Virus Neuraminidase and Hemagglutinin Confer Resistance against a Broadly Neutralizing Hemagglutinin Stem Antibody. Journal of Virology, 2019. 93(2). 99. Kosik, I., et al., Neuraminidase inhibition contributes to influenza A virus neutralization by anti-hemagglutinin stem antibodies. J Exp Med, 2019. 216(2): p. 304-316. 100. Mifsud, E.J., et al., Prophylaxis of ferrets with nitazoxanide and oseltamivir combinations is more effective at reducing the impact of influenza a virus infection compared to oseltamivir monotherapy. Antiviral Res, 2020. 176: p. 104751. 101. Numata, M., et al., Pulmonary surfactant lipids inhibit infections with the pandemic H1N1 influenza virus in several animal models. J Biol Chem, 2020. 295(6): p. 1704- 1715. 102. Toots, M., et al., Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia. Sci Transl Med, 2019. 11(515). 103. Beigel, J.H., et al., Advances in respiratory virus therapeutics - A meeting report from the 6th isirv Antiviral Group conference. Antiviral Res, 2019. 167: p. 45-67.

195 104. Chen, Y.-Q., et al., Influenza Infection in Humans Induces Broadly Cross-Reactive and Protective Neuraminidase-Reactive Antibodies. Cell, 2018. 173(2): p. 417- 429.e10. 105. Koutsakos, M., et al., Human CD8(+) T cell cross-reactivity across influenza A, B and C viruses. Nat Immunol, 2019. 20(5): p. 613-625. 106. Amarante-Mendes, G.P., et al., Pattern Recognition Receptors and the Host Cell Death Molecular Machinery. Frontiers in Immunology, 2018. 9. 107. Krieg, A.M. and J. Vollmer, Toll-like receptors 7, 8, and 9: linking innate immunity to autoimmunity. Immunol Rev, 2007. 220: p. 251-69. 108. Martínez, I., et al., Apoptosis, Toll-like, RIG-I-like and NOD-like Receptors Are Pathways Jointly Induced by Diverse Respiratory Bacterial and Viral Pathogens. Frontiers in Microbiology, 2017. 8. 109. Lawrence, T., The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol, 2009. 1(6): p. a001651. 110. Hatesuer, B., et al., Deletion of Irf3 and Irf7 Genes in Mice Results in Altered Interferon Pathway Activation and Granulocyte-Dominated Inflammatory Responses to Influenza A Infection. J Innate Immun, 2017. 9(2): p. 145-161. 111. Garcia-Sastre, A., Induction and evasion of type I interferon responses by influenza viruses. Virus Res, 2011. 162(1-2): p. 12-8. 112. Klinkhammer, J., et al., IFN-lambda prevents influenza virus spread from the upper airways to the lungs and limits virus transmission. Elife, 2018. 7. 113. Horvath, C.M., The Jak-STAT pathway stimulated by interferon alpha or interferon beta. Sci STKE, 2004. 2004(260): p. tr10. 114. Fu, X.Y., et al., ISGF3, the transcriptional activator induced by interferon alpha, consists of multiple interacting polypeptide chains. Proc Natl Acad Sci U S A, 1990. 87(21): p. 8555-9. 115. Di Pietro, A., et al., TRIM22 Inhibits Influenza A Virus Infection by Targeting the Viral Nucleoprotein for Degradation. Journal of Virology, 2013. 87(8): p. 4523-4533. 116. Wang, X., E.R. Hinson, and P. Cresswell, The Interferon-Inducible Protein Viperin Inhibits Influenza Virus Release by Perturbing Lipid Rafts. Cell Host & Microbe, 2007. 2(2): p. 96-105. 117. Hale, B.G., R.A. Albrecht, and A. Garcia-Sastre, Innate immune evasion strategies of influenza viruses. Future Microbiol, 2010. 5(1): p. 23-41. 118. Lacy, P., Editorial: Secretion of Cytokines and Chemokines by Innate Immune Cells. Frontiers in Immunology, 2015. 6. 119. Rosales, C. and E. Uribe-Querol, Phagocytosis: A Fundamental Process in Immunity. Biomed Res Int, 2017. 2017: p. 9042851. 120. Hughes, C.E., et al., Antigen-Presenting Cells and Antigen Presentation in Tertiary Lymphoid Organs. Frontiers in Immunology, 2016. 7. 121. Tremblay-McLean, A., et al., Expression of ligands for activating natural killer cell receptors on cell lines commonly used to assess natural killer cell function. BMC Immunology, 2019. 20(1). 122. Jegaskanda, S., et al., Influenza Virus Infection Enhances Antibody-Mediated NK Cell Functions via Type I Interferon-Dependent Pathways. Journal of Virology, 2019. 93(5). 123. Krzewski, K., et al., LAMP1/CD107a is required for efficient perforin delivery to lytic granules and NK-cell cytotoxicity. Blood, 2013. 121(23): p. 4672-83. 124. Vanderven, H.A., et al., What Lies Beneath: Antibody Dependent Natural Killer Cell Activation by Antibodies to Internal Influenza Virus Proteins. EBioMedicine, 2016. 8: p. 277-290.

196 125. Condon, T.V., et al., Lung dendritic cells at the innate-adaptive immune interface. J Leukoc Biol, 2011. 90(5): p. 883-95. 126. Chen, X., et al., Host Immune Response to Influenza A Virus Infection. Front Immunol, 2018. 9: p. 320. 127. Silverstein, A.M., The Clonal Selection Theory: what it really is and why modern challenges are misplaced. Nature Immunology, 2002. 3(9): p. 793-796. 128. Li, L.X., et al., A Flt3- and Ras-dependent pathway primes B cell development by inducing a state of IL-7 responsiveness. J Immunol, 2010. 184(4): p. 1728-36. 129. Busslinger, M., Transcriptional Control of Early B Cell Development. Annual Review of Immunology, 2004. 22(1): p. 55-79. 130. Lutz, J., et al., Pro-B cells sense productive immunoglobulin heavy chain rearrangement irrespective of polypeptide production. Proc Natl Acad Sci U S A, 2011. 108(26): p. 10644-9. 131. Hewitt, S.L., et al., RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci. Nat Immunol, 2009. 10(6): p. 655-64. 132. Martensson, I.L. and R. Ceredig, Role of the surrogate light chain and the pre-B-cell receptor in mouse B-cell development. Immunology, 2000. 101(4): p. 435-441. 133. Otero, D.C. and R.C. Rickert, CD19 Function in Early and Late B Cell Development. II. CD19 Facilitates the Pro-B/Pre-B Transition. The Journal of Immunology, 2003. 171(11): p. 5921-5930. 134. Grawunder, U., et al., Down-regulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement. Immunity, 1995. 3(5): p. 601-8. 135. Lin, W.C. and S. Desiderio, Cell cycle regulation of V(D)J recombination-activating protein RAG-2. Proc Natl Acad Sci U S A, 1994. 91(7): p. 2733-7. 136. Levin-Klein, R. and Y. Bergman, Epigenetic regulation of monoallelic rearrangement (allelic exclusion) of antigen receptor genes. Front Immunol, 2014. 5: p. 625. 137. Winkler, T.H. and I.-L. Mårtensson, The Role of the Pre-B Cell Receptor in B Cell Development, Repertoire Selection, and Tolerance. Frontiers in Immunology, 2018. 9. 138. Wabl, M. and C. Steinberg, A theory of allelic and isotypic exclusion for immunoglobulin genes. Proc Natl Acad Sci U S A, 1982. 79(22): p. 6976-8. 139. Geisberger, R., M. Lamers, and G. Achatz, The riddle of the dual expression of IgM and IgD. Immunology, 2006. 118(4): p. 429-37. 140. Jackson, K.J., et al., Exonuclease activity and P nucleotide addition in the generation of the expressed immunoglobulin repertoire. BMC Immunol, 2004. 5: p. 19. 141. Baumgarth, N. and Y.S. Choi, Dual role for B-1a cells in immunity to influenza virus infection. Journal of Experimental Medicine, 2008. 205(13): p. 3053-3064. 142. Baumgarth, N., How specific is too specific? B-cell responses to viral infections reveal the importance of breadth over depth. Immunol Rev, 2013. 255(1): p. 82-94. 143. Jayasekera, J.P., E.A. Moseman, and M.C. Carroll, Natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity. J Virol, 2007. 81(7): p. 3487-94. 144. Holodick, N.E., N. Rodriguez-Zhurbenko, and A.M. Hernandez, Defining Natural Antibodies. Front Immunol, 2017. 8: p. 872. 145. Yuseff, M.-I., et al., How B cells capture, process and present antigens: a crucial role for cell polarity. Nature Reviews Immunology, 2013. 13(7): p. 475-486. 146. Okada, T., et al., Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol, 2005. 3(6): p. e150. 147. Coffey, F., B. Alabyev, and T. Manser, Initial clonal expansion of germinal center B cells takes place at the perimeter of follicles. Immunity, 2009. 30(4): p. 599-609.

197 148. Chan, T.D., et al., Antigen affinity controls rapid T-dependent antibody production by driving the expansion rather than the differentiation or extrafollicular migration of early plasmablasts. J Immunol, 2009. 183(5): p. 3139-49. 149. MacLennan, I.C., et al., Extrafollicular antibody responses. Immunol Rev, 2003. 194: p. 8-18. 150. Allen, C.D., et al., Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nat Immunol, 2004. 5(9): p. 943-52. 151. Steele, E.J., Reverse Transcriptase Mechanism of Somatic Hypermutation: 60 Years of Clonal Selection Theory. Front Immunol, 2017. 8: p. 1611. 152. Stavnezer, J., J.E. Guikema, and C.E. Schrader, Mechanism and regulation of class switch recombination. Annu Rev Immunol, 2008. 26: p. 261-92. 153. Mayer, C.T., et al., The microanatomic segregation of selection by apoptosis in the germinal center. Science, 2017. 358(6360). 154. Kepler, T.B. and A.S. Perelson, Cyclic re-entry of germinal center B cells and the efficiency of affinity maturation. Immunol Today, 1993. 14(8): p. 412-5. 155. Suan, D., C. Sundling, and R. Brink, Plasma cell and memory B cell differentiation from the germinal center. Curr Opin Immunol, 2017. 45: p. 97-102. 156. Palm, A.-K.E. and C. Henry, Remembrance of Things Past: Long-Term B Cell Memory After Infection and Vaccination. Frontiers in Immunology, 2019. 10. 157. Lightman, S.M., A. Utley, and K.P. Lee, Survival of Long-Lived Plasma Cells (LLPC): Piecing Together the Puzzle. Frontiers in Immunology, 2019. 10. 158. Kobinger, G.P., et al., Comparison of Antiviral Activity between IgA and IgG Specific to Influenza Virus Hemagglutinin: Increased Potential of IgA for Heterosubtypic Immunity. PLoS ONE, 2014. 9(1). 159. Graham, B.S. and D.M. Ambrosino, History of passive antibody administration for prevention and treatment of infectious diseases. Curr Opin HIV AIDS, 2015. 10(3): p. 129-34. 160. Lunemann, J.D., I. Quast, and M.C. Dalakas, Efficacy of Intravenous Immunoglobulin in Neurological Diseases. Neurotherapeutics, 2016. 13(1): p. 34-46. 161. Kohler, G. and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975. 256(5517): p. 495-7. 162. Singh, S., et al., Monoclonal Antibodies: A Review. Curr Clin Pharmacol, 2018. 13(2): p. 85-99. 163. Puligedda, R.D., et al., Human IgA Monoclonal Antibodies That Neutralize Poliovirus, Produced by Hybridomas and Recombinant Expression. Antibodies (Basel), 2020. 9(1). 164. Goto, M., et al., High prevalence of human anti-mouse antibodies in the serum of colorectal cancer patients. Anticancer Res, 2010. 30(10): p. 4353-6. 165. LoBuglio, A.F., et al., Mouse/human chimeric monoclonal antibody in man: kinetics and immune response. Proc Natl Acad Sci U S A, 1989. 86(11): p. 4220-4. 166. Smith, G.P., Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 1985. 228(4705): p. 1315-7. 167. Kennedy, P.J., et al., Monoclonal antibodies: technologies for early discovery and engineering. Critical Reviews in Biotechnology, 2017. 38(3): p. 394-408. 168. Aghebati-Maleki, L., et al., Phage display as a promising approach for vaccine development. J Biomed Sci, 2016. 23(1): p. 66. 169. Hammers, C.M. and J.R. Stanley, Antibody phage display: technique and applications. J Invest Dermatol, 2014. 134(2): p. 1-5.

198 170. Tiller, T., et al., Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. Journal of Immunological Methods, 2008. 329(1-2): p. 112-124. 171. Tiller, T., C.E. Busse, and H. Wardemann, Cloning and expression of murine Ig genes from single B cells. J Immunol Methods, 2009. 350(1-2): p. 183-93. 172. Lei, L., et al., Antigen-Specific Single B Cell Sorting and Monoclonal Antibody Cloning in Guinea Pigs. Frontiers in Microbiology, 2019. 10. 173. Shah, H.B., et al., Insights From Analysis of Human Antigen-Specific Memory B Cell Repertoires. Front Immunol, 2018. 9: p. 3064. 174. Hobeika, E., et al., Conditional Selection of B Cells in Mice With an Inducible B Cell Development. Frontiers in Immunology, 2018. 9. 175. Hu, Z., et al., Mouse-adapted H9N2 avian influenza virus causes systemic infection in mice. Virology Journal, 2019. 16(1). 176. Blazejewska, P., et al., Pathogenicity of different PR8 influenza A virus variants in mice is determined by both viral and host factors. Virology, 2011. 412(1): p. 36-45. 177. Hofer, C.C., P.S. Woods, and I.C. Davis, Infection of mice with influenza A/WSN/33 (H1N1) virus alters alveolar type II cell phenotype. Am J Physiol Lung Cell Mol Physiol, 2015. 308(7): p. L628-38. 178. Thangavel, R.R. and N.M. Bouvier, Animal models for influenza virus pathogenesis, transmission, and immunology. J Immunol Methods, 2014. 410: p. 60-79. 179. Pekosz, A., et al., A Novel Nonhuman Primate Model for Influenza Transmission. PLoS ONE, 2013. 8(11). 180. Itoh, Y., Translational research on influenza virus infection using a nonhuman primate model. Pathol Int, 2016. 66(3): p. 132-141. 181. van der Lubbe, J.E.M., et al., Mini-hemagglutinin vaccination induces cross-reactive antibodies in pre-exposed NHP that protect mice against lethal influenza challenge. NPJ Vaccines, 2018. 3: p. 25. 182. Smith, W., C.H. Andrewes, and P.P. Laidlaw, A Virus Obtained from Influenza Patients. The Lancet, 1933. 222(5732): p. 66-68. 183. Carrat, F., et al., Time Lines of Infection and Disease in Human Influenza: A Review of Volunteer Challenge Studies. American Journal of Epidemiology, 2008. 167(7): p. 775-785. 184. Beigel, J.H., Influenza. Critical Care Medicine, 2008. 36(9): p. 2660-2666. 185. Ng, P.S., et al., Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors. Nat Commun, 2014. 5: p. 5750. 186. Johnson-Delaney, C.A. and S.E. Orosz, Ferret Respiratory System: Clinical Anatomy, Physiology, and Disease. Veterinary Clinics of North America: Exotic Animal Practice, 2011. 14(2): p. 357-367. 187. Shinya, K., et al., Influenza virus receptors in the human airway. Nature, 2006. 440(7083): p. 435-436. 188. van Riel, D., et al., Human and Avian Influenza Viruses Target Different Cells in the Lower Respiratory Tract of Humans and Other Mammals. The American Journal of Pathology, 2007. 171(4): p. 1215-1223. 189. Hemmink, J.D., C.J. Whittaker, and H.A. Shelton, Animal Models in Influenza Research, in Influenza Virus. 2018. p. 401-430. 190. Zitzow, L.A., et al., Pathogenesis of avian influenza A (H5N1) viruses in ferrets. J Virol, 2002. 76(9): p. 4420-9. 191. Belser, J.A., et al., Complexities in Ferret Influenza Virus Pathogenesis and Transmission Models. Microbiology and Molecular Biology Reviews, 2016. 80(3): p. 733-744.

199 192. Buhnerkempe, M.G., et al., Mapping influenza transmission in the ferret model to transmission in humans. Elife, 2015. 4. 193. Frise, R., et al., Contact transmission of influenza virus between ferrets imposes a looser bottleneck than respiratory droplet transmission allowing propagation of antiviral resistance. Sci Rep, 2016. 6: p. 29793. 194. Lowen, A.C., et al., Characterising viable virus from air exhaled by H1N1 influenza- infected ferrets reveals the importance of haemagglutinin stability for airborne infectivity. PLOS Pathogens, 2020. 16(2). 195. Richard, M., et al., Influenza A viruses are transmitted via the air from the nasal respiratory epithelium of ferrets. Nat Commun, 2020. 11(1): p. 766. 196. van den Brand, Judith M.A., et al., Severity of Pneumonia Due to New H1N1 Influenza Virus in Ferrets Is Intermediate between That Due to Seasonal H1N1 Virus and Highly Pathogenic Avian Influenza H5N1 Virus. The Journal of Infectious Diseases, 2010. 201(7): p. 993-999. 197. Moore, I.N., et al., Severity of clinical disease and pathology in ferrets experimentally infected with influenza viruses is influenced by inoculum volume. J Virol, 2014. 88(23): p. 13879-91. 198. Belshaw, R., et al., Influenza Infectious Dose May Explain the High Mortality of the Second and Third Wave of 1918–1919 Influenza Pandemic. PLoS ONE, 2010. 5(7). 199. Allen, J.D. and T.M. Ross, H3N2 influenza viruses in humans: Viral mechanisms, evolution, and evaluation. Hum Vaccin Immunother, 2018. 14(8): p. 1840-1847. 200. Lefeuvre, C., et al., Fatal Myopericarditis Following an Influenza A (H3N2) Infection. Am J Case Rep, 2018. 19: p. 540-544. 201. Figueredo, A.L., et al., Clinical and epidemiological features in H1N1 and H3N2 influenza A virus, in 10.1 Respiratory Infections. 2015. 202. Ryan, K.A., et al., Cellular immune response to human influenza viruses differs between H1N1 and H3N2 subtypes in the ferret lung. PLoS One, 2018. 13(9): p. e0202675. 203. Hensley, S.E., et al., Identification of Low- and High-Impact Hemagglutinin Amino Acid Substitutions That Drive Antigenic Drift of Influenza A(H1N1) Viruses. PLOS Pathogens, 2016. 12(4). 204. Sandbulte, M.R., et al., Discordant antigenic drift of neuraminidase and hemagglutinin in H1N1 and H3N2 influenza viruses. Proceedings of the National Academy of Sciences, 2011. 108(51): p. 20748-20753. 205. Pearce, M.B., et al., Seasonal trivalent inactivated influenza vaccine protects against 1918 Spanish influenza virus infection in ferrets. J Virol, 2012. 86(13): p. 7118-25. 206. Sambhara, S., et al., Randomized Controlled Ferret Study to Assess the Direct Impact of 2008–09 Trivalent Inactivated Influenza Vaccine on A(H1N1)pdm09 Disease Risk. PLoS ONE, 2014. 9(1). 207. Jefferson, T., et al., Vaccines for preventing influenza in the elderly. Cochrane Database of Systematic Reviews, 2010. 208. Arriola, C.S., et al., Does Influenza Vaccination Modify Influenza Severity? Data on Older Adults Hospitalized With Influenza During the 2012−2013 Season in the United States. Journal of Infectious Diseases, 2015. 212(8): p. 1200-1208. 209. Matsuzaki, Y., et al., Epitope Mapping of the Hemagglutinin Molecule of A/(H1N1)pdm09 Influenza Virus by Using Monoclonal Antibody Escape Mutants. Journal of Virology, 2014. 88(21): p. 12364-12373. 210. Guo, Z., et al., Biosensor-based epitope mapping of antibodies targeting the hemagglutinin and neuraminidase of influenza A virus. J Immunol Methods, 2018. 461: p. 23-29.

200 211. Flannery, B., et al., Influenza Vaccine Effectiveness Against Pediatric Deaths: 2010– 2014. Pediatrics, 2017. 139(5). 212. Blyth, C.C., et al., Influenza Vaccine Effectiveness and Uptake in Children at Risk of Severe Disease. The Pediatric Infectious Disease Journal, 2016. 35(3): p. 309-315. 213. Arriola, C., et al., Influenza Vaccination Modifies Disease Severity Among Community-dwelling Adults Hospitalized With Influenza. Clinical Infectious Diseases, 2017. 65(8): p. 1289-1297. 214. Alfelali, M., et al., Mismatching between circulating strains and vaccine strains of influenza: Effect on Hajj pilgrims from both hemispheres. Hum Vaccin Immunother, 2016. 12(3): p. 709-15. 215. Sah, P., et al., Optimizing the impact of low-efficacy influenza vaccines. Proceedings of the National Academy of Sciences, 2018. 115(20): p. 5151-5156. 216. Paessler, S. and V. Veljkovic, Prediction of influenza vaccine effectiveness for the influenza season 2017/18 in the US. F1000Res, 2017. 6: p. 2067. 217. Sautto, G.A., G.A. Kirchenbaum, and T.M. Ross, Towards a universal influenza vaccine: different approaches for one goal. Virol J, 2018. 15(1): p. 17. 218. Nachbagauer, R., et al., Hemagglutinin Stalk Immunity Reduces Influenza Virus Replication and Transmission in Ferrets. Journal of Virology, 2016. 90(6): p. 3268- 3273. 219. Krammer, F., et al., Assessment of Influenza Virus Hemagglutinin Stalk-Based Immunity in Ferrets. Journal of Virology, 2014. 88(6): p. 3432-3442. 220. Yassine, H.M., et al., Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection. Nature Medicine, 2015. 21(9): p. 1065-1070. 221. Korenkov, D.A., et al., Safety, immunogenicity and protection of A(H3N2) live attenuated influenza vaccines containing wild-type nucleoprotein in a ferret model. Infect Genet Evol, 2018. 64: p. 95-104. 222. Rosendahl Huber, S.K., et al., Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets. PLoS One, 2015. 10(6): p. e0127969. 223. Price, G.E., et al., Vaccination focusing immunity on conserved antigens protects mice and ferrets against virulent H1N1 and H5N1 influenza A viruses. Vaccine, 2009. 27(47): p. 6512-21. 224. Ross, T.M., et al., A trivalent virus-like particle vaccine elicits protective immune responses against seasonal influenza strains in mice and ferrets. PLoS One, 2009. 4(6): p. e6032. 225. Music, N., et al., Supplementation of H1N1pdm09 split vaccine with heterologous tandem repeat M2e5x virus-like particles confers improved cross-protection in ferrets. Vaccine, 2016. 34(4): p. 466-473. 226. Holzer, B., et al., Comparison of Heterosubtypic Protection in Ferrets and Pigs Induced by a Single-Cycle Influenza Vaccine. J Immunol, 2018. 200(12): p. 4068- 4077. 227. Si, L., et al., Generation of influenza A viruses as live but replication-incompetent virus vaccines. Science, 2016. 354(6316): p. 1170-1173. 228. Baz, M., et al., Nonreplicating influenza A virus vaccines confer broad protection against lethal challenge. MBio, 2015. 6(5): p. e01487-15. 229. Romanova, J., et al., Preclinical evaluation of a replication-deficient intranasal DeltaNS1 H5N1 influenza vaccine. PLoS One, 2009. 4(6): p. e5984. 230. Wang, L., et al., Generation of a Live Attenuated Influenza Vaccine that Elicits Broad Protection in Mice and Ferrets. Cell Host Microbe, 2017. 21(3): p. 334-343.

201 231. Cheng, X., et al., Evaluation of the humoral and cellular immune responses elicited by the live attenuated and inactivated influenza vaccines and their roles in heterologous protection in ferrets. J Infect Dis, 2013. 208(4): p. 594-602. 232. Min, J.Y., et al., A live attenuated H7N7 candidate vaccine virus induces neutralizing antibody that confers protection from challenge in mice, ferrets, and monkeys. J Virol, 2010. 84(22): p. 11950-60. 233. Ann, J., et al., Adjuvanted inactivated influenza A(H3N2) vaccines induce stronger immunogenicity in mice and confer higher protection in ferrets than unadjuvanted inactivated vaccines. Vaccine, 2014. 32(43): p. 5730-9. 234. Clegg, C.H., et al., GLA-AF, an emulsion-free vaccine adjuvant for pandemic influenza. PLoS One, 2014. 9(2): p. e88979. 235. Bodewes, R., et al., A single immunization with CoVaccine HT-adjuvanted H5N1 influenza virus vaccine induces protective cellular and humoral immune responses in ferrets. J Virol, 2010. 84(16): p. 7943-52. 236. Christensen, D., et al., Seasonal Influenza Split Vaccines Confer Partial Cross- Protection against Heterologous Influenza Virus in Ferrets When Combined with the CAF01 Adjuvant. Front Immunol, 2017. 8: p. 1928. 237. Yang, J., et al., Poly-gamma-glutamic acid/chitosan nanogel greatly enhances the efficacy and heterosubtypic cross-reactivity of H1N1 pandemic influenza vaccine. Sci Rep, 2017. 7: p. 44839. 238. Skibinski, D.A., et al., Enhanced neutralizing antibody titers and Th1 polarization from a novel Escherichia coli derived pandemic influenza vaccine. PLoS One, 2013. 8(10): p. e76571. 239. Petsch, B., et al., Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection. Nat Biotechnol, 2012. 30(12): p. 1210-6. 240. Pillet, S., et al., Cellular immune response in the presence of protective antibody levels correlates with protection against 1918 influenza in ferrets. Vaccine, 2011. 29(39): p. 6793-801. 241. Kreijtz, J.H., et al., A single immunization with modified vaccinia virus Ankara-based influenza virus H7 vaccine affords protection in the influenza A(H7N9) pneumonia ferret model. J Infect Dis, 2015. 211(5): p. 791-800. 242. Allen, J.D., et al., Elicitation of Protective Antibodies against 20 Years of Future H3N2 Cocirculating Influenza Virus Variants in Ferrets Preimmune to Historical H3N2 Influenza Viruses. Journal of Virology, 2019. 93(3). 243. Bodewes, R., et al., Infection of the upper respiratory tract with seasonal influenza A(H3N2) virus induces protective immunity in ferrets against infection with A(H1N1)pdm09 virus after intranasal, but not intratracheal, inoculation. J Virol, 2013. 87(8): p. 4293-301. 244. Bird, N.L., et al., Oseltamivir Prophylaxis Reduces Inflammation and Facilitates Establishment of Cross-Strain Protective T Cell Memory to Influenza Viruses. PLoS One, 2015. 10(6): p. e0129768. 245. Cameron, C.M., et al., Gene expression analysis of host innate immune responses during Lethal H5N1 infection in ferrets. J Virol, 2008. 82(22): p. 11308-17. 246. Maines, T.R., et al., Local innate immune responses and influenza virus transmission and virulence in ferrets. J Infect Dis, 2012. 205(3): p. 474-85. 247. Carolan, L.A., et al., Characterization of the Localized Immune Response in the Respiratory Tract of Ferrets following Infection with Influenza A and B Viruses. J Virol, 2015. 90(6): p. 2838-48. 248. Leon, A.J., et al., Sequencing, annotation, and characterization of the influenza ferret infectome. J Virol, 2013. 87(4): p. 1957-66.

202 249. Kobinger, G.P., et al., Assessment of the efficacy of commercially available and candidate vaccines against a pandemic H1N1 2009 virus. J Infect Dis, 2010. 201(7): p. 1000-6. 250. Carolan, L.A., et al., TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses. J Virol Methods, 2014. 205: p. 38-52. 251. Maier, T., M. Guell, and L. Serrano, Correlation of mRNA and protein in complex biological samples. FEBS Lett, 2009. 583(24): p. 3966-73. 252. Martel, C.J. and B. Aasted, Characterization of antibodies against ferret immunoglobulins, cytokines and CD markers. Vet Immunol Immunopathol, 2009. 132(2-4): p. 109-15. 253. Rutigliano, J.A., et al., Screening monoclonal antibodies for cross-reactivity in the ferret model of influenza infection. J Immunol Methods, 2008. 336(1): p. 71-7. 254. Reber, A.J., et al., Extensive T cell cross-reactivity between diverse seasonal influenza strains in the ferret model. Scientific Reports, 2018. 8(1). 255. Hayashi, S., et al., Cross-reactivity of Monoclonal Antibodies on Peripheral Blood Cells from Common Marmosets by Flow Cytometry. Cytometry Research, 2007. 17(1): p. 21-28. 256. Rees, J., et al., Characterisation of monoclonal antibodies specific for hamster leukocyte differentiation molecules. Vet Immunol Immunopathol, 2017. 183: p. 40-44. 257. Layton, D.S., et al., Development of an anti-ferret CD4 monoclonal antibody for the characterisation of ferret T lymphocytes. J Immunol Methods, 2017. 444: p. 29-35. 258. DiPiazza, A., et al., Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection. J Virol, 2016. 90(17): p. 7991-8004. 259. Albrecht, R.A., et al., Moving Forward: Recent Developments for the Ferret Biomedical Research Model. mBio, 2018. 9(4). 260. Vidana, B., et al., Heterogeneous pathological outcomes after experimental pH1N1 influenza infection in ferrets correlate with viral replication and host immune responses in the lung. Vet Res, 2014. 45: p. 85. 261. Vidana, B., et al., Immune system cells in healthy ferrets: an immunohistochemical study. Vet Pathol, 2014. 51(4): p. 775-86. 262. Aasted, B., Mink infected with aleutian disease virus have an elevated level of CD8- positive T-lymphocytes. Veterinary Immunology and Immunopathology, 1989. 20(4): p. 375-385. 263. Kirchenbaum, G.A. and T.M. Ross, Generation of Monoclonal Antibodies against Immunoglobulin Proteins of the Domestic Ferret (Mustela putorius furo). Journal of Immunology Research, 2017. 2017: p. 1-13. 264. DiPiazza, A.T., et al., Analyses of Cellular Immune Responses in Ferrets Following Influenza Virus Infection. Methods Mol Biol, 2018. 1836: p. 513-530. 265. Richards, K.A., F.A. Chaves, and A.J. Sant, The memory phase of the CD4 T-cell response to influenza virus infection maintains its diverse antigen specificity. Immunology, 2011. 133(2): p. 246-56. 266. Angeletti, D. and J.W. Yewdell, Understanding and Manipulating Viral Immunity: Antibody Immunodominance Enters Center Stage. Trends Immunol, 2018. 39(7): p. 549-561. 267. Vidaña, B., et al., Immune System Cells in Healthy Ferrets. Veterinary Pathology, 2013. 51(4): p. 775-786. 268. Kirchenbaum, G.A., et al., Infection of Ferrets with Influenza Virus Elicits a Light Chain–Biased Antibody Response against Hemagglutinin. The Journal of Immunology, 2017. 199(11): p. 3798-3807.

203 269. Music, N., et al., Influenza vaccination accelerates recovery of ferrets from lymphopenia. PLoS One, 2014. 9(6): p. e100926. 270. Music, N., et al., Peripheral Leukocyte Migration in Ferrets in Response to Infection with Seasonal Influenza Virus. PLoS One, 2016. 11(6): p. e0157903. 271. O, E., et al., Roles of Major Histocompatibility Complex Class II in Inducing Protective Immune Responses to Influenza Vaccination. Journal of Virology, 2014. 88(14): p. 7764-7775. 272. Frank, G.M., et al., A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus. MBio, 2015. 6(4): p. e01156. 273. Angeletti, D., et al., Defining B cell immunodominance to viruses. Nat Immunol, 2017. 18(4): p. 456-463. 274. Jegaskanda, S., The Potential Role of Fc-Receptor Functions in the Development of a Universal Influenza Vaccine. Vaccines (Basel), 2018. 6(2). 275. Schultz-Cherry, S., Viral Interference: The Case of Influenza Viruses. Journal of Infectious Diseases, 2015. 212(11): p. 1690-1691. 276. Laurie, K.L., et al., Evidence for Viral Interference and Cross-reactive Protective Immunity Between Influenza B Virus Lineages. J Infect Dis, 2018. 217(4): p. 548-559. 277. Chan, K.F., et al., Investigating Viral Interference Between Influenza A Virus and Human Respiratory Syncytial Virus in a Ferret Model of Infection. J Infect Dis, 2018. 218(3): p. 406-417. 278. Lai, C., et al., C-C Motif Chemokine Ligand 2 (CCL2) Mediates Acute Lung Injury Induced by Lethal Influenza H7N9 Virus. Front Microbiol, 2017. 8: p. 587. 279. Schultz-Cherry, S., Role of NK Cells in Influenza Infection, in Influenza Pathogenesis and Control - Volume II. 2014. p. 109-120. 280. Waithman, J. and J.D. Mintern, Dendritic cells and influenza A virus infection. Virulence, 2012. 3(7): p. 603-8. 281. Fouchier, R., et al., The Role of Neutrophils during Mild and Severe Influenza Virus Infections of Mice. PLoS ONE, 2011. 6(3). 282. Cline, T.D., D. Beck, and E. Bianchini, Influenza virus replication in macrophages: balancing protection and pathogenesis. J Gen Virol, 2017. 98(10): p. 2401-2412. 283. Auladell, M., et al., Recalling the Future: Immunological Memory Toward Unpredictable Influenza Viruses. Frontiers in Immunology, 2019. 10. 284. Manocha, S., K.R. Walley, and J.A. Russell, Severe acute respiratory distress syndrome (SARS): A critical care perspective. Critical Care Medicine, 2003. 31(11): p. 2684-2692. 285. van den Brand, J.M., et al., The pathology and pathogenesis of experimental severe acute respiratory syndrome and influenza in animal models. J Comp Pathol, 2014. 151(1): p. 83-112. 286. Martina, B.E., et al., Virology: SARS virus infection of cats and ferrets. Nature, 2003. 425(6961): p. 915. 287. Roberts, A., et al., Animal models and vaccines for SARS-CoV infection. Virus Res, 2008. 133(1): p. 20-32. 288. Czub, M., et al., Evaluation of modified vaccinia virus Ankara based recombinant SARS vaccine in ferrets. Vaccine, 2005. 23(17-18): p. 2273-9. 289. Weingartl, H., et al., Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets. J Virol, 2004. 78(22): p. 12672-6.

204 290. Kobinger, G.P., et al., Adenovirus-based vaccine prevents pneumonia in ferrets challenged with the SARS coronavirus and stimulates robust immune responses in macaques. Vaccine, 2007. 25(28): p. 5220-31. 291. Cameron, M.J., et al., Lack of innate interferon responses during SARS coronavirus infection in a vaccination and reinfection ferret model. PLoS One, 2012. 7(9): p. e45842. 292. Danesh, A., et al., Early gene expression events in ferrets in response to SARS coronavirus infection versus direct interferon-alpha2b stimulation. Virology, 2011. 409(1): p. 102-112. 293. Chu, Y.K., et al., The SARS-CoV ferret model in an infection-challenge study. Virology, 2008. 374(1): p. 151-63. 294. Shi, J., et al., Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS–coronavirus 2. Science, 2020. 368(6494): p. 1016-1020. 295. Muñoz-Fontela, C., et al., Animal models for COVID-19. Nature, 2020. 296. Blanco-Melo, D., et al., 2020. 297. Zhu, F.-C., et al., Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non- randomised, first-in-human trial. The Lancet, 2020. 395(10240): p. 1845-1854. 298. Geisbert, T.W., H. Feldmann, and C.C. Broder, Animal Challenge Models of Henipavirus Infection and Pathogenesis, in Henipavirus. 2012. p. 153-177. 299. Leon, A.J., et al., Host gene expression profiles in ferrets infected with genetically distinct henipavirus strains. PLoS Negl Trop Dis, 2018. 12(3): p. e0006343. 300. Pallister, J.A., et al., Vaccination of ferrets with a recombinant G glycoprotein subunit vaccine provides protection against Nipah virus disease for over 12 months. Virol J, 2013. 10: p. 237. 301. MacPhail, M., et al., Identification of small-animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hMPV) and implications for hMPV vaccine design. J Gen Virol, 2004. 85(Pt 6): p. 1655-63. 302. Stittelaar, K.J., et al., Ferrets as a Novel Animal Model for Studying Human Respiratory Syncytial Virus Infections in Immunocompetent and Immunocompromised Hosts. Viruses, 2016. 8(6). 303. Chan, K.F., et al., Pathogenesis, humoral immune responses and transmission between co-housed animals in a ferret model of human RSV infection. Journal of Virology, 2017. 304. Collins, P.L. and B.S. Graham, Viral and host factors in human respiratory syncytial virus pathogenesis. J Virol, 2008. 82(5): p. 2040-55. 305. de Waal, L., et al., Transmission of Human Respiratory Syncytial Virus in the Immunocompromised Ferret Model. Viruses, 2018. 10(1). 306. Bukreyev, A.A., et al., Discussions and decisions of the 2012-2014 International Committee on Taxonomy of Viruses (ICTV) Filoviridae Study Group, January 2012- June 2013. Arch Virol, 2014. 159(4): p. 821-30. 307. Kozak, R., et al., Ferrets Infected with Bundibugyo Virus or Ebola Virus Recapitulate Important Aspects of Human Filovirus Disease. J Virol, 2016. 90(20): p. 9209-23. 308. Cross, R.W., et al., The Domestic Ferret (Mustela putorius furo) as a Lethal Infection Model for 3 Species of Ebolavirus. J Infect Dis, 2016. 214(4): p. 565-9. 309. de La Vega, M.-A., et al., Modeling Ebola Virus Transmission Using Ferrets. mSphere, 2018. 3(5). 310. Cross, R.W., et al., Comparative Transcriptomics in Ebola Makona-Infected Ferrets, Nonhuman Primates, and Humans. J Infect Dis, 2018. 218(suppl_5): p. S486-S495.

205 311. Gerritsen, B., et al., Characterization of the ferret TRB locus guided by V, D, J, and C gene expression analysis. Immunogenetics, 2020. 72(1-2): p. 101-108. 312. Peng, X., et al., The draft genome sequence of the ferret (Mustela putorius furo) facilitates study of human respiratory disease. Nat Biotechnol, 2014. 32(12): p. 1250- 5. 313. Montague, M.J., et al., Comparative analysis of the domestic cat genome reveals genetic signatures underlying feline biology and domestication. Proceedings of the National Academy of Sciences, 2014. 111(48): p. 17230-17235. 314. Lindblad-Toh, K., et al., Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature, 2005. 438(7069): p. 803-819. 315. Lefranc, M.P., et al., IMGT(R), the international ImMunoGeneTics information system(R) 25 years on. Nucleic Acids Res, 2015. 43(Database issue): p. D413-22. 316. Cui, A., et al., A Model of Somatic Hypermutation Targeting in Mice Based on High- Throughput Ig Sequencing Data. The Journal of Immunology, 2016. 197(9): p. 3566- 3574. 317. Allen, R.L., et al., Soluble T-Cell Receptors Produced in Human Cells for Targeted Delivery. Plos One, 2015. 10(4). 318. Fonville, J.M., et al., Antigenic Maps of Influenza A(H3N2) Produced With Human Antisera Obtained After Primary Infection. J Infect Dis, 2016. 213(1): p. 31-8. 319. Liu, S.T.H., et al., Antigenic sites in influenza H1 hemagglutinin display species- specific immunodominance. J Clin Invest, 2018. 128(11): p. 4992-4996. 320. Skowronski, D.M., et al., Prevalence of seroprotection against the pandemic (H1N1) virus after the 2009 pandemic. Canadian Medical Association Journal, 2010. 182(17): p. 1851-1856. 321. Zacour, M., et al., Standardization of Hemagglutination Inhibition Assay for Influenza Serology Allows for High Reproducibility between Laboratories. Clinical and Vaccine Immunology, 2016. 23(3): p. 236-242. 322. Jersmann, H.P., Time to abandon dogma: CD14 is expressed by non-myeloid lineage cells. Immunol Cell Biol, 2005. 83(5): p. 462-7. 323. Liyanage, S.E., et al., Flow cytometric analysis of inflammatory and resident myeloid populations in mouse ocular inflammatory models. Experimental Eye Research, 2016. 151: p. 160-170. 324. Sánchez-Torres, C., et al., CD16+ and CD16− human blood monocyte subsets differentiate in vitro to dendritic cells with different abilities to stimulate CD4+ T cells. International Immunology, 2001. 13(12): p. 1571-1581. 325. Stocks, S.C., et al., CD66: role in the regulation of neutrophil effector function. Eur J Immunol, 1996. 26(12): p. 2924-32. 326. Dreyfus, C., et al., Highly conserved protective epitopes on influenza B viruses. Science, 2012. 337(6100): p. 1343-8. 327. Tiller, T., et al., Autoreactivity in human IgG+ memory B cells. Immunity, 2007. 26(2): p. 205-13. 328. Wardemann, H. and C.E. Busse, Expression Cloning of Antibodies from Single Human B Cells. Methods Mol Biol, 2019. 1956: p. 105-125. 329. Liao, H.X., et al., High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. J Virol Methods, 2009. 158(1-2): p. 171-9. 330. Starkie, D.O., et al., Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells. PLoS One, 2016. 11(3): p. e0152282.

206 331. Sundling, C., et al., Single-cell and deep sequencing of IgG-switched macaque B cells reveal a diverse Ig repertoire following immunization. J Immunol, 2014. 192(8): p. 3637-44. 332. Mason, R.D., et al., Targeted Isolation of Antibodies Directed against Major Sites of SIV Env Vulnerability. PLoS Pathog, 2016. 12(4): p. e1005537. 333. Chaudhary, N. and D.R. Wesemann, Analyzing Immunoglobulin Repertoires. Front Immunol, 2018. 9: p. 462. 334. Boyd, S.D. and J.E. Crowe, Jr., Deep sequencing and human antibody repertoire analysis. Curr Opin Immunol, 2016. 40: p. 103-9. 335. Enkirch, T. and V. von Messling, Ferret models of viral pathogenesis. Virology, 2015. 479-480: p. 259-70. 336. Afgan, E., et al., The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Research, 2018. 46(W1): p. W537- W544. 337. Grabherr, M.G., et al., Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol, 2011. 29(7): p. 644-52. 338. Boratyn, G.M., et al., Magic-BLAST, an accurate RNA-seq aligner for long and short reads. BMC Bioinformatics, 2019. 20(1): p. 405. 339. Kirkham, P.M., et al., Immunoglobulin VH clan and family identity predicts variable domain structure and may influence antigen binding. EMBO J, 1992. 11(2): p. 603-9. 340. Bao, Y., et al., Molecular characterization of the VH repertoire in Canis familiaris. Vet Immunol Immunopathol, 2010. 137(1-2): p. 64-75. 341. Steiniger, S.C., et al., Comparative analysis of the feline immunoglobulin repertoire. Biologicals, 2017. 46: p. 81-87. 342. Olivieri, D.N., et al., The immunologic V-gene repertoire in mammals. bioRxiv, 2014. 343. Wu, T.T., G. Johnson, and E.A. Kabat, Length distribution of CDRH3 in antibodies. Proteins: Structure, Function, and Genetics, 1993. 16(1): p. 1-7. 344. Shi, B., et al., Comparative analysis of human and mouse immunoglobulin variable heavy regions from IMGT/LIGM-DB with IMGT/HighV-QUEST. Theor Biol Med Model, 2014. 11: p. 30. 345. Leon, A.J., et al., Sequencing, Annotation, and Characterization of the Influenza Ferret Infectome. Journal of Virology, 2012. 87(4): p. 1957-1966. 346. Bergeron, L.M., et al., Comparative functional characterization of canine IgG subclasses. Veterinary Immunology and Immunopathology, 2014. 157(1-2): p. 31-41. 347. Steiniger, S.C.J., et al., Fundamental characteristics of the expressed immunoglobulin VH and VL repertoire in different canine breeds in comparison with those of humans and mice. Molecular Immunology, 2014. 59(1): p. 71-78. 348. Andris, J.S., et al., The human antibody repertoire: heavy and light chain variable region gene usage in six alloantibodies specific for human HLA class I and class II alloantigens. Mol Immunol, 1995. 32(14-15): p. 1105-22. 349. Larijani, M., et al., The recombination difference between mouse kappa and lambda segments is mediated by a pair-wise regulation mechanism. Mol Immunol, 2006. 43(7): p. 870-81. 350. Arun, S.S., W. Breuer, and W. Hermanns, Immunohistochemical examination of light- chain expression (lambda/kappa ratio) in canine, feline, equine, bovine and porcine plasma cells. Zentralbl Veterinarmed A, 1996. 43(9): p. 573-6. 351. Mole, C.M., et al., Light chains of immunoglobulins in human secretions. Clin Chim Acta, 1994. 224(2): p. 191-7. 352. Kügler, J., et al., Generation and analysis of the improved human HAL9/10 antibody phage display libraries. BMC Biotechnology, 2015. 15(1).

207 353. Schubert, M., et al., NGS-based analysis of the mouse B-cell receptor repertoire. Protocol Exchange, 2019. 354. Rizzetto, S., et al., B-cell receptor reconstruction from single-cell RNA-seq with VDJPuzzle. Bioinformatics, 2018. 34(16): p. 2846-2847. 355. Lindeman, I., et al., BraCeR: B-cell-receptor reconstruction and clonality inference from single-cell RNA-seq. Nat Methods, 2018. 15(8): p. 563-565. 356. Upadhyay, A.A., et al., BALDR: a computational pipeline for paired heavy and light chain immunoglobulin reconstruction in single-cell RNA-seq data. Genome Med, 2018. 10(1): p. 20. 357. Canzar, S., et al., BASIC: BCR assembly from single cells. Bioinformatics, 2017. 33(3): p. 425-427. 358. Ramesh, A., et al., Structure and Diversity of the Rhesus Macaque Immunoglobulin Loci through Multiple De Novo Genome Assemblies. Frontiers in Immunology, 2017. 8. 359. Cirelli, K.M., et al., Slow Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance. Cell, 2019. 177(5): p. 1153-1171.e28. 360. Corcoran, M.M., et al., Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity. Nature Communications, 2016. 7(1). 361. Buhler, J., D.K. Ralph, and F.A. Matsen, Per-sample immunoglobulin germline inference from B cell receptor deep sequencing data. PLOS Computational Biology, 2019. 15(7). 362. Cyster, J.G. and C.D.C. Allen, B Cell Responses: Cell Interaction Dynamics and Decisions. Cell, 2019. 177(3): p. 524-540. 363. Tabel, H. and D.G. Ingram, Immunoglobulins of mink. Evidence for five immunoglobulin classes of 7S type. Immunology, 1972. 22(6): p. 933-42. 364. Schultz, R.D., et al., Feline immunoglobulins. Infect Immun, 1974. 9(2): p. 391-3. 365. Jegaskanda, S., et al., Antibody-dependent cellular cytotoxicity is associated with control of pandemic H1N1 influenza virus infection of macaques. J Virol, 2013. 87(10): p. 5512-22. 366. Jegaskanda, S., P.C. Reading, and S.J. Kent, Influenza-Specific Antibody-Dependent Cellular Cytotoxicity: Toward a Universal Influenza Vaccine. The Journal of Immunology, 2014. 193(2): p. 469-475. 367. Wheatley, A.K., et al., HIV-dependent depletion of influenza-specific memory B cells impacts B cell responsiveness to seasonal influenza immunisation. Scientific Reports, 2016. 6(1). 368. Lee, C.-C., et al., An Effective Neutralizing Antibody Against Influenza Virus H1N1 from Human B Cells. Scientific Reports, 2019. 9(1). 369. Davis, A.K.F., et al., Sera from Individuals with Narrowly Focused Influenza Virus Antibodies Rapidly Select Viral Escape Mutations In Ovo. Journal of Virology, 2018. 92(19). 370. Li, C., et al., Selection of antigenically advanced variants of seasonal influenza viruses. Nat Microbiol, 2016. 1(6): p. 16058. 371. Gould, V.M.W., et al., Nasal IgA Provides Protection against Human Influenza Challenge in Volunteers with Low Serum Influenza Antibody Titre. Frontiers in Microbiology, 2017. 8. 372. Nachbagauer, R., et al., A universal influenza virus vaccine candidate confers protection against pandemic H1N1 infection in preclinical ferret studies. npj Vaccines, 2017. 2(1).

208 373. Wilson, J.R., et al., Diversity of the murine antibody response targeting influenza A(H1N1pdm09) hemagglutinin. Virology, 2014. 458-459: p. 114-24. 374. Krammer, F., et al., Influenza. Nature Reviews Disease Primers, 2018. 4(1). 375. Nelson, S.A. and A.J. Sant, Imprinting and Editing of the Human CD4 T Cell Response to Influenza Virus. Frontiers in Immunology, 2019. 10. 376. Vincek, V., et al., A Tissue Fixative that Protects Macromolecules (DNA, RNA, and Protein) and Histomorphology in Clinical Samples. Laboratory Investigation, 2003. 83(10): p. 1427-1435. 377. Hassane, M. and C. Paget, "Universal Flu Vaccine": Can NK Cell-mediated ADCC Tip the Scales? EBioMedicine, 2016. 8: p. 18-19. 378. von Boehmer, L., et al., Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat Protoc, 2016. 11(10): p. 1908-1923. 379. Liu, Y., et al., Cross-lineage protection by human antibodies binding the influenza B hemagglutinin. Nature Communications, 2019. 10(1). 380. Sievers, F., et al., Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol, 2011. 7: p. 539. 381. Schwede, T., et al., SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res, 2003. 31(13): p. 3381-5. 382. Del Nagro, C.J., et al., CD19 Function in Central and Peripheral B-Cell Development. Immunologic Research, 2005. 31(2): p. 119-132. 383. Cherukuri, A., P.C. Cheng, and S.K. Pierce, The Role of the CD19/CD21 Complex in B Cell Processing and Presentation of Complement-Tagged Antigens. The Journal of Immunology, 2001. 167(1): p. 163-172. 384. Ashkenazy, H., et al., ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Research, 2016. 44(W1): p. W344-W350. 385. Chen, K. and A. Cerutti, New insights into the enigma of immunoglobulin D. Immunol Rev, 2010. 237(1): p. 160-79. 386. Depoil, D., et al., CD19 is essential for B cell activation by promoting B cell receptor- antigen microcluster formation in response to membrane-bound ligand. Nat Immunol, 2008. 9(1): p. 63-72. 387. Hebditch, M., R. Curtis, and J. Warwicker, Sequence composition predicts immunoglobulin superfamily members that could share the intrinsically disordered properties of antibody CH1 domains. Sci Rep, 2017. 7(1): p. 12404. 388. Mann, M., R.C. Hendrickson, and A. Pandey, Analysis of Proteins and Proteomes by Mass Spectrometry. Annual Review of Biochemistry, 2001. 70(1): p. 437-473. 389. Halliley, J.L., et al., Long-Lived Plasma Cells Are Contained within the CD19(- )CD38(hi)CD138(+) Subset in Human Bone Marrow. Immunity, 2015. 43(1): p. 132- 45. 390. Leonova, E.I. and O.V. Galzitskaya, Cell communication using intrinsically disordered proteins: what can syndecans say? Journal of Biomolecular Structure and Dynamics, 2014. 33(5): p. 1037-1050. 391. Saunders, S., et al., Molecular cloning of syndecan, an integral membrane proteoglycan. J Cell Biol, 1989. 108(4): p. 1547-56. 392. Unal, E.S., et al., N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT). Biochim Biophys Acta, 2008. 1778(6): p. 1407-14. 393. Alter, G., J.M. Malenfant, and M. Altfeld, CD107a as a functional marker for the identification of natural killer cell activity. Journal of Immunological Methods, 2004. 294(1-2): p. 15-22.

209 394. Stils, H.F., Adjuvants and Antibody Production: Dispelling the Myths Associated with Freund's Complete and Other Adjuvants. ILAR Journal, 2005. 46(3): p. 280-293. 395. Dogan, I., et al., Multiple layers of B cell memory with different effector functions. Nat Immunol, 2009. 10(12): p. 1292-9. 396. Reynaud, C.A., et al., IgM memory B cells: a mouse/human paradox. Cell Mol Life Sci, 2012. 69(10): p. 1625-34. 397. De-Simone, S.G., et al., IgE and IgG epitope mapping by microarray peptide- immunoassay reveals the importance and diversity of the immune response to the IgG3 equine immunoglobulin. Toxicon, 2014. 78: p. 83-93. 398. Chiuppesi, F., et al., Comparison of homologous and heterologous prime-boost vaccine approaches using Modified Vaccinia Ankara and soluble protein to induce neutralizing antibodies by the human cytomegalovirus pentamer complex in mice. PLoS One, 2017. 12(8): p. e0183377. 399. Brynjolfsson, S.F., et al., Long-Lived Plasma Cells in Mice and Men. Frontiers in Immunology, 2018. 9. 400. Rettig, T.A., et al., Characterization of the naive murine antibody repertoire using unamplified high-throughput sequencing. PLoS One, 2018. 13(1): p. e0190982. 401. Wentink, M.W.J., et al., Deficiencies in the CD19 complex. Clinical Immunology, 2018. 195: p. 82-87. 402. Xu, Y., et al., CD19 differentially regulates BCR signalling through the recruitment of PI3K. Autoimmunity, 2014. 47(7): p. 430-437. 403. Orlova, M.A., et al., Conformational differences between native and recombinant horseradish peroxidase revealed by tritium planigraphy. Biochemistry (Mosc), 2003. 68(11): p. 1225-30. 404. Kuroki, K., et al., Polymorphisms of human CD19 gene: possible association with susceptibility to systemic lupus erythematosus in Japanese. Genes & Immunity, 2002. 3(S1): p. S21-S30. 405. Goding, J.W., G.W. Warr, and N.L. Warner, Genetic polymorphism of IgD-like cell surface immunoglobulin in the mouse. Proc Natl Acad Sci U S A, 1976. 73(4): p. 1305-9. 406. Rath, A., et al., Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proceedings of the National Academy of Sciences, 2009. 106(6): p. 1760-1765. 407. Ridderstad, A. and D.M. Tarlinton, Kinetics of establishing the memory B cell population as revealed by CD38 expression. J Immunol, 1998. 160(10): p. 4688-95. 408. Gitlin, Alexander D., et al., Independent Roles of Switching and Hypermutation in the Development and Persistence of B Lymphocyte Memory. Immunity, 2016. 44(4): p. 769-781. 409. Vences-Catalán, F. and L. Santos-Argumedo, CD38 through the life of a murine B lymphocyte. IUBMB Life, 2011. 63(10): p. 840-846. 410. Soria-Guerra, R.E., et al., An overview of bioinformatics tools for epitope prediction: implications on vaccine development. J Biomed Inform, 2015. 53: p. 405-14. 411. Leyendeckers, H., et al., Correlation analysis between frequencies of circulating antigen-specific IgG-bearing memory B cells and serum titers of antigen-specific IgG. Eur J Immunol, 1999. 29(4): p. 1406-17. 412. Khodadadi, L., et al., The Maintenance of Memory Plasma Cells. Frontiers in Immunology, 2019. 10. 413. Wu, L., et al., Long-Lived Plasma Cells Secrete High-Affinity Antibodies Responding to a T-Dependent Immunization in a Teleost Fish. Frontiers in Immunology, 2019. 10.

210 414. Minges Wols, H.A. and P.L. Witte, Plasma cell purification from murine bone marrow using a two-step isolation approach. J Immunol Methods, 2008. 329(1-2): p. 219-24. 415. Collins, A.M., et al., The mouse antibody heavy chain repertoire is germline-focused and highly variable between inbred strains. Philos Trans R Soc Lond B Biol Sci, 2015. 370(1676). 416. Gretebeck, L.M. and K. Subbarao, Animal models for SARS and MERS coronaviruses. Current Opinion in Virology, 2015. 13: p. 123-129. 417. Harding, J.D., Progress in genetics and genomics of nonhuman primates. Introduction. ILAR J, 2013. 54(2): p. 77-81. 418. Keck, Z.-Y., et al., Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein. Journal of Virology, 2016. 90(1): p. 279-291. 419. Parra, D., F. Takizawa, and J.O. Sunyer, Evolution of B cell immunity. Annu Rev Anim Biosci, 2013. 1: p. 65-97. 420. Steiniger, S.C.J., et al., Comparative analysis of the feline immunoglobulin repertoire. Biologicals, 2017. 46: p. 81-87. 421. Sun, W., et al., Antibody Responses toward the Major Antigenic Sites of Influenza B Virus Hemagglutinin in Mice, Ferrets, and Humans. J Virol, 2019. 93(2). 422. Xie, H., et al., H3N2 Mismatch of 2014–15 Northern Hemisphere Influenza Vaccines and Head-to-head Comparison between Human and Ferret Antisera derived Antigenic Maps. Scientific Reports, 2015. 5: p. 15279. 423. Lee, M.S. and C.F. Yang, Cross‐Reactive H1N1 Antibody Responses to a Live Attenuated Influenza Vaccine in Children: Implication for Selection of Vaccine Strains. The Journal of Infectious Diseases, 2003. 188(9): p. 1362-1366. 424. Ana-Sosa-Batiz, F., et al., Influenza-Specific Antibody-Dependent Phagocytosis. PLoS One, 2016. 11(4): p. e0154461. 425. de Vries, R.D., et al., Influenza virus-specific antibody dependent cellular cytoxicity induced by vaccination or natural infection. Vaccine, 2017. 35(2): p. 238-247. 426. Vanderven, H.A., et al., Fc functional antibodies in humans with severe H7N9 and seasonal influenza. JCI Insight, 2017. 2(13). 427. Ochi, A., et al., Cloning, expression and immunoassay detection of ferret IFN-gamma. Dev Comp Immunol, 2008. 32(8): p. 890-7. 428. Gasteiger, E., et al., Protein Identification and Analysis Tools on the ExPASy Server, in The Proteomics Protocols Handbook. 2005. p. 571-607. 429. Teng, F., et al., Homology modeling and epitope prediction of Der f 33. Braz J Med Biol Res, 2018. 51(5): p. e6213. 430. Chatchatee, P., et al., Identification of IgE- and IgG-binding epitopes on alpha(s1)- casein: differences in patients with persistent and transient cow's milk allergy. J Allergy Clin Immunol, 2001. 107(2): p. 379-83. 431. Lu, S., Heterologous prime-boost vaccination. Curr Opin Immunol, 2009. 21(3): p. 346-51. 432. Le Grand, R., et al., Heterologous Prime-Boost Regimens with a Recombinant Chimpanzee Adenoviral Vector and Adjuvanted F4 Protein Elicit Polyfunctional HIV- 1-Specific T-Cell Responses in Macaques. Plos One, 2015. 10(4). 433. Snounou, G., et al., Distinct Kinetics of Memory B-Cell and Plasma-Cell Responses in Peripheral Blood Following a Blood-Stage Plasmodium chabaudi Infection in Mice. PLoS ONE, 2010. 5(11). 434. Jones, D.D., J.R. Wilmore, and D. Allman, Cellular Dynamics of Memory B Cell Populations: IgM+ and IgG+ Memory B Cells Persist Indefinitely as Quiescent Cells. The Journal of Immunology, 2015. 195(10): p. 4753-4759.

211 435. Lim, C.C., P.C.Y. Woo, and T.S. Lim, Development of a Phage Display Panning Strategy Utilizing Crude Antigens: Isolation of MERS-CoV Nucleoprotein human antibodies. Sci Rep, 2019. 9(1): p. 6088. 436. Henson, J., G. Tischler, and Z. Ning, Next-generation sequencing and large genome assemblies. Pharmacogenomics, 2012. 13(8): p. 901-15. 437. Wang, T., et al., Comparative analysis of differential gene expression analysis tools for single-cell RNA sequencing data. BMC Bioinformatics, 2019. 20(1): p. 40. 438. Lee, C.M.Y., et al., Selection of human antibody fragments by phage display. Nature Protocols, 2007. 2(11): p. 3001-3008. 439. Rouet, R., et al., Next-Generation Sequencing of Antibody Display Repertoires. Frontiers in Immunology, 2018. 9. 440. Setliff, I., et al., High-Throughput Mapping of B Cell Receptor Sequences to Antigen Specificity. Cell, 2019. 179(7): p. 1636-1646 e15.

212 Appendices

Chapter 3 Appendix

Appendix 3.1 Amino acid sequence alignments of ferret IGKV genes

213 Appendix 3.2 Amino acid sequence alignments of ferret IGLV genes

214

Appendix 3.3 Ferret germline heavy variable genes

CAGGTGCAGCTGGTGCAATCTGAGGCTGAGGTAAGGAAGCCTGGGGAATCCGTGAAGGTCTCCTGCAAGGCATCTGGGTACACCTTCAC CAGCTATGCAATGAACTGGGTGCAACAGGCACCAGGAAAGAGCCTGCAGTACATGGGATGGATCGACACTAACACTGGGAAACCAACATA I-HV1 TGCCCCGGGCTTCTCCGGTCGATTTGTTTTCTCCACGGACACCTCTGTCAGCACAGCCTATCTACAGATGAACAGCCTGAACTCTGAGGAC ACGGCCGTGTATTACTGT CAGGTCCATCTGTTACAGTCTGGGGCTGAGGTGAGGAATCCTGGAGCATCTGTGAAGGTCTCCTGCAAGGCATCTGGATACACATTCACT GACTACTATATGCACTGGGTGCGACAGGCCCCAGAACGAGGGCTTGAGTGGATGGGACGAATTGACCCTGAAGATGGTGCCACAAATATT I-HV2 GCACAGAAATTCCAGGCTAGAGTCACGCTTATGGCAGACACATCCACAAGCATGGCCTACATGGAACTGAGAAGTCTGAGGTCTGAGGAC ACGGCTTTGTATTACTGT CAGGTCCATCTGTTACAGTCTGGGGCAGAGGTGAGGAATCCTGGAGCATCTGTGAAGGTCTCCTGCAAGGCATCTGGATACACATTCACT AACTACTATATGCACTGGGTGCAACAGGCCCCAGAACGAGGGCTTGAGTGGATGGGACAAATTGACCCTGAAGATGGTGCCACAAATATT I-HV3 GCACAGAAATACCAGGCTAGAGTCACGCTTATGGCAGACACATCCACAAACATGGCCTACATGGAACTGAGAAGTCTGAGGCCTGAGGAC ACGGCTTTGTATTACTGT CAACTTACACTTCAGGAGTCAGGCCCAGGACTGGTGAAGCCCTCACAGACCCTCTCTCTCACATGTGTTGTCTCCGGGGGCTCCGTCACC AGCAGTTACTATTGGAACTGGATCCGCCAGCGCCCTGGAAAAGCCCTGGAGTGGATGGGCTACTGGACGGGTAGTACAAGGTACAACCC II-HV1 GGCTTTCCAGGGCCGCATCTCCATCACTGCTGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAGTTCCATGACCACTGAGGACACGGC CGTGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGGAGGGTCTCTGAGACTTTCCTGTACAGCCTCTGGATTCACCTTCAG CAGCTACAGCATGCAATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGGTCGCATATATACGCTATGATGGAGGTAGCACAAGCT III-HV1 ACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGGCAAGAACACGCTGTACCTGCAGATGAACAGCCTGAGAGCCGAG GACACGGCCCTGTATTACTGT GAGATGCAGTTGGTGGAGTCTGGGGGAGACTTGGTGAAGCCTGGGGGTTCTCTGAGACTCTCCTGTGAAGCCTCTGGTTTCACCTTCAGT GGCTATGGAATGAGCTGGGTCCGCCAGGCACCAGGAAAGCGGATAGAGTTGGTCTCAAATATTGATGCTGGTGGAGGTAGCACAAGCTAC III-HV2 ACAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCAAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAACCGAGGAC ACAGCCATGTATTTCTGT GAGATGAAGCTAGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGT AGCTATGGCATGACCTGGGTCTGCCAGGATCCAGGGAAGGGGCCGCAGTGGGTTGCAGGTATTTGGATTGATGGAAGTTTCACAAGCTAT III-HV3 GTAGACTCTGTGAAGGGCCAATTCACCATCTCCAGAGACAATGGCAAGAACACGCTGTATCTTCAGATGAACAGCCTGAGATCCAATGACA TGGACGTGTATTATTGT GAGATGCAGCTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGAAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG TAGCTACGGCATGAGCTGGGTCCGCCAGGCTCCAGGGAATGGGCTGCAGTGGGTCGCAGGTATTAGCTATGATGGAAGTAGCACATACTA III-HV4 CGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGAGACAACGGCAAGAACACGCTGTATCTTCAGATGAACAGCCTGAGAGCCGAGGA CACGGCCGTGTATTACTGT

215 GAGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGTTGAAGCCTGGGGGTTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG TAGCTATGGCATGAGCTGGGTCCACCAGGCCCCAGGGAAGGGGCTGCAGTGGGTCGCAGATATTAGCAAGGGTGGTAGTTACACATACT III-HV5 ACACAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGGCAAGAACATGCTGTATCTGCAGATGAACAGCCTGAGAGCTGAGG ACACGGCCGTGTATTACTGTGC GAGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTTTCCTGTGCAGCCTCTGGATTCATCTTCAG TAGCTACTGGATGAGATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGAAGTGGGTCACAAGTATTAGCAATACTGGTAGTAACACATACTA III-HV6 TGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGGCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCTGAGGA CACGGCCGTGTATTATTGT GAGGTGCAGTTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG TAACTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGGTCACAAGTATTAGCAATACTGGTAGTAACACATACTA III-HV7 TGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGGCAAGAACATGCTGTATCTTCAGATGAATAGCCTGAAAGCCGAGGAC ACAGCCGTTTATTACTGT GAGGTGAAGCTTGTGGAATCTGGGGGAGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCGTGTGCAGCCTCTGGATTCACCTTCAG TAACTATGACATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGACTGCAGTGGGTCGCATACATTAGCAGTGGTGGAAGTAGCACATACTA III-HV8 TGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGACAAGGACATGCTGTATCTGCAGATGAACAGCCTGAGAGCAGAGGA CACGGCCATGTATTATTGT GAGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAG TAACAATGAAATGAACTGGGTCCGCCAGGCTCCAGGGATGGGGCTGCAGTGGGTCGCATATATTAACAGTGTTGGAAGCACATACTATGC III-HV9 AGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACAGCAAGAACACGCTGCATCTGCATATGAGCAACCTGAATGCTGAGGACAC GGCTGTGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGTGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCACCTTCAG TAACTACGGCATGAGCCGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGGTCGCATGGATTAGCAGTAGAAGTAGTTACACATACT III-HV10 ACACAGACTCTGTGAAGGGCCAATTCACCATCTCCAGAGACAACAGCAAGAACACGCTGTATCTGCAGATGAGCAGCCTGAGAGCCGAGG ACACGGCTGTATATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGGCGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG TAACTATGGCATGAGCTGGGTCAGGCAGACTCCAGGGAAGGGGCTGCAGTGGGTCGCATATATTAGCACTAGTAGTAGTTACACATACTAT III-HV11 GCAGACTCTGTGAAGGGCCAATTCACTGTCTCCAGAGACAATGGGAAGAACACGCTGTATCTGCAGATGAGCAGCCTGAGAGCTGAGGAC ACGGCCGTGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGAGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG CAACTACTACATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGGTCGCAGAAATTAGCAATACTGGTAGTAGCACATACTA III-HV12 CACAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGGCAAGAATACACTTTATCTTCAGATGAACAGCCTGAGAGCTGAGGAC ACAGCCATGTATTACTGT GTGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTAGGGGTTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG CAACTACCACATGGACTGGGTCCACCAGGCTCCAGGGAAGGGGCTGCAGTGGGTCGCAGAAATTAGCAATACTGGTAGTAGCACAAACTA III-HV13 CGCAGACTCTGTGAAAGGCCAAATCACCATCTCCAGAGACAACGGCAAGAACACACTGTATCTGCAGATGAACAGCCTGAGAGTCGAGGA CACAGCCGTGTATTACTGT

216 GAGGTGCAGCTGGTGGAGTCTGGGGAAGACCTGGTGAAGCCTGGGGGATCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG CAACTACTACATGGACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGGTCGCAGATATTAGCGGTGGTGGAAGTAGCACAAGCT III-HV14 ACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGGCAAGAATATGCTGTATCTGCAGATGAATAGCCTGAGAGCAGAGG ACACGACCCTGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGTGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG CAACTACTACATGGAACGGGTCCGCCAGGCTCCAGGGAAGGGACTGCAGTGGGTCGCAGGTATTAGCAGGGATGGAAGTAGCACAAGCT III-HV15 ATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGGCAAGAACACACTGTACCTGCAGATTAACAGCCTGAGAAGTGAGG ACACGGCCATGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG CAACTACTACATGTACTGGGTCCGCCAGGCACCAGGGAAGGGGCTGCAGTGGGTCGCAGGTATTAGCAGGGATGGAAGTAGCACAAGCT III-HV16 ATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGGCAAGAACACACTGTACCTGCAGATTAACAGCCTGAGAAGTGAGG ACACGGCTGTGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGTGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG III- TAGCTACGACATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGTGTCGCATGGATTAATACTGGTGGAAGTAGCACAAGCTA HV17a CGCAGACTCTGTGAAGGGTCGATTCACCATCTCCAGAGACAACGGCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGA CACGGCCATGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGTGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG III- TAGCTACGACATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGTGTCGCATGGATTAATACTGGTGGAAGTAGCACAAGCTA HV17b CGCAGACTCTGTGAAGGGTCGATTCACCATCTCCAGAGACAACGACAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGTCGAGGA CACGGCCATGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGTGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG III- TAGCTACGACATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGTGTCGCATGGATTAATACTGGTGGAAGTAGCACAAGCTA HV17c CGCAGACTCTGTGAAGGGTCGATTCACCATCTCCAGAGACAACGGCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGTCGAGGA CACGGCCATGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG III- CAACTACTACATGGACTGGGTCCGCCAGGCACCAGGGAAGGGGCTGCAGTGGGTCGCACGTATTAGCAGTGATGGAAGTAGCACATACT HV18a ACGCAGACTCTGTGAAGGGCCGATTCGCCATCTCCAGAGACAACGGCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAACTGAGG ACACGGCCGTGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG III- CAACTACTACATGGACTGGGTCCGCCAGGCACCAGGGAAGGGGCTGCAGTGGGTCGCACGTATTAGCAGTGATGGAAGTAGCACATACT HV18b ACGCAGACTCTGTGAAGGGCCGATTCGCCATCTCCAGAGACAACGGCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAACTGAGG ACACGGCCGTGTTTTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGTGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG III- CAACTACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGGTCGCATGGATTAATACTGGTGGAAGTAGCACATACTA HV19a CGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGGCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGA CACGGCCATGTATTACTGT

217 GAGGTGCAGCTGGTGGAGTCTGGGGGTGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTTTCCTGTGCAGCCTCTGGATTCACCTTCAG III- CAACTACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGGTCGCATGGATTAATACTGGTGGAAGTAGCACATACTA HV19b CGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGGCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGA CACGGCCATGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG CAACTACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGGTCGCATATATTAGCAGTGATGGAAGTAGCACAAGCTA III-HV20 CGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGGCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGA CACGGCCATGTATTACTGT GAGGTGCAGCTGGTGGAGTCTGGGGGAGACCTGGTGAAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG TAGCTATGGCATGCACTGGGTCCACCAGGCTCCAGGGAAGGGACTGCAGTGGGTCGCAGGTATTAGCAATGACGGAAGTGGCACATACT III-HV21 ACGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGGCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGG ACACGGCCGTGTACTACTGT

218 Appendix 3.4 Ferret germline kappa variable genes

GACATTGTGCTGACCCAGTCTCCAGCCTCTTTGACTATGTCTCCAGGGCAGAGGACCACCATCTCTTGCAAGACCAGTCAGGAAGTCAGTG ACATTTTGGGCATTACACACTGTATTACATGGTATCAACAAAAATCAGGACAGTGTCCGAGAATCCTGATTTATAAAGCATCTAGTGGAGTGT III-KV1 GTGGGGTCCCGGTCCGGTTCAATGGTGGTAGGTCTGGGACGGAATTCAGTCTCACGACTGGTCTGGTGGAGGATGGGGATGCTGCCAGT TATTACTGC GACATTGTGCTGACCCAGTCTCCAGCCTCTTTGACTGTGTCTCCAGGGCAGAGGGCCACCATCTCTTGTAAGACCAGTCAGAACGTTGGTG ACATTTGGGGCATGACACACTATATTACATGGTATCAACAAAAATCAGGACAGCGTCCAAGAATGCTGATTTATAAAGCATCCAGTCAAGCA III-KV2 TCTGGGGCCCCGGCCCGCTTCAGTGGCAGTGGGTCTGGGACTGAATTCAGTCTCACAATTGATCCCGTGGAGGATGGGGATGCTGCCAAC TATTACTGC GACATCGTGATGACCCAGTCTCCAGCCTCCCTGAACGGGTCTCCAGGAGAACGCATTGCCATGAACTGCAAGTCTAGCCAGAGTCTTCTAT ACAGCTCCAACCAGAAGAACTACTTAGCTTGGTACCAGCAGAAGCCAGGGCAGGCTCCTAAGCTACTCATCATCTGGGCATCCACCCGGG II-KV1 CATCTGGGGTTCCCGACCGGTTCAGCGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAACCTCCAGGCTGAAGATGTGGGG GACTATTACTGC GAAATCACGATGACTCAGTCTCCAGGCTCCCTGGCTGTGTCTCCAGGTCAACGGGTCACCCTGAACTGCAAAGCCAGTCAGAGTGTTAGCA II-KV2 ACTATGTAAACTGGTACCAGCAGAAACCTGGGCAGTCTCCCAGGCTCCTCATCTATGATGCATCCAGCAGGGCCACGGGCATCCCAGACC GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAACCTCCAGGCCGAAGATGTGGGAAGCTATTACTGC GATATCACGATGACTCAGTCTCCAGGCTCCCTGGCTGTGTCTCCAGGTCAGCGGGTCACCATAAACTGCAGAGCCAGTCAGAGTGTTAGCA II-KV3 ACTATGTAGCCTGGTACCAGCAGAAACCTGGGCAGTCTCCCAGGCTCCTCATCTATTATGCATCCAGCAGGGCCACGGGTATCCCAGACC GGTTCAGCGGCAGTGGGTCTGGGACAGACTTCACCCTCAGCATCAGCAACCTCCAGGCCGAAGATGTGGGGGACTATTACTGC GACATCACGATGACTCAGTCTCCAGGCTCGCTGGCTGTGTCTCCAGGTCAACGGATCACCATGAACTGCAGAGCCAGTCAGAGTGTTAGC II-KV4 AACTATGTAGCCTGGTACCAGCAGAAACCTGGGCAGGCTCCCAGGCTCCTCATCTATTATGCATCCAGCAGGGCCACGGGGATCCCAGAC CGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAACCTCCAGGCTGAAGATGTGGGAAACTATTACTGCC GACATCACGATGACTCAGTCTCCAGGCTCCCTGGCTGTGTCTCCAGGTCAGCGGGTCACCCTGAACTGCAGAGCCAGTCAAAGTGTTAGC II-KV5 AACTATGTAGCCTGGTACCAGCAGAAACCTGGGCAGTCTCCCAGGCTCCTCATCTATTATGCATCCAGCAGGGCCACGGGTATCCCAGACC GGTTCAGCGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAACCTCCAGGCCGAAGATGTGGGAAACTATTACTGT GACATCACAATGACTCAGTCTCCAGGCTCCCTGGCTGTGTCTCCAGGTCAGCGGGTCACCATGAACTGCAGAGCCAGTCAGAGTGTTAGC II-KV6 AACTATGTAGCCTGGTACCAGCAGAAACCTGGGCAGGCTCCCAGGCTCCTCATCTATGCTGCATCCAGCAGGGCCACGGGCATACCAGAC CGGTTCAGCGGCAGTGGGTCTGGGACAGACTTCACCTTCAGCATCAGCAACCTCCAGGCCGAAGATGTGGGGGACTATTACTGT GACATCATGATGACTCAGTCTCCAGGCTCCCTGGCTGTGTCTCCAGGTCAGCGGGTCACCCTGAACTGCAGAGCCAGTCAGAGTGTTAGC II-KV7 AACTATGTAGCCTGGCACCAGCAGAAACCTGGGCAGGCTCCCAGGCTCCTCATCTATCGTGCATCCAACAGGGCCACGGACATCCCAGGC CGTTTCAGCGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAACCTCCAGGCCGAAGATGTGGGAAACTATTACTGC

219 GACATCACGATGACTCAGTCTCCAGGCTCCCTGGCTGTGTCTCCAGGTCAGCGGGTCACCATGAACTGCAGAGCCAGTCAGAGTGTTAGC II-KV8 AAATATGTAGCCTGGTACCAGCAGAAACCTGGGCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAGCAGGGCCACGGGCATCCCAGAC CGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAACCTCCAGGCCGAAGATGTGGGAAGCTATTATTGC GACATCACGATGACTCAGTCTCCAGGCTCATTGGCTGTGTCTCCAGGTCAGCGGGTCACCATGAACTGCAGAGCCAGTCAGAGTGTTAACA II-KV9 ACTATGTAGCCTGGTACCAACAGAAACCTGGGCAGGCTCCCAGGCGCCTCATCTATGCTGCATCCAGCAGGGACACGGGCATCCCAGACC GGTTCAGCGGCAGTGGGTCTGGGACAGACTACACCCTCACCATCAGCAACATCCAGGCTGAAGATGTGGGAAGCTACTACTGC GACATCACGATGACTCAGTCTCCAGGCTCGCTGGCTGTGTCTCCAGGTCAGCGGGTCACCATGAACTGCAGAGCCAGTCAGGGTGTTTAT II-KV10 AGCAATGTAGCCTGGTACCAGCAGAAACCTGGGCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAGCAGGGACACGGGCATCCCAGAC CGGTTCAGTGGCAGTGGGTCTGGGACAGACTACACCCTCACCATCAGCAACCTCCAGGCTGAAGATGTGGGAAACTATTACTGC GACGTCACGATGACTCAGTCTCCAGGCTCACTGGCTGTGTCTCCAGGTCAGCGGGTCACCATGAACTGCAGAGCCAGTCAGGGTGTTTAC II-KV11 AACAATGTAGCATGGTACCAGCAGAAACCTGGGCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAGCAGGGCCACGGGCATCCCAGAC CGGTTCAGCGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAACCTCCAGGCTGAAGATGTGGGGGACTATTACTGC GACATCACGATGACTCAGTCTCCAGGCTCTCTGGCTGTGTCTCTAGGTCAACGGGTCACCATGAATTGCAGAGCCAGTCAGAATATTTACA II-KV12 GCAACGTAAACTGGTACCAGCAGAAACCTGGGCAGGCTCCCAGGCTCCTCATCTATGCTGCATCCAGCAGGGCCACGGGCATCCCAGGC CGGTTCAGCGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAACCTCCAGGCCGAAGATGTGGGGGACTATTACTGC CACACGGAGCTGACCCAGTCTCCAGCCTTCCTCTCCGTGTCCCTGGGAGACAGCGTGTCCATCACCTGCAGGGTCAACGAGAGCGTCAGC II-KV13 GATTACTTGAGCTGGTATCAGCAGAAACCAGGCCAGGCTCCTAAGCATCTCATCTATGATGCTGATAACCTCAAGTCTGGTGTCCCAGCCC GGTTCTCTGCGATTCAGTCGGACAAAGAATTCACCCTCAAAATCAGCGCAGTAGAGGCTGAGGATGCTGCCAGTTATTACTGC CACACGGGGCTGACCCAGTCTCCGGCCTTCCTCTCGGTGTCCCTCAGAGAGGGAGTGTCCATCACCTGCAGGGCTAAAGAGAGTGTCAGC II-KV14 GATTACTTGAGCTGGTATCAGCAGAAACCAGGCCAGGATCCTCAGCTTCTCATCTATGATGCTGATAACCTCAAGTCTGGAGTCCCAGACC GGTTCTCTGTGATTCAGTTGGACAGAGAATTCATCCTCAATATCAGCTCAGTAGAGGCTGAGGATGCTGCCAGTTATTACTGC CACGTGGGGCTGACCCAGTCTCCGGCCTTCCTCCAGGTGTCCCTGAGAGAGGGCGTGTCCATCACCTGCAGGGCCAACGAGAGCGTCAG II-KV15 CGATTACTTGAGCTGGTATCAGCAGAAACCAGGCCAGGCTCCTCAGCTTCTCATCTATGATGCTGATAACCTCAAGTCTGGAGTCCCAGAC CGGTTCTCTGCGATTCAATCGGACAAAGAATTCATCCTCAAGATCAGCGCAGTAGAGGCTGAGGATGCTGCCAGTTATTACTGC GATATTGTGCTGACACAGACCCCACGGTCCCTGTCCCTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCCTG CACAGAGATGGAAACACCTATTTGTATTGGTATCTGCAGAAACCAGGCCAGTCCCCACAGCTCCTGATCTATGAGGTTTCCATCCATAAATC II-KV16 TGGGGTCCCAGACAAGTTCAGTGGCAGCAGGTCAGGGACAGATTTCACCCTGAAACTCAGCAGGGTGGAGGATGGCGATGTGGGAGTTTA TTACTGC GATATCATGCTGACACAGACCCCACTGTCCCTGTCCATCACCCCCAGAGAGCTGGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCCTG CACAGAGATGGAAACACCTATTTGTATTGGTACCTGCAGAAACCAGGCCAGTCCCCACAGCTCCTGATCTATGAGGTTTCCATCCATAAATC II-KV17 TGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCACCCTGAGAATCAGCAAGGTGGAGGATGACGATGTGGGAGTTTA TTACTGC

220 GATATTGTGATGACACAGACCCCACTCTCACTGTCGGTTACTCCTGGAAAGCCGGTTTCCATCTCCTGCAGGGCCAGTCAGAGCCTCCTGC ACAGTAATGGAATGAACTATTTGTATTGGTACCTGCAGAAGCCAGGCCAGTCTCCACAGAGCCTGATCTACTTGGCCTCCAGCCGTTACCC II-KV18 TGGGGTTCCAGACAGGTTCAGTGGCAGGGGGTCAGGGACAGATTTCACCCTGACTATCAGCAGTGTGGAGGCTGAGGATGTGGGAGTTTA TTACTGC GAAATTGTATTGACACAGACCCCACTGTCCCTGTCTGTCAGTTCTAGAGAGCCGGCTTCCATCTCCTGCCGGGCCAGTCAGAGCCTCCTGC ACAGTAATGGAAACAATTATTTGCATTGGTACCTGCAGAAGCCAGGCCAGTCTCCACAGCTTCTGATCTTCTTTGCCACTAACCGTTTCAGT II-KV19 GGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACCCTGAGGATCAGCAGGGTGGAGGCTGACGATGTGGGGGTTTA TTACTGT GAAGTTGTATTGACACAGACCCCACTGTCCCTGTCCGTCACTCTGGGAGAGCCGGCTTCCATCTCTTGCCGGGCCAGTCAGAGCCTCCTG CACAGTACTAGATACAATTTTTTGCATTGGTACCTGCAGAAGCCAGGCCAGTCTCCACAGCTTCTGATCTACTTGGCCACTAACCGTTTCAC II-KV20 TGGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACCCTGAGGATCAGCAGGGTGGAGGCTGACGATGTGGGGGTCT ATTACTGT GAAGTTGTATTGACACAGAACCCACTGTCCCTGTCCATCACTCTGGGAGAGCCGGCTTCCATCTCCTGCCGGGCCAGTCAGAGCCTCCTG CACAGTAATAGATACAATTACTTGCATTGGTACCTGCAGAAGCCAGGCCAGTCTCCACAGCTTCTGATCTACTTGGCCACTAACCGTTTCAG II-KV21 TGGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACCCTGAGGATCAGCAGGGTGGAGGCTGACGATGTGGGGGTTT ATTACTGT GAAATCGTGATGACACAGACCCCGCTGTCACTGTCCGTCACCCCCGGAGAGCCGGCCTCCATCTCCTGCAGGGCTAGTCAGAGCCTCCTA CATAGTGATGGAAACACCTATTTAAATTGGTACCGGCAGAAGCCAGGCCAGTCCCCACAGCTCCTCATCTACTTGGTTTCCAACCGTTTCAC II-KV22 TGGAGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACGTTGAGGATCAGCAGGGTGGAGGCTGACGATATGGGAGTTTA TTACTGC GAAATCATGATGACACAGACCCCACTGTCCCTGTCTGTCACCCCCGGAGAGCCGGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCGTA CACAGTAATGGAAACACCTATTTGAGTTGGTTCCGGCAGAAGCCAGGCCAGTCCCCACAGGGCCTGATCTACAAGGTTTCCAACCGTTTCA II-KV23 CTGGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACGCTGAGAATCAGCAGGGTGGAGGCTGACGATGCGGGAGTT TATTACTGC AAAATCATGATGACACAGACCCCACTGTCCCTGCCCATCACCCCTGGAGAGCCAGCCTCCATCTCCTGCCAGGCCAGTCAGAGCCTCGTA CACAATAATGAAAACACCTATTTGCATTGGTACCTGCAGAAGCCAGGCCAGTCCCCACAGGGCCTGATCTACAGGGTTTCCAACCATTTCAC II-KV24 TTGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACCCTGAGAATCAGCACGGTGGAGGGTGACGGTGTTGGGGTTT ATTACTGC GAAATTGTGATGACACAGACCCCACTGTCCCTGCCCATCAGCCCTGGAGAGCCGGCCTCCATCTCCTGCAAGGCCAGTCAGAGCCTCCTG TACAGTAATGGAAAAACCTATTTGCATTGGTACCTGCAGAAGCCAGGCCAGTCCCCACAGCTCCTGATCTATGAGGTTTCCAACCATTTCTC II-KV25 TGGGGTCTCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCAACCTGAGAATCAGCAGGGTGGAGGCTGACGATATGGGAGTTTA TTACTGT GAAATCGTGATGACACAGACCCCACTGTCCCTGCCCATCACCCCTGGAGAGTCAGCTTCCATCTCCTGCAGGGCCAGTCAGAGCCTCCTG CATAGTAATGGAAACACCTATTTGTATTGGTTCCGGCAGAAGCCTGGCCAGTCCCCACAGGATCTGATCTATAAGGTTTCGAACCAGTATTC II-KV26 CTGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTTACCCTGAGAATCAGCAGGGTGGAGGCTGACGATGTGGGAGTTTA TTACTGT

221 GAAATCGTGATGGCACAGACCCCACTCTCACTGCCCATCACACCTGGAGAGCCGTCCTCCATCTCCTGCAGGGCCAGTCGGAGCCTCCTG CACAGTGATGGAAATACCTGTTTGTATTGGTACCTGCAGAAGCCAGGCCAGTCTCCACAAGGCCTCATCTACTTGGTTTCCAACCGTTACTC II-KV27 CTGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACCCTGACCATCAGCATGGCGGAGGCTGATGATGTGGGGGTCT ATTACTGC GAAATTGTGATGACACAGACCCCACTGTCCCTGCCCATCAGCCCTGGAGAGCCGGCCTCCATCTCCTGCAAGGCCAATCAGAACCTCCTG CACAGTGATGGAAAAACCTATTTGACTTGGTTCCGGCAGAAGCCAGGCCAGTCTCCACAGGGCCTGATCTACTTGCTTTCCAAACGTTTCTC II-KV28 TGGGGTCTCAGACAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCACTTTGACCATCAGCCGGGTGGAAGCTGATGATGTGGGAATTTA TTACTGC GAGGTCGTGCTGACACAGACCCCGCTCTTGCTGTCCGTCACCCCTGGAGAACCGGCCTCCATCTCCTGCAAGGCCAGTCAGAGCCTTGTA CACAGTAATGGAAATACCTATTTGGATTGGTACCTGCAGAAACCAGGCCAGTCCCCACAGCTCCTCATCTATGAGGTTTCCAACCGTTACAC II-KV29 TGGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACCCTGAGAATCAGCAGGGTGGAGGCTGATGATGTGGGAGTTT ATTACTGC GAGGTTGTGATTACACAGACCCCACACTTGCTGCCCGTCATCCCCGGAGAGTCAGCCTCCATCTCTTGCAGGGCCAGTCAGAGCCTCGTA CACAGTACTGGAAACACCTATTTGTATTGGTACCTGCAGAGGCCAGGCCATTCCCCACAGGGCCTGATCTATAAGGTTTCCAACCGTTTCAC II-KV30 TGGGGTCCCTGACAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCTCCCTGAGAATCAGCAGGGTGGAGGCTGACGATGTGGGAGTTTA TTACTGC GAGGTTGTGATGACACAGACCCCACTCTTCCTGCCCGTCACCCCCGGAGAACCAGCCTCCATTTCCTGCAGGGCCAGTCAGAGCCTACTA CACAGTGATGGAAACACTTATTTGTATTGGTACCTGCAAAAGCCAGGCCAGTCTCCACAGCTTCTGATCTATGGGGTTTCCTACCGTTTCCT II-KV31 TCGGGTTCCGGGCAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCACCCTGAGAATCAGCAGGGTGGAGGCTGACGATATGGGAGTTTA TTACTGC GAGGTTGTGATGACACAGACCCCACTCTCACTGACTGTCACACCCAGAGAGCCGGCCTCCATCTCCTACAGGGCCAGTCAGAGCCTTGTA II-KV32 CACAGTGATGGAAACACCTATTTGCATTGGTACCTGCAGAAGCCAGGCCAGTCCCCACAGCTCCTGATCTATAGTCATTTCACTGGGGTCC CAGACAGGTTCAGTGGTAGCGGGTCAGGGACAGATTTTACCCTGACAATCAACAGGGTGGAGGCTGATGATGTGGGAGTGTATTACTGT GAGGTTGTGATGACACAGACCCCACTCTCACTGACTGTCACACCCGGAGAGCCAGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTTGTA II-KV33 CACAGTGATGGAAACACCTATTTGCATTGGTACCTGCAGAAGCCAGGCCAGTCCCCACAGCTCCTGATCTACAGCCATTTCACTGGGGTCC CAGACAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCACCCTGACAATCAACAGGGTGGAGGCTGATGATGTGGGAGTTTATTACTGT GAAGCTGTGCTGACACAATCCCCACTCACCCTGTCTGTCTCCCCAGGAGAGTCGGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTTGTA CACAGTAATGGAAACACCTATCTGCATTGGTACCTGCAGAAGCCAGGCCAGTCCCCACAGGGCCTGATCTACAGGGTTTCCAACCATTACA II-KV34 CTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGAGACAGATTTCACCCTGAGAATCAGCAGAGTGGAGGCTAATGATGTGGGAGTTT ATTACTGC GAGGCCGTGCTGACACAGACCACCCCACTCACCCTGTCTGTCACCTCCGGAGAGCCGGCCTCCAACTCCTGCAGGGCCAGTCAGAGCCT TGTACACAGTAATGGAAACACCTCTTTAAATTGGTACCTGCAGAAGCCAGGCCAGTCACCACAGATCCTGATCTACTTGGTTTCCAGCCGTG II-KV35 TCTCTGGAATCCCAGACAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCACCCTGACAATCAGCAGGGTGGAGGCTGGCGATGTGGGA GTTTATTACTGT

222 GATATAGTGCTGACACAGACCCCACTGTCTCTGTCCATCACCCCCGGAGAGTCAGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTTGTAC ACAGTAATGGAAACACCCATTTGAGTTGGTACCTGCAGAAACCAGGCCAGTCCCCACAGCTCCTGATCTACAAGATTTCCAACCATTTCACT II-KV36 GGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACCCTGACAGTCAGCAGGGTGGAGGCTGATGATGTGGGAGTTTA TTACTGC GAGGTCGTGCTGACACAGACCCCACTCTCACTGTCCATCACCCCCGGAGAGTCAGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTTGTA CACAGTAATGGAAACACCTATTTGAGTTGGTACCTGCAGAAGCCAGGCCAGTCGCCACAGCTCCTCATCTATAAGGTTTCCAACCGTTTCAC II-KV37 TGGAGTCCCAGACAGGTTCACTGGCAGCGGGTCAGGGACAGATTTCACCCTGAGAATCAGCAGGGTGGAGGTTGATGACGTGGGAATTTA TTACTGT AAGGTTGTACTAACTCAGACCCCACTCTCACTGTCCGTCACCCCCGGAGAGTCGGGCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCGTA II- CACAGTGATGGAAACACATATTTGAGTTGGTACCTGCAAAAGCCAGACCAGTCACCACAGCTCCTCATCTATATGGTTTCCAACCGTTTCAC KV38/39 TGGGCTCCCAGAAAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACCCTGACAATCAGCAGGGTAGAAGCTGATGATGTGGGGGTTTA TTACTGT GAGGTCGTGCTGACACAGACCCCACTCTCACTGTCAGTCACGCCCGGAGAGCCAGTCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCGTA CACAGTAATGGAAACACCTATTTGCACTGGTACCTGCAGAAGCCAGGCCAGTCCCCACAGCTCCTGATCTACAAGGTTTCCAACCGTTTCA II-KV40 CTGGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACCGATTTCACCCTGAGAATCAGCAGGGTGGAGGCTGACGATGTGGGAGTT TATTACTGT GAGGTCGTGCTGACACAGACCCCACTCTCACTGTCAGTCACCCCCGGAGAGTCAGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCGTA CACAGTGATGGAAACACGTATTTAAATTGGTACCTGCAGAAGCCAGGCCAGTCCCCACAGCTCCTGATCTACAGGGTGTCCAACCGTTTCA II-KV41 CTGGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACCCTGAGAATCAGCAGGGTGGAGGCTGATGATGTGGGAGTT TATTACTGT GAGGTCGTGCTGACACAGACTCCACTCTCGCTGTCCGTCACCCCCGGAGAGCCAGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCGTA CACAGTAATGGAAACACATATTTGAGCTGGTTCCGGCAGAAGCCAGGCCAGTCCCCACAGGGCCTGATCTACAAGGTTTCCAACCGTTTCA II-KV42 CTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCACCCTGAGAATCAGCAGAGTGGAGGCTGATGATGTGGGAGTTT ATTACTGC GAGGTCGTGCTGACTCAGACCCCACTCTTGCTGTCCGTCACTCCTGGAGAGCCAGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCGTA CACAGTAATGGAAACACCTATTTGCATTGGTACCTGCAGAAGCCAGGCCAGTCCCCACAGCTCCTGATCTACAAGGTTTCCAACCGTTACA II-KV43 CTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCACCCTGAGAATCAGCAGGGTGGAGGCTGATGATGTGGGAGTTT ATTACTGT GAGGTCGTGCTGACTCAAACCCCACTCTTGCTGCCCGTCACCCCCGGAGAGCCAGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCATA CACAGTGATGGAAACACCTATTTGCATTGGTACCTACAGAAGCCAGGCCAGTCCCCACAGGGCCTGATCTACAAGGTTTCCAACCGTTTCA II-KV44 CTGGGGTCCCAGACAGGTTCAGTGGCAGCGGATCAGGGACAGATTTCACCCTGAGAATCAGCAGGGTGGAGGCTGATGATGTAGGAGTTT ATTACTGT GAGGTCGTGCTGACACAGACCCCACTCTCACTGTCAGTCACCCCCGGGGAGCCAGTCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCGTA II- CACAGTAATGGAAACACGTACGTGAGTTGGTTCCGGCAAAAGCCAGGCCAGTCCCCACAGCTCCTGATCTACAAGGTTTCCAACCGTTTCA KV45a CTGGGGTCCCAGACAGGTTCAGTGGCAGCGGATCAGGAACAGATTTCACCCTGAGAATCAGCAGGGTGGGGGCTGATGATGTGGGAGTTT ATTACTGT

223 GAGGTCGTGCTGACACAGACCCCACTCTCACTGTCAGTCACCCCCGGGGAGCCAGTCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCGTA II- CACAGTAATGGAAACACGTACGTGAGTTGGTTCCGGCAAAAGCCAGGCCAGTCCCCACAGCTCCTGATCTACAAGGTTTCCAACCGTTTCA KV45b CTGGGGTCCCAGACAGGTTCAGTGGCAGCGGATCAGGAACAGATTTCACCCTGAGAATCAGCAGGGTGGAGGCTGATGATGTGGGAGTTT ATTACTGT GAGGTCGTGCTGACACAGACCCCACTCTCACTGTCAGTCACCCCCGGGGAGCCAGTCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCGTA CACAGTAATGGAAACACGTACTTGAGTTGGTTCCGGCAAAAGCCAGGCCAGTCCCCACAGCTCCTGATCTACAAGGTTTCCAACCGTTTCA II-KV45c CTGGGGTCCCAGACAGGTTCAGTGGCAGCGGATCAGGAACAGATTTCACCCTGAGAATCAGCAGGGTGGAGGCTGATGATGTGGGAGTTT ATTACTGT GAGGTCGTGCTGACACAGACTCCACTCTCACTGTCAGTCACCCCTGGAGAGCCGGTCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCGTA CACAGTAATGGAAACACCTATTTGCACTGGTACCTGCAGAAGCCAGGCCAGTCCCCACAGGGCCTGATCTACAAGGTTTCCAACCGTTTCA II-KV46 CTGGGGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACCGATTTCACCCTGAGAATCAGCAGGGTGGAGGCTGACGATATGGGAGTT TATTTCTGC GAAATCGTGATGACACAGACCCCGCTGTCACTGTCCGTCACCCCCGGAGAGCCGGCCTCCATCTCCTGCAGGGCTAGTCAGAGCCTCCTA CATAGTAATGGAAACACCTATTTAAATTGGTACCGGCAGAAGCCAGGCCAGTCCCCACAGCTCCTCATCTACTTGGTTTCCAACCGTTTCAC II-KV47 TGGAGTCCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACGGATTTCACGTTGAGGATCAGCAGGGTGGAGGCTGACGATATGGGAGTTT ATTACTGC GAGGTCGTGCTGACACAGACTCCACTCTCACTGTCAGTCACCCCCGGAGAGCCGGCCTCCATCTCCTGCAGGGCCAGTCAGAGCCTCGTA CACAGTAATGGAAACACGTACTTGAGTTGGTACCTGCAGAAGCCAGGCCAGTCCCCACAGGGCCTAATCTACAAGGTTTCCAACCGTTTCA II-KV48 CTGGGGTCCCAGACAGGTTCAGTGGCAGCGGATCAGGGACAGATTTCACCCTGAGAATCAGCAGGGTGGAGGCTGACGATGTGGGAGTT TATTACTGT GAGATTGTCCTGACCCAGTCTCCTGCCGTCCTGTCCATGGCTCCAAAAGAAAGAGTCACCATCACCTGCCAGGCCAGTCAGAACATTAACA I-KV1 AGTGGTTAGCCTGGTATCATCAGGAGCCAGGGAGAGCTCCTAAGCTCCTCATCTATGAGGCATCCAAGTTGATAACTGGGGTCCCATCACG GTTCAGCGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTGGAGCCTGAAGATGCTGGGACCTACTATTGC GAAAATGTGCTGACCCAGTCTTCTGCCATCCTATCCATGGTTCCAAAAGAAAGAGTCAGCATCACCTGCAGGGCCAGTCAGAACATTAACAA I-KV2 GTGGTTAGCCTGGTATCAGCAGGAGCCAGGGAGAGCTCCTAAACTCTTGATCTGTGAAACATCCAAGTTGGAAACTGGGGTCACGTCGCG GTTCAGTGGCAGTGAGTCTGGAACAGACATCACTCTCATCAGCAGCAGCCTGGAGCCTGAAGATGCTGGGACCTACTACTGC

224

Appendix 3.5 Ferret germline lambda variable genes

II- TCCAGTGAGCTGACACAGCTGCCCTCAGTGTCAGTGGCCCTGGGTCAGACGGCTACAATCACCTGCACTGGAGAGGAACTGGACTATA LV1_(GL CTGTGCAGTGGTACCAGAAGAAGCCAGGCCAAGCCCCTTTGACAATCATTTATGATGAGAGCGAGAGGCCCTCAGGGATCCCAGAGC 897406.1) GATTCTCTGGCTCCAGATCGGGGAACACAGCCACCCTGACCATCAGTGGGGTCCAGGCTGAGGATGAGGCTGACTATTACTGT II- TCCAGTGAACTGACTCAGCCTCCCTCAGTGTCAGTTGCCCTGGGTCAGACGGCTACAGTCACCTGCACTGGAGGGGAGTTAGAGTATT LV2_(AE TTTATGTACACTGGTACCAGCAGAAGCCAGGCCAAGCCCCTTTGACAATCATTTATGGTGATAAGGAGAGGCCCTCAGGGATCCCAGA YP01111 GCGATTCTCTGGCTCCAAGTCGGGGAGCACGGCTACCCTGACCATCAGCAGCGCACAGGCTGAGGACGAGGCTGACTATTACTGT 698.1) II- ACCAGTGAACTGACTCAGCCTCCCTCAGTGTCAGCTGCCCTGGGTCAGACCGCTACAATCACCTGCACTGGAGAGGAAGTAGAATATA LV3_(GL TTTATGTCTACTGGTACCATCAGAAGCCAGGCCAAGCCCCTCTGACGGTCACTTACGATGATAGTGATCAGCCTTCAGGGATCCCAGAG 896906.1) AAATTCTCTGGCTCCAAGTCAGGGAACACGGCCACCCTGACCATCAGCAGCGCACAGGCCAAGGACGAGGCTGACTATTACTGT II- TCCAGTGAGCTGACTCAGACGCCCTTTGTGTCAGTGGCCCCGGGACAGACCGCTACAATCACCTGCTCTGGAGAAGAACTGGACTATG LV4_(AE GTTATGTACAATGGTACCAACAGAAGCCAGGGCAAGCCCCTCTGATGGTAATTTATTTGGATAATGAGCGGGCCTCTGGGATCCCAGA YP01112 GCGATTCTCTGGCTCCAAGTCAGGGAACACGGCCACCCTCACTATCAGCAGCGTACAGGCCGAGGACGAGGCTGACTATTTCTGT 098.1) II- TCCAGTGAGGTGACTCAGCCACCCTTTGTGTCAGTGGCCCTGGGACAGACCGCTACAATCACCTGCACCGGAGAGAAGGTGAAATATA LV5_(GL GCTCTATACACTGGTACCAGCAGAAGCCAGGGCAAGCCCCTCTGACCGTCATTTATGCAGAGATTGAGCGGCCCTCTGGGATCCCAGA 896906.1) GCGATTCTCTGGCTCCAGGTCAGGGAACACGGCCACCCTCACCATCAGCAGTGTACAGGCCGAGGACGAGGCTGACTATTACTGT II- TCCAGTGTGCTGACTCAGTCTCCCTCAGTATCAGTGTCCCTGGGACAGACAGCTACCATCACCTGCTCTGGAGAGATACTGAGCAAAA LV6_(GL GTTATGCACGATGGCACCAGCAGAAGCCAGGCCAATCCCCTGTGAACATTATTTATAAGGACACCCAGCGTCCCTCTGGGATCCCTGA 897285.1) CCGATTCTCTGGGTCCAGCTCAGGGAGCACACACACCCTGACCATCAGCAGGGCTCAGGCTGAGGACGAGGCTGACTACTACTGT II- TCCAGTGTGCTGACTCAGTCTCCCTCGGTGTCTGTGTCCCTGGGACAAACGGCTACAATCACCTGCTCTGGAGAAGTACTGAACAAAAA LV7_(GL ATACGCACAGTGGTTCCAGCAGAAGCCAGGCCAAGCACCCATGCTGGTCATTTATAAGGACACTGAGCGTCCTTCTGGGATCCCTGAC 897285.1) CGATTCTCTGGCTCCAGTTCAGGGACCACACACACCCTGACCATTAGTGGGACCCGGGCTGAGGACGAGGCTGACTATTACTGT II- TCCAGTGTGTTGACTCAGCCTCATTCAGTGTCAGTGTCCCTGGGACAGATGGCTACCATCAGCTGCTCTGGAGAGATATTGTCAGGAAG LV8_(GL TTATGCATATTGGTACCAGAAGAAGCCAGGCCAACCCCCTGTGCAGGTCATTTATAAGGACACTGAGCGTCCTTCTGGGATCCCGGAC 897406.1) CGATTCTCTGGCTCCAGTTCTGGCACCACACACACCCTGACCATCAACGGGGCCCAGGCCGAGGATGAGGCTGACTATTACTGC II- TTCAGTGTGTTGACTCAGCCTCATTCGGTGTCAGTGTCCCTGGGACAGACAGCTACCATCATCTGCTCTGGGGAAGTACTGAGCAAATA LV9_(GL CTATGCACAATGGTACCAGCAGAAGGCAGGTCAAGCCCCCAAGCAGGTCATTTATAAGGACACTGAGTGTCCCTCTGGGATCCCTGAC 897344.1) CAATTCTCTGGTTCCAGTTCAGGGACCACACACACCCTGACCATCAACGGAGCCCAGGCCGAGGATGAGGCTGACTATTACTGT

225 II- TCCAGTGTGTTGACTCAGCCTCATTTGGTGTCAGTGTCCCTGGGAGAGACAGCTACCATCACCTGCTCTGGGGAAGTACTGAGCAAGT LV10_(G ACTATGCACAATGGTACCAGCAGAAGGCAGGTCAAGCCCCCAAGCAGGTCATTTATAAGGACACTGAGCGTCCTTCTGGGATCCCTGA L897406. CCGATTCTCTGGTTCCAGTTCAGGGACCACACACACCCTGACCATCAACAGGGTCCAGGCTGAGGATGAGGCTGACTATTACTGT 1) II- GCCTATGAGCTGACTCAGCCTCCCTTAATGTCGGTGAACCTGGGACGGACGGCCAGCATCACCTGCAGTAGAGACAACATTGGAAATA LV11_(G CATATGTTTCCTGGTACCAACAGAAGCTGGGCAAGGCACCCATGATGATTATCTATAGTGGTAGCAACCGGCCCTCAGGGATCCCAGA L897565. CCGGTTCTCTGGCTCCAATTCAGGGAACACGGCCACCCTGACCATCAGCGGGGCTCAGGCAGAGGACGAGGCTGACTATTACTGTTT 1) II- GCCTACGTGCTGACTCAGCCTCCCTCAATGTCGGTGAACCTGGGACGGACGGCCAGCATCACCTGCAGTGGAGACAACATTGGAAGT LV12_(G ACATATGTTTCCTGGAACCAACAGAAGCCGGGCCAGGCACCAGTGACAATTATCTATAGTGATAGCAGCCGGCCCTCAGGAATCCCTG L897406. ACAGGTTCTCTGGCTCCAACTCGGGGAACATGGCCACCCTGACCATCAGCGGGGCCCGGGCTGAGGATGAGGCTGACTATTACTGT 1) II- GCCTACGTGCTGACTCAGCCTCCCTCAATGTCGGTGAACCTGGGACAGATGGCCAGCATCACCTGCAGTGGAGACAACATTGGAGGAA LV13_(G TATATGTTTCCTGGCAACAACTGAAGTTGGGACAGACACCCATGACAATTATATATAGTGATAGCAACCAGCCCTCAGGAATCCCTGACA L897418. GGTTCTCTGGCTCCAACTCAGGGAACATGGCCACCCTGACCATCAGCGGGGCCCAGGCTGAAGATGAGGCTATTACTGACTATTACTG 1) T II- TCCTATGTGCTGACACAGCCCCCATCTGTGTCGGTGGCCCTGGGGCAGACGGCCCAGGTCACATGTACGGGAAACAACATTGGAAGT LV14_(G AAACATGTTTACTGGTACCAGCAGCAGCCAGGCCATATCCCTGTGCTGATCATCTATAATAGCAACAAACGGCCCTCAGGGACTCCTGA L897418. GCGATTTTCAGGCACCGGCTCTGGGAACACGGCCACCCTGACCATCAGTGGGGCCCAGGCTGAGGACGAGGCTGACTATTACTGT 1) II- TCCTATGTGCTGACTCAGCCCCCATCTGTGTCAGTGGCTCTGGGAAAGACAGCCCAGGTCACCTGTGGGGGAAACAACATTGGAAATA LV15_(G AATATGTTCACTGGTACCAGCAGAAGGCAGGCCAGGCTCCTGTGCTGATCATCTATGAAAGCAACAAACGGCCCTCAGGGATTCCTGA L897285. GCGATTTTCAGGCACCAACTCTGGGAACACGGCCACCCTGACTATCAGTGGGGCCCGGGCTGAGGACGAGGCTGACTATTACTGT 1) II- TCCTATGTGCTGACTCAGCCTCCATCTGTGTCAGTGGCTCTGGGACAGACAGCCCAGGTTACCTGCGGGGGAAACAACATTGGAAATA LV16_(G AATTTGTTCACTGGTACCAGCAGAAGCCGGGCCAGGCTCCTGTGCTGATCATCTATAACAGCAACAAACGGCCCTCAGGGATTCCTGA L897418. GCGATTTTCAGGCACCAAATCTGGGAACACGGCCACCCTGACCATCAGTGGGTCCCGGGCTGAGGATGAGGCTGACTATTACTGT 1) II- GCTTATGAGCTGACTCAGGACTCCTCGGTGTCAGTGAACCTGGGACAGACGGCCAGGATCACGTGTCGGGGAGACAACATTGGTAGT LV17_(G AAATATGCTTACTGGTACCAGCAGAAGCCAAGCCAGGCCCCTCTGCTAATTATCTATGGTGATAGCAACCGGCCCTCAGGGATCCCTGA L896906. GCGATTCTCAGGAACGAACTCGGGGAACACGGCCACCCTGACCATCAGCGGTGCCCAGGCTGAGGATGAGGCTGACTATTACTGT 1) II- TCCTATGTGCTGACTCAGCCTCCCTCGATGTCAGTGAACCTGGGACAGACGGTCAGGATGACTTGTGGGGGAAACAACATTGGAAGAA LV18_(A AAAGTGTTCCGTGGTACCAGCAGAAGCCAGGTCTGGCCCCTGTGATGATTATCTACGGTGATAGCAGCCGGCCCTCAGGGATCCCTGA EYP0111 CCGGTTCTCAGGCACCAACTCGGGGAACACAGCCACCCTGACCATCAGCGGGGTCCGGGCTGAGGACGAGGCTGACTATTACTGT 0728.1)

226 II- TCATATGTGCTGACTCAACCACCCTCAATATCGGTGAACCTGGGACAGACGGCCAAGATCACATGTGAGGGAAACAACATTGGAAGAAA LV19_(G AAGTATTTACTGGTACCAGCAGAAGCTGGACCAGTCCCCCGTATTGATTATCTATAGGAATAGCAACCGGCCCTCAGGGATCCCTGACC L897406. GGTTCTCAGGCACCAACTCGGGAAACATGGCCACCCTGACCATCAGCGGAGCCTGTGCTGAGGACGAGGCTGACTATTACTGT 1) II- TCGTATGTGCTGACTCAACCACCCTCAATATCGGTGAAACTGGGACAGACGGCCAAGATCACGTGTGGGGGAGACAACATTGGGAATA LV20_(G AATATGCTTACTGGTACCAGCAGAAGCCAGGCAGGGTCCCTGTGCTGATTATCTACGAGGATAGCAAAAGGCCGTCAGGGATTCCTGA L896906. CCAGTTCTCAGGCACCAACTCAGGGAATACAGCCACCCTGACCATCAGCGGGGCCCGTGCTGAGGACGAGGCTGACTATTACTGT 1) II- TCATATGTGCTGACTCAACCACCCTCACTATCGGTGAACCTGGGACAGACGGCCAAGATCACGTGTGGGGGAGACAACATTGGGTGTG LV21_(A AAAATGCTCACTGGTACCAGCAGAAGCCAGGTCAGGCACCTGTGCTGATTATCTATACTGATAAAAACCAGCCTTCAGGGATCCCTGAC EYP0110 CGGTTCTCAGGCTCCAACTCGGGGAATACAGCCACCCTGACCATCAGCAGGGCCCATGCTAAGGATGAATCTGACTATTACTGT 8526.1) TCATATGTGCTGACTCAACCACCCTCACTATCGGTGAACCTGGAACAGACGGCCAAGATCACATGTGGGGGAAACAACATTGGGGGTA II-LV22a AAAGTGTTCACTGGTACCAGCAGAAGCCAGCACAGGTCCCTGTGCTGATTATCTATAATGGTAAAAACCGGCCCTCAGGGATCCCTGAC CGGTTCTCAGGCACCAACTCGGGGAACACAGCCACCCTGACCATCAGCGGCACCCGCGCTGAGGACGAGGCTGACTATTACTGT TCATATGTGCTGACTCAACCACCCTCACTATCGGTGAACCTGGAACAGACGGCCAAGATCACATGTGGGGGAAACAACATTGGGGGTA II-LV22b AAAGTGTTCACTGGTACCAGCAGAAGCCAGCACAGGTCCCTGTGCTGATTATCTATAATGATAAAAACCGGCCCTCAGGGATCCCTGAC CGGTTCTCAGGCACCAACTCGGGGAACACAGCCACCCTGACCATCAGCGGCACCCGCGCTGAGGACGAGGCTGACTATTACTGT TCCAGTGAACTGACTCAGCCTCCCTCAGTGTCAGTTGCCCTGGGTCAGACGGCTACAGTCACCTGCACTGGAGGGGAATTAGAGTATT II-LV23 TTTATGTACACTGGTACCAGCAGAAGCCAGGCCAAGCCCCTTTGACAATCATTTATGGTGATAAGGAGAGGCCCTCAGGGATCCCAGA GCGATTCTCTGGCTCCAAGTCGGGGAGCACGGCCACCCTGACCATCAGCAGCGCACAGGCTGAGGACGAGGCTGACTATTACTGT TGGGCTGTGCTGACTCAGCCTCCCTATGTGTCTGGGGCCCTAGGTGAGAGTGTCACCATCTCCTGCACTGGAATCCCCACCAGCATAG I- ATTATGATGAAGAGGAATACACATATAATGTGAACTGGTACCAACAGCTCCAAGGAAAGGTACCCATTCTACTCATCTATGGAGATAATA LV1_(GL ACAGAAATCCTGGAGTCCCTGATCGATTCTCTGGTTCCAAGTCAGGCAGCTCAGCCTCCCTGACCATCAGTGGCCTTCAGGCTGAAGA 897285.1) TGAGGCTGATTATTACTGC CAGTCTATGCTGACTCAGCCACCCTCTGTGTCTGGGGCCCTGGGACAGAGGGTCACCATCTCCTGCACTGGAAGCAGCTCCAATATTG I- GGAGTGGTAATTATGTGTACTGGTACCAACAACTTCCAGGAATAACCCCCAAACTCATCATCTATGGTAGTAGCAACCGACCCTCTGGT LV2_(GL GTCCCAGATCGATTCTCTGGCTCCAGGTCTGGCAGCTCAGGCACCCTGACCATCACTGGGCTCCACACTGAGGATGAGGCTGATTATT 897638.1) ACTGC GCACCTGTGCTGACTCAGCCTCCATCTGCATCTGCCTCTCCAGGAGCCTCTGTCAAGCTCACCTGCACCCTGAGCAGGGAGCACAGCA IV- ATTACTATATTCACTGGTATCAACAGGAACCAGGGAAGGCCCCTCGGTATGTGATGATGGTTAATAGTGACGGAAGCCACAACAAGGG L1_(GL89 GGACGGAATCCCCAGTCGCTTCTCAGGATCCAGCTCTGGGGTTGACCGCTACTTAACCATCTCCGACATCCAGTCTGAGGATGAGGCT 7406.1) GAGTATTACTGT IV- CTGCCTGTGCTGACCCAGCTTTCATCTGCATCTGCCTCCCTGGGAGCCTCAGTCAAGCTCACCTGCACCCTGAGCAGTGAGCACAGCA L2_(GL89 ATTATTTGGTTCACTGGTATCAACAGCGACCAGGGAAGGCTCCCCTGTATATGATGACAGTTAATAGAGATGGAAGCCACAGCAATGGG 7418.1)

227 GATGGGATCCCCAACCGCTTCTCAGGCTCCAGCTCTGGAGTTGACCGCTATTTAGCCATCTCCAACATCCAGTCTGAAGATGAGGCTG AGTATTACTGT

CAGCTTTTGGTGACCCAGCCTCCCTTCCTCTCTGCATCTCTGGAAACAACAGCCAGACTCACCTGCACCCTGAGCAGTGACATCAGTGT V- TGATATTTACCCGATATTCTGGTACCAGCAGAAGCCAGGGAGCCCTCCTCGTTACCTCCTTACCTATGTAACAGACTCAAACAAGCACC LV1_(GL AGGGCTCTGGGGTCCCGAGCCGCTTCTCTGGATCCAAAGATGCCTCAGCCAGTGCAGGGATCCTGCTCATCTCTGGGCTCCAGGCAG 897285.1) AGGATGAGGCTGATTATTACTGT CAGCCAGTGCTGACTCAGCCACCATCCCTCTCTGCCTCTCTCGGAACAACAGCCAGACTCACCTGCACCCTGAGCAGTGGCTTCAGTG V- TCAGGAGTTATAACATATACTGGTACCAGCAGAAGCCAGGGAGCCCTCCCAGGTATCTCCTGTGGTTCTACTCAGACTCAGATAAGCAC LV2_(GL CAGGGCTCGGGGGTCCCCAGCCGCTTCTCTGGCTCCAAAGATGCCAATGCAGGGCTTCTACTCATCTCTGGGCTCCAGCCTGAGGAC 897484.1) GAGGCTGACTATTACTGT CAGCCTGTGCTGACCCAGCCGCCCTCACTCTCTGCATCTCCGGGAACAACAGCCAGACTCACCTGCACCTTGAGCAGGGATATCAGTG V- TTGGTAGTAAATACATGCACTGGTACCAGCAGAAGCCAGGGACCCCTCCCAGGTATCTCCTGTACTACTACTCAGACTCAAGTACACAG LV3_(GL CTGGGACCTGGGATTCCCAGTCGCTTCTCTGGCTCCAAGGATACCTCAGCCAATGCAGGGATTCTGCTCATCTCTGGGCTGCAGCCTG 897019.1) AGGACGAGGCTGACTATTACTGT CAGCCTGTGCTGACCCAGCCGCCCTCACTCTCTGCCTCTCTGGGAACAATAGCCAGACTCACCTGCACCTTGAGCAGGGACATCAGTG V- TTGGTAGTAAATACATGTACTGGTACCAGCAGAAGCCAGGGACCCCTCCCCGGGTTCTCCTGTACTACTACTCAGACTCAAATACACAG LV4_(GL CTGGGACCTGGGATTCCCAGTCGCTTCTCTGGCTCCAAGGACACTTCAGCCAATGCAGGGATTCTGCTCATCTCTGGGCTGCAGCCTG 897400.1) AGGACGAGGCTGACTATTACTGT CAGCCTGTGCTGACCCAGCCAACGTCCCTCTCTGCATCTGTAGGAGCAACAGCCAGACTCACCTGTACCTTGAGCAGGGACATAAGTG V- TCAGTGGGAAAAACATGTACTGGTATCAGCAGAAGCCTGGAAGCCCTCCCCGGTTTTTCCTGTACTACTACTCAGACTCAGACAAGCAG LV5_(GL CTGGGACCTGGGGTCCCCAGTCGAGTCTCTGGATCCAAAGATACCCCCACAAACACAGCAATTTTGTTCATCTCTGGGCTCCAGCCTG 897285.1) AGGACGAGGCTGATTATTACTGT CAGCCTGTGCTGACCCAACCTCCCTCCCTCTCTGCTTCCCTGGGATCATCAGCCAGACGCACCTGCACCCTGAGCAGGGACATCAATG V- TTGGTGGTAAAAACATGTTCTGGTACCAGCAGAAGCCCGGAAGCCCTCCCCGTTATTTCCTGTACTACTACTCAGACTCAGATAAGCAG LV6_(GL CTGGGACCTGGGGTCCCCAGTCGTGTCTCCGGATCCAAAGATACCTCCACCAATACGGCAATTATGCTCATCTCTGGGCTTCAGCCTG 897400.1) AGGATGAGGCTGACTATTACTGT

228

Chapter 4 Appendix

Appendix 4.1 Ferret Immunoglobulin heavy chain clonal families recovered from influenza infected ferrets

Clonal CDR1- CDR2- CDR3 FR1-IMGT FR2-IMGT FR3-IMGT CDR3-IMGT family IMGT IMGT length EVQLVESGGDLVKPGGSLRLS MSWVRQAPGKGLQW ISTDEGG SYADSVKGRFTVSRDNGKNTLYLQMNSLRAEDT GFIFSNYY ATPYVPFEY 9 CAAS VAY T AMYYC EVQLVESGGDLVKPGGSLRLS MSWVRQAPGKGLQW TYADSVKGRFTVSRDDDKNTLYLQMNSLRAEDT GFTFINYY ISNDGSIT ATPYVPFEY 9 CAAS VAY AMYYC EVQLVESGGDLVKPGGSLRLS GFTFNDY MSWVRQAPGKGLQW ISNDGDT YYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ATPYVPFEY 9 CAAS Y VAY T ALLGC EVQLVESGGDLVKPGGSLRLS GFTFSDY MSWVRQAPGKGLQW ILPGGDD NYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ARDVTYFEY 9 CAAS Y VAW F AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSDY MSWVRQAPGKGLQW ILPGGND NYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT 1 ARDVTYFDY 9 CAAS Y VAW F AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSDY MSWVRQAPGKGLQW ISNDGSS SYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ATPYVPFEY 9 CAAS Y VAY T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSDY MSWVRQAPGKGLQW ISNDGSS SYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ATPYVPFEY 9 CAAS Y VAY T AMYYC EVQLVESGGDLVKPGGSLRLS MSWVRQAPGKGLQW ISNDGSS SYADSVKGRFTISRDNGKNTLYLRMNSLRAEDT GITFNNYY ATPYVPFEY 9 CAAS VAY T AMYYC EVQLVESGGDLVKPGGSLRLS MSWVRQAPGKGLQW ISNDGSS SYADSVKGRFTISRDNGKNTLYLRMNSLRAEDT GITFNNYY ATPYVPFEY 9 CAAS VAY T AMYYC

EVRLVESGGDLVKPGASLRISC GSTLSTY MHWVRQAPGKGLQW TFTEGSG YYADSVKGRFTISRDNGKNTLYLQMNSLRTEDT ARDDWNSPRY 10 AGS G VAV T AVYYC 2 EVRLVESGGDLVKPGASLRISC GSTLSTY MHWVRQAPGKGLQW TFTEGSG YYADSVKGRFTISRDNGKNTLYLQMNSLRTEDT ARDDWNSPRY 10 AGS G VAV T AVYYC

EVQLVESGGDLVKPGGSLRLS GFTFSNY MYWVRQAPGKGLQC IDPDGSS YYADSVKGRFTISRDDGWNTLYLQMNSLRVEDT ATGGYDNRIEY 11 CAAS D VAW T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY IYWVRQAPGKGLQCV IDTDGST SYADSVRGRFTISRDNGKNTLYLQMNSLRVKDT AIGGYDNRIEY 11 CAAS D AW T AMYYC EVQLVESGGDLMKPGGSLRLS GFTFSNY MYWVRQAPGKGLQFV IDTGGST SYADSVKGRFTISRDNGKNTLYLQMNSLRVEDT AIGGYDNRIEY 11 CAAS D AW T AMYYC EVQLVESGGDLMKPGGSLRLS GFTFSNY MYWVRQAPGKGLQFV IDTGGST SYADSVKGRFTISRDNGKNTLYLQMNSLRVEDT 3 AIGGYDNRIEY 11 CAAS D AW T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY IYWVRQAPGKGLQFV IDTGGYT SYADSVKGRFTISRDIGNTTLYLQMNSLRVEDTA TIGGYDNRIEY 11 CAAS D AW T MYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY IYWVRQAPGKGLQFV IDTGGYT SYADSVKGRFTISRDIGNTTLYLQMNSLRVEDTA TIGGYDNRIEY 11 CAAS D AW T MYYC EVQLVESGGDLVKPGGSLRLS GFTFSRY MYWVRQAPGKGLQY IDTGGSS SYADSVKGRFTISRDNGKNTLYLQMNSLRVEDT AIGGYDNRIEY 11 CAAS D VAW T AMYYC

229 EVQLVESGGDLVKPGGSLRLS GFTFSRY MYWVRQAPGKGLQY IDTGGSS SYADSVKGRFTISRDNGKNTLYLQMNSLRVEDT AIGGYDNRIEY 11 CAAS D VAW T AMYYC EVQLVESGGDLVKPGGSLRLS MYWVRQAPGKGPQY IDTDGGT TYADSVKGRFTISRDNGKNTLYLQMNSLRVEDT KFTFSSYD AIGGYDNRIEY 11 CAAS VAL T AMYYC EVQLVESGGDLVKPGGSLRLS MYWVRQAPGKGPQY IDTDGGT TYADSVKGRFTISRDNGKNTLYLQMNSLRVEDT KFTFSSYD AIGGYDNRIEY 11 CAAS VAL T AMYYC

EVQLVESGGDLVKPGASLRLS IHWVRQAPGKGLQWV TSTDGS YYADSVKGRFTISRDNGKNTLYLQMNSLRTEDT GFTFSNYI ARDWYDNAFAY 11 CTAS AV GT AVYYC 4 EVQLVESGGDLVKPGASLRLS MFTFSSY MHWVRQAPGKGLQW ITTDGDG YYADSVKGRFTISRDNGKNTLYLQMNSLRPEDT ATDWYDNAFAY 11 CAAS G VAF T AVYYC

EVQLVESGGDLVKPGGSLRLS GFTFSSY MYWVRQAPGKGLQC INTGEMN NYADSVKGRFTISRDNGKNTLYLQMNSLRVEDT GTGGYDNRIEY 11 CAAS D VAW T AMYFC EVQLVESGGDLVKPGGSLRLS GFTFSSY MYWVRQAPGKGLQC INTGEMN NYADSVKGRFTISRDNGKNTLYLQMNSLRVEDT 5 GTGGYDNRIEY 11 CAAS D VAW T AIYYC EVQLVESGGDLVKPGGSLRLS GFTFSSY MYWVRQAPGKGLQC INTGEMN NYADSVKGRFTISRDNGKNTLYLQMNSLRVEDT GTGGYDNRIEY 11 CAAS D VAW T AIYYC

EVQLVESGGDLVKPGGSLRLS GFTFNNY MHWVRQAPGKGLQW IRYDGSD TYADSVKGRFTISRDNGKNTLYLQMNSLRTEDT ARTDWKWGYFEY 12 CAAS W VAF T AVYYC EVQLVESGGDLVKPGGSLRLS GFTFNNY MHWVRQAPGKGLQW IRYDGSD TYADSVKGRFTISRDNGKNTLYLQMNSLRTEDT ARTDWKWGYFEY 12 CAAS W VAF T AVYYC EVQLVESGGDLVKPGGSLRLS GFTFNNY MHWVRQAPGKGLQW ITYDGSS SYADSVKGRFTISRDNGKNTLYLQMSSLRTEDT 6 ARTDWKWGYFEY 12 CAAS W VAV T AVYYC EVQLVESGGDLVKPGGSLRLS GFTFNNY MHWVRQAPGKGLQW ITYDGSS SYADSVKGRFTISRDNGKNTLYLQMSSLRTEDT ARTDWKWGYFEY 12 CAAS W VAV T AVYYC EVQLVESGGDLVKPGGSLRLS GFTFNTY MHWVRQAPGKGLQW IRYDGSS NYADSVKGRFIISRDNGKNTLYLQMSSLITEDTA ARTDWKWGYFEY 12 CAAS W VAV T VYYC

EVKLVESGGDLVKPGGSLRLS GFTFSNF MNWVRQAPGKGLQW ISSGGSS FYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT VQNGREDYAMDY 12 CAAS D VAY I AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNF MNWVRQAPGKGLQW ISSGGSS FYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT VQNGREDYAMDY 12 CAAS D VAY I AMYYC 7 EVKLVESGGDLVKPGGSLRLS GFTFSNF MNWVRQAPGKGLQW ISSVGSS FYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT VQNGREDYVMDY 12 CAAS D VAY M AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNF MNWVRQAPGKGLQW ISSVGSS FYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT VQNGREDYVMDY 12 CAAS D VAY M AMYYC

EVQLVESGGDLVKPGASLRLS GFTFSNF IHWVRQAPGKGLQWV TSTDGS YYADSVKGRFTVSRDNGRNTLYLQMNSLRTDDT ARDGYDTSHFEY 12 CAAS V AV GT AVYYC EVQLVESGGDLVKPGASLRLS GFTFSNF IHWVRQAPGKGLQWV TSTDGS YYADSVKGRFTVSRDNGRNTLYLQMNSLRTDDT 8 ARDGYDTSHFEY 12 CAAS V AV GT AVYYC EVQLVESGGDLVKPGASLRLS GFTFSNF IHWVRQAPGKGLQWV TSTDGS YYADSVKGRFTVSRDNGKNTLYLQMNSLRTDDT ARDGYDTSHFEY 12 CAAS V AV GT AVYYC

230

EVKLVESGGDLVKPGGSLSLS GFTFSNH MNWVRQAPGKGLQW ISSGGSA YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT ARIPSYSVYLDV 12 CAAS D VAY T AMYYC 9 EVKLVESGGDLVKPGGSLSLS GFTFSNH MNWVRQAPGKGLQW ISSGGSA YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT ARIPSYSVYLDV 12 CAAS D VAY T AMYYC

EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISDTDSD YYADSVKGRFTISRDNGKNVLYLQMNSLKAEDT VKEEYDNSDFEY 12 CAAS A VTR T AVYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISDTDSD YYADSVKGRFTISRDNGKNVLYLQMNSLKAEDT 10 VKEEYDNSDFEY 12 CAAS A VTR T AVYYC EVQLVESGGDLVKPGGSLRLS GFSFSNY MSWVRQAPGKGLQW ISDPDSD YYADSVKGRFTISRDNGKNMLYLQMKSLKTEDT VKEEYDNSDFDY 12 CAAS A VTR T AVYYC

EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW SYAGSVKGRFTISRDNGKNTLYLKMNSLRDEDT ISIDGSST ASWGGNDNQIAY 12 CAAS D VAY AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW SNADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ISIDGSST ASWGGYDNQIDY 12 CVAS D VAY AKYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW SNADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ISIDGSST ASWGGYDNQIDY 12 CVAS D VAY AKYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW SYADSVKGRFTISRDNGKNTLYLKMNSLRDEDT ISIDGSTT ASWGGNDNQIEY 12 CAAS D VAY AMYYC EVQLVESGGDLVKPGGSLTLS GFTFSNY MSWVRQAPGKGLQW SYADSVKGRFTISRDNGKDTLYLKMNSLRGEDT ISIDGTST ATWGGNDNQIEY 12 CAAS D VAY AMYHC 11 EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISSDGSS SNADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ASWGGSDNRIDY 12 CAAS D VAY T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISSDGSS SNADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ASWGGSDNRIDY 12 CAAS D VAY T AMYYC EVKLVESGGDLEKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISSDGSS SYADSVKGRFTISRDNGKNTLYLKMNSLRGEDT ATWGGNDNQIEY 12 CAAS D VAY T AMYHC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISSDGSS SYADSVKGRFTISRDNGKNTLYLKMNSLRGEDT ATWGGNDNQIEY 12 CAAS D VVY T AMYHC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISSDGSS SYADSVKGRFTISRDNGKNTLYLKMNSLRGEDT ATWGGNDNQIEY 12 CAAS D VVY T AMYHC

EVQLLEAGGDLVKPGGSLRLS GFTFSNY IYWVRQAPGKGLQWV ISDGESS YYADSVKGRFTISRDNGKNTLYLQMNSLTTEDT ARDGYDDSHFEY 12 CAAS N AG T GVYFC EVQLVESGGDLVKPGASLRLS GFTFSNY MHWVRQAPGKGLQW TSTDGS YYADSVKGRFTISRDDGKNTLYLQMNSLRTEDT 19 ARDNYDDSNFEY 12 CAAS V VAV GT AVYYC EVQLVESGGDLVKPGGSLRLS MNWVRQAPGKGLQW ISTDDSS SYADSVKGRFTISRDNGKNTLYLHMNSLRAEDT GFTYSTT ARDNYDDSQFEY 12 CAAS VAY T AMYFC

GVQLVESGGDLVKPGASLRLS GFTFSSY MHWVRQAPGKGLQW INTDGSG YRADSVKGRFTISRDDGKNTLYLQMNSLRIEDTA ARDRWDWNSPGD 12 CAAS G VAF T VYYC 20 EVQLVESGGDLVKPGASLRLS GFTFSSY MHWVRQAPGKGLQW INTDGSG YRADSVKGRFTISRDDGKNTLYLQMNSLRIEDTA ARDRWDWNSPGD 12 CAAS G VAF T VYYC

231

EVQLVESGGDLVKPGGSLRLS GFTFSSY MHWVRQAPGKGLQW ISFDGSS SYVDSVKGRFTISRDDGKNTLYLQMSSLRTEDT ARTDWKWGYFEY 12 CAGS W VAV T AVYYC EVQLVESGGDLVKPGGSLRLS GFTFSYY MHWVRQAPGKGLQW IRYDGSS SYADSVKGRFTISRDNGKNTLYLQMNSLRTEDT ARTDWKWGYFEY 12 CAAS W VAV T AVYYC EVQLVESGGDLVKPGGSLRLS GFTFSYY MHWVRQAPGKGLQW IRYDGSS SYADSVKGRFTISRDNGKNTLYLQMNSLRTEDT 21 ARTDWKWGYFEY 12 CAAS W VAV T AVYYC EVQLVESGGDLVKPGGSLRLS VFTFNNY MHWVRQAPGKGLQS IRYDGSS GYADSVKGRFTISRDNGKNTLYLQMNSLRTEDT ARTDWKWGYFEY 12 CAAS W VAV T AVYYC EVQLVESGGDLVKPGGSLRLS VFTFNNY MHWVRQAPGKGLQS IRYDGSS GYADSVKGRFTISRDNGKNTLYLQMNSLRTEDT ARTDWKWGYFEY 12 CAAS W VAV T AVYYC

EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISSDGDD SYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ASWGGNDNQIDY 12 CAAS Y VAY T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISSDGDS TYADSVKGRFTISRDNGKNTLYLQMSSLRAEDT ATWAGYDNQIEY 12 CAAS Y VAY T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFTNY MSWVRQAPGKGLQW ISSDGDS TYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ATWGGSDNQIDY 12 CAAS Y VAY T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISSDGDS TYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT 22 ATWGGYDNQIDY 12 CAAS Y VAY T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISSDGDS TYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ATWGGYDNQIDY 12 CAAS Y VAY T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISSDGDS TYADSVKGRFTISRDNGKNTLYLQMNSLRDGHG ATWGGYDNQIEY 12 CAAS Y VAY T HVLL EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISSDGDS TYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ATWGGYDNQVEY 12 CAAS Y VAY T AMYYC

EVKLVESGGDLVKPGGSLRLS GFTFSYF MNWVRQAPGKGLQW FYADSVKGRFTISRDNDKDMLYLQMDSLRAEDT ISSGGSTI VQNGREDYAMDY 12 CAAS D VAY AMYYC EMKLVESGGDLVKPGGSLRLS GFTFTNY MNWVRQAPGKGLQW ISSGGSS FYADSVKGRFTISRDNDKDMLYLLMNSLRADDT VQNGREDYAMDY 12 CAAS D VAY T AMYYC 23 EMKLVESGGDLVKPGGSLRLS GFTFTNY MNWVRQAPGKGLQW ISSGGSS FYADSVKGRFTISRDNDKDMLYLLMNSLRADDT VQNGREDYAMDY 12 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFTNY MNWVRQAPGKGLQW ISSGGSS YHVDSVKGRFTISRDDAKDMLYLLMNSLRADDT VQNGREDYAMDY 12 CAAS D VAY T AMYYC

QLTLQESGPGLVKPSQTLSLTC GGSVTSS WNWIRQRPGKALEW RYNPAFQGRISITADTSKNQFSLQLSSMTTEDTA 13 VVS YY MGY WTGST VYYC ARGPGAPEPYLDV 24 QLTLQESGPGLVKPSQTLSLTC GGSVTSS WNWIRQRPGKALEW RYNPAFQGRISITADTSRNQFSLQLSSMTTEDTA 13 VVS YY MGY WTGST VYYC ARGPGAPEPYLDV

EVKLVESGGDLVKPGGSLRLS GFTFSAY MSWVRQAPGKGLQW INSGGST YSADSVKGRFTISRDDDKDMLYLQMNSLRAEDT AQGETTEGYAMDY 13 CAAS D VAY T AMYYC 25 EVKLVESGGDLVKPGGSLRLS GFTFSAY MNWVRQAPGKGLQW ISSGGSS YSADSVKGRFTISRDDDKDMLYLQMNSLRAEDT AQGETTEGYAMDY 13 CAAS D VAY T AMYYC

232 EVKLVESGGDLVKPGGSLRLS GFTFSAY MNWVRQAPGKGLQW ISSGGST YSADSVKGRFTISRDDDKDMLYLQMNSLRAEDT AQGETTEGYAMDY 13 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSAY MNWVRQAPGKGLQW ISSGGST YSADSVKGRFTISRDDDKDMLYLQMNSLRAEDT AQGETTEGYAMDY 13 CAAS D VAY T AMYYC

EVKLVESGGDLVKPGGSLRLS MNWVRQAPGRGLQW ISSGGGD YYVDSVKGRFTISRDDGKDMLYLQMNSLRAEDT GFTFINYD ARDPWAGTKYFEY 13 CAAS VAY T AMYYC EVKLVESGGDLVKPGGSLRLS MNWVRQAPGRGLQW ISSGGGD YYVDSVKGRFTISRDDGKDMLYLQMNSLRAEDT GFTFINYD ARDPWAGTKYFEY 13 CAAS VAY T AMYYC 26 EVKLVESGGDLVKPGGSLRLS MNWVRQAPGKGLQW ISSGGGD YYSDSVKGRFTISRDDGKDMLYLQMNSLTAEDT GFIFSNYD ARDPWAGTKYFQY 13 CAAS VAY T AMYYC EVKLVESGGDLVKPGGSLRLS MNWVRQAPGKGLQW ISSGGGD YYSDSVKGRFTISRDDGKDMLYLQMNSLTAEDT GFIFSNYD ARDPWAGTKYFQY 13 CAAS VAY T AMYYC

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGGD YYTDSVKGRFTISRDDGKDMLYLQMNSLRAEDT ARDPWAGTKYFQY 13 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGGD YYTDSVKGRFTISRDDGKDMLYLQMNSLRAEDT ARDPWAGTKYFQY 13 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGGD YYADSVKGRFTISRDDGKDMLYLQMSSLRAEDT ARDPWAGTKYFQY 13 CAAS D VAY T AMYYC 27 EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGGD YYADSVKGRFTISRDDGKDMLYLQMSSLRAEDT ARDPWAGTKYFQY 13 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFTNY MNWVRQAPGKGLQW ISSGGGD YYTDSVKGRFTISRDDGKDMLYLQMNSLRAEDT ARDPWAGTKYFQY 13 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFTNY MNWVRQAPGKGLQW ISSGGGD YYTDSVKGRFTISRDDGKDMLYLQMNSLRAEDT ARDPWAGTKYFQY 13 CAAS D VAY T AMYYC

EVQLVESGGDLVKPGGSLRLS GFTFSNY MTWVRQAPGKGLQW ISGFENS TYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT AREGDNYDNSFVY 13 CAAS F VAY T AMYIC 28 EVQLVESGGDLVKPGGSLRLS GFTFSNY MTWVRQAPGKGLQW ISGFENS TYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT AREGDNYDNSFVY 13 CAAS F VAY T AMYIC

EVQLVESGGDLVKPGGSLRLS GFTFSDY MYWVRQAPGKGLQW INRDGST SYADSVKGRFTISRDDGKDTLYLQINSLRSEDTA AREVNYDNYAMGY 13 CAAS Y VAG T VYYC EVQLVESGGDLVKPGGSLRLS GFTFTNY MYWVRQAPGKGLQW VNRDGS SYADSVKGRFTISRDDGKNTLYLQINSLRSEDTA AREVNYDNYAMGY 13 CAAS Y VAG TT VYYC 29 EVQLVESGGDLVKPGGSLRLS GFTFTNY MYWVRQAPGKGLQW VNRDGS SYADSVKGRFTISRDDGKNTLYLQINSLRSEDTA AREVNYDNYAMGY 13 CAAS Y VAG TT VYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLPW ISSGGST YYADSVKGRFTISRDDDKDMLYLQMNSLRAEDT AREVNYDNYTMGY 13 CAAS D VAY T AIYYC

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW VSSDRIS YFADSVKGRFTISRDDDKDMLYLQMNSLRPEDT ARGESIEGYAMDY 13 CAAS D VAY T AMYYC 30 EVKLVESGGDLVKPGGSLRLS GFTFNNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDDGKDMLFLQMNSLRAEDT ARGETGIGYAMDY 13 CAAS D VAY T AMYHC

233 EVKLVESGGDLVKPGGSLRLS GFTFNNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDDGKDMLFLQMNSLRAEDT ARGETGIGYAMDY 13 CAAS D VAY T AMYHC EVKLLESGGDLVKPGGSLRLS MNWVRQAPGKGLQW INSDGSY FYADSVKGRFTISRDNGKDMLYLHMNSLRAEDT GFTFSIYD ARGETGIGYAMDY 13 CAAS VAY T AMYYC EVKLLESGGDLVKPGGSLRLS MNWVRQAPGKGLQW INSDGSY FYADSVKGRFTISRDNGKDMLYLHMNSLRAEDT GFTFSIYD ARGETGIGYAMDY 13 CAAS VAY T AMYYC EIKLVESGGDLVKPGGSLRLSC MNWVRQAPGKGLQR INSGGST YYADSVKGRFTISSDNDKDILYLQMNSLRAEDTA GFIFSNYD ARGETISGYAMDY 13 TAS VAF T MYYC EIKLVESGGDLVKPGGSLRLSC MNWVRQAPGKGLQR INSGGST YYADSVKGRFTISSDNDKDILYLQMNSLRAEDTA GFIFSNYD ARGETISGYAMDY 13 TAS VAF T MYYC EVKLVESGGDLVKPGGSLRLS GFTFSDC MNWVRQAPGKGLQR ISRGGTT YYTDSVRGRFTISRDDDKDMLYLQMNGLRAEDT ARGETISGYAMDY 13 CVAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSDY MNWVRQAPGKGLQR ISRGGTT YYADSVRGRFTISRDDDKDMLYLQMNGLRAEDT ARGETISGYAMDY 13 CVAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSDY MNWVRQAPGKGLQR ISRGGTT YYVDSVRGRFTISRDDDKDMLYLQMNGLRAEDT ARGETISGYAMDY 13 CVAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSDY MNWVRQAPGKGLQR ISRGGTT YYVDSVRGRFTISRDDDKDMLYLQMNGLRAEDT ARGETISGYAMDY 13 CVAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSDY MNWVRQAPGKGLQR ISRGGTT YYADSVRGRFTISRDDDKDMLYLQMNGLRAEDT ARGETISGYAMDY 13 CVAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSDY MNWVRQAPGKGLQR ISRGGTT YYADSVRGRFTISRDDDKDMLYLQMNGLRAEDT ARGETISGYAMDY 13 CVAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSDY MNWVRQAPGKGLQR ISRGGTT YYADSVRGRFTISRDDDKDMLYLQMNGLRAEDT ARGETISGYAMDY 13 CVAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW FSSDGS FYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT ARGETISGYAMDY 13 CAAS A VAY ST AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW FSSDGS FYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT ARGETISGYAMDY 13 CAAS A VAY ST AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQTPGKGLQR ITVGGSS YYADSVKGRFTISRDDDKDILYLQMNSLRAEDTA ARGETISGYAMDY 13 CAAS D VAF T MYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW IYADSVKGRFTISRDNGKNTLYLQMNSLRAEDTA ISGDGITT ARGETISGYAMDY 13 CAAS Y VSS MYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW IYADSVKGRFTISRDNGKNTLYLQMNSLRAEDTA ISGDGITT ARGETISGYAMDY 13 CAAS Y VSS MYYC EVKLVESGGDLVRPGGSLRLS GFTFSNY MNWVRQAPGKGLQW INSDGST YYADSVTGRFTISRDDDKDMLYLQMNSLRAEDT ARGETISGYVMDY 13 CAAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW YFADSVKGRFTISRDDDKDTLHLQMNSLRPEDT ISSDRIST ARGETTEGYAMDY 13 CAAS G VAY AIYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW YFADSVKGRFTISRDDDKDTLHLQMNSLRPEDT ISSDRIST ARGETTEGYAMDY 13 CAAS G VAY AIYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLHW ISSGGSS YYADSVKGRFTISRDDDKDILYLHMNSLRAEDTA ARGETTSGYAMDY 13 CAAS D VAY T MYHC

EVKLVESGGDLVKPGGSLRLS GFTFTNY MKWVRQAPGKGLQW ISSGGDS YYADSVKGRFTISRDNDKDMLYLQMNSLRTEDT PRGETISGYAMDY 13 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFTNY MKWVRQAPGKGLQW ISSGGDS YYADSVKGRFTISRDNDKDMLYLQMNSLRTEDT 31 PRGETISGYAMDY 13 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSDY MSWVRQAPGKGLQR ITRGGTT YYADSVRGRFTISRDDDKDMLYLQMNGLRAEDT TRGETISGYAMDY 13 CVAS D VAF T AMYYC

234 EVRLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGVS YYADSMQGQFTISRDDDRDMLSLQMNSLRAED TRGETISGYAMDY 13 CAAS D VAY T MAMYYC EVRLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGVS YYADSMQGQFTISRDDDRDMLSLQMNSLRAED TRGETISGYAMDY 13 CAAS D VAY T MAMYYC EVRLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGVS YYADSVQGRFTISRDDDRDMLSLQMNSLRAED TRGETISGYAMDY 13 CAAS D VAY T MAMYYC

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISNDGSS YYADSVRGRFTMSRDNGKDMLYLQMNSLRAED TRVYDHEGYAMGY 13 CAAS D VAY T TAMYHC 32 EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISNDGSS YYADSVRGRFTMSRDNGKDMLYLQMNSLRAED TRVYDHEGYAMGY 13 CAAS D VAY T TAMYHC

EVQLVESGGDLVKPGASLRLS GFTFSTY MHWVRQAPGKGLQW VGADES YYADSVKGRFTIFRDDGKNTLYLQMNSLRIEDTA VLGSISWSWAMDY 13 CAAS G VVF GT VYYC EVKLVESGGDLVKPGGSLRLS GFTFSAY MNWVRQAPGKGLQW ISSGGST YSADSVKGRFTISRDDDKDMLYLQMNSLRAEDT 33 VQGETTEGYAMDY 13 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSAY MNWVRQAPGKGLQW ISSGGST YSADSVKGRFTISRDDDKDMLYLQMNSLRAEDT VQGETTEGYAMDY 13 CAAS D VAY T AMYYC

EVQLVESGGDLVKPGGSLRLS GFTFNNY MSWVRQAPGKGLQW INTGGSS FYADSVKGRFTISRDNAKNTLYLQMNGLRAEDT VRAGGSSWYNWLDY 14 CAAS Y VAW T AMYYC EVQLVESGSDLVRPGGSLRLS GFTFTNY MSWVRQAPGKGLQW INTGGSN YYADSVKGRFTISRDNGKNTLYLQMNSLSAEDT ARAGGSSWYNWLDF 14 CAAS Y VAW T AIYYC EVQLVESGSDLVRPGGSLRLS GFTFTNY MSWVRQAPGKGLQW INTGGSN YYADSVKGRFTISRDNGKNTLYLQMNSLSAEDT ARAGGSSWYNWLDF 14 CAAS Y VAW T AIYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW INTGGSN FYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ARAGGSSWYNWLDY 14 CAAS Y VAW T AMYYC 34 EVLLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW INTGGSS FYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ARAGGSSWYNWLDY 14 CAAS Y VAW T AMYYC EVLLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW INTGGSS FYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ARAGGSSWYNWLDY 14 CAAS Y VAW T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW INTGGSS FYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ARAGGSSWYNWLDY 14 CAAS Y VAW T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFNNY MSWVRQAPGKGLQW INTGGDN FYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ARAGSSSWYNWLDY 14 CAAS Y VAW T AMYYC

EVKLVESGGDLVKPGGSLRLS AFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRAEDT ARQMEYWGDYAMGY 14 CAAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS AFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRAEDT 35 ARQMEYWGDYAMGY 14 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS FYGDSVKGRFTISRDNGKDILYLQMNSLRAEDTA ARQMEYWGDYAMGY 14 CAAS D VAY T MYYC

EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ISGDGSS YYADSVKGRLIISRDDAKNTVYLQMNGLRVEDTA 36 ATEDITTISWAFEY 14 CAAS Y VAS T MYYC

235 EVQLVESGGDLVKPGGSLRLS GFTFNNY MNWVRQAPGKGLQW ISGDGSS SYTDSVKGRFTISRDNGKNTLYLQMNSLRAEDT ATEGISTISWAFEY 14 CAAS Y VAC T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFNNY MNWVRQAPGKGLQW ISGDGSS SYTDSVKGRFTISRDNGKNTLYLQMNSLRAEDT ATEGISTISWAFEY 14 CAAS Y VAC T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFTNY MNWVRQAPGKGLQW ISGDGSS SYADSVKGRFTISRDNGKNTLYLQMNRLRAEDT ATEGISTISWAFEY 14 CAAS Y VAC T AMQGC EVQLVESGGDLVKPGGSLRLS GFTFTNY MNWVRQAPGKGLQW ISGDGSS SYADSVKGRFTISRDNGKNTLYLQMNRLRAEDT ATEGISTISWAFEY 14 CAAS Y VAC T AMQGC EVQLVESGGDLVKPGGSLRLS GFTFSNY LNWVRQAPGKGLQW VSGDGT SYADSLKGRFTISRDNGKNTLYLQMNSLRAEDT ATEGITAISWAFEY 14 CAAS Y VAC YT AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW IYADSVKGRFTISRDNGKNTLYLQMNSLRAEDTA ISGDGITT ATEGITTISWAFEY 14 CAAS Y VSS MYYC EVLLVESGGDLVKPGGSLRLS GFTFSNY INWVRQAPGKGLQWV ISGDRST SYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ATEGITTISWAFEY 14 CAAS Y AS T AMYYC EVLLVESGGDLVKPGGSLRLS GFTFSNY INWVRQAPGKGLQWV ISGDRST SYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ATEGITTISWAFEY 14 CAAS Y AS T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW VSGDGS SFADSMKGRFTISRDNGKSTLYLQMHTLRAEDT ATEGITTISWAFEY 14 CAAS Y VAS FT AMYYC EVQLVESGGDLVKPGGSLRLS GFTFTNY MNWVRQAPGKGLQW ISGDGGS YYADSVKGRFTISRDNGKNTLYLRMNSLRAEDT ATEGITTISWAFEY 14 CAAS Y VAS T AMYYC EVQLLESGGDLVKPGGSLRLS GFTFTNY MNWVRQAPGKGLQW VSGDGS SYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT ATEGITTISWAFEY 14 CAAS Y VAH TT AIYYC EVQLVESGGNLVKPGGSLRLS GFTFSDY LNWVRQAPGKGLQW VSGDGT SYADSLKGRFTISRDNGKNTLYLQMNSLRAEDT ATESITTISWAFEY 14 CAAS Y VAC YT AVYYC EVQLVESGGNLVKPGGSLRLS GFTFSDY LNWVRQAPGKGLQW VSGDGT SYADSLKGRFTISRDNGKNTLYLQMNSLRAEDT ATESITTISWAFEY 14 CAAS Y VAC YT AVYYC EVQLVESGGDLVKPGGSLRLS MNWVRQAPGKGLHW ISGDGST GYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT GFTFTNFY ATETITTISWAFEY 14 CAAS VAS T AMYYC

QLTLQESGPGLVKPSQTLSLTC GDSVTST WNWIRQRPGKALEW RYNPAFQGRISITADTSKNQFSLQLSSMTTEDTA WTGST YYCAVDSILIRIGS 14 VVS YY MGY VYYC 37 QLTLQESGPGLVKPSQTLSLTC GDSVTST WNWIRQRPGKALEW RYNPAFQGRISITADTSKNQFSLQLSSMTTEDTA WTGST YYCAVDSILIRIGS 14 VVS YY MGY VYYC

EVQLVESGGDLVKPGGSLRLS GFTFSSY LYWVRQAPGKGLQCV INVGGGS TYADSVKGRFTISRDNDKNTLYLQMNSLRVEDT ATIGNWGDSYAMGY 14 CAAS D AW T AMYYC 38 EVQLVESGGDLVKPGGSLRLS GFTFSSY LYWVRQAPGKGLQCV INVGGGS TYADSVKGRFTISRDNDKNTLYLQMNSLRVEDT ATIGNWGDSYAMGY 14 CAAS D AW T AMYYC

EEQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ITSDGSS SYADSVKGRFTISRDDGKNTLYLRMNSLRAEDT AREDYRNFDNAMGY 14 CAAS Y VAS T AMYYC EEQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW ITSDGSS SYADSVKGRFTISRDDGKNTLYLRMNSLRAEDT 39 AREDYRNFDNAMGY 14 CAAS Y VAS T AMYYC EVQLVESGGDLVKPGGSLRLS GFTFSNY MYWVRQAPGKGLQW INRDGSS SYADSVKGRFTISRDNGKNTLYLRINSLTSEDTA AREWDYASDYAMGY 14 CAAS Y VAG T VYYC

236 EVKLVESGGDLVKPGGSLRLS GFTFSDY MNWVRQAPGKGLQW INRDGSN YYADSVKGRFTISRDDDEDILFLQMNSLRAADTA ARGDTYYSGYAMDY 14 CATA D VAF T MYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW INRGGSS YYADSVKGRFTISRDDDEDILFLQMNSLRAADTA ARGDTYYSGYGMDY 14 CATA D VAF T MYYC 40 EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNDNHMLYLQMNSLRAEDP ARGLDHEPYYAMDY 14 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNDNHMLYLQMNSLRAEDP ARGLDHEPYYAMDY 14 CAAS D VAY T AMYYC

EVILVESGGDLVKPGGSLRLSC GFTFSNY MNWVRQAPGKGLQW INSDGSS YYADSVKGRFAISRDNGKDMLYLQMNSLRAEDT ARAETISPHYYGMGS 15 AAS D VAY A AMYYC 41 EVILVESGGDLVKPGGSLRLSC GFTFSNY MNWVRQAPGKGLQW INSDGSS YYADSVKGRFAISRDNGKDMLYLQMNSLRAEDT ARAETISPHYYGMGS 15 AAS D VAY A AMYYC

EVKLVESGGDLVKPGGSLRLS GFTFSHY MNWVRQAPGKGLQW INSDGSS YYSDSVKGRFTISRDSGKDMLYLQMNSLRAEDT TRAGTISPHYYAMDS 15 CAPS D VAF T AIYYC EVKLVESGGDLVKPGGSLRLS GFTFSHY MNWVRQAPGKGLQW INSDGSS YYSDSVKGRFTISRDSGKDMLYLQMNSLRAEDT TRAGTISPHYYAMDS 15 CAPS D VAF T AIYYC EVKLVESGGDLVKPGGSLRLS GFTFSHY MNWVRQAPGKGLQW INSDGSS YYTDSVKGRFTISRDYGKDILYLQMNSLRAEDTA TRAGTISPHYYAMDY 15 CAAS D VAY T MYYC EVKLVESGGDLVKPGGSLRLS GFTFSHY MNWVRQAPGKGLQW INSDGSS YYADSVKGRFTISRDSGKDMLYLQMNSLRPEDT TRAGTISPHYYAMDY 15 CAAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSHY MNWVRQAPGKGLQW INSDGSS YYADSVKGRFTISRDSGKDMLYLQMNSLRPEDT TRAGTISPHYYAMDY 15 CAAS D VAF T AMYYC EVILVESGGDLVKPGGSLRLSC GFTFSNY MNWVRQAPGKGLQW INSDGSS YYADSVKGRFAISRDNGKDMLYLQMNSLRAEDT TRAGTISPHYYAMGY 15 AAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSTY MNWVRQAPGKGLQW INSDGSS YYADSVKGRFTISRDYGKDMLYLQMNSLRAVDT TRAGTISPHYYAMGY 15 CAAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSHY MNWVRQAPGKGLQW INSDGTS YYADSVKGRFTISRDSGKDMLYLQMNSLRPEDT 42 TRAGTISPHYYTMDY 15 CAAS D VAF T AIYYC EVKLVESGGDLVKPGGSLRLS GFTFSHY MNWVRQAPGKGLQW INSDGTS YYADSVKGRFTISRDSGKDMLYLQMNSLRPEDT TRAGTISPHYYTMDY 15 CAAS D VAF T AIYYC EVKLVESGGDLVKPGGSLRLS GFTFSHY MNWVRQAPGKGLQW INSDGSS YYADSVKGRFTISRDSGKDMLYLQMNSLRPEDT TRAGTMSPHYYAMDY 15 CAAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSHY MNWVRQAPGKGLQW INSDGSS YYADSVKGRFTISRDSGKDMLYLQMNSLRPEDT TRAGTMSPHYYAMDY 15 CAAS D VAF T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS HYADSVKGRFTISRDNGKDMLYLQMNSLRAEDT TRGGTYDPKYYAMDY 15 CAAS D VAY T AMYYC EVKLVQSGGDLVKPGGSLRLS GFPFSNY MNWVRQAPGKGLQW INSDGSS YFADSVKGRFTISRDYGKDMLYLQMNSLRAEDT TRGGTYDPKYYGMDY 15 CAAS D VAY T AIYYC EVKLVQSGGDLVKPGGSLRLS GFPFSNY MNWVRQAPGKGLQW INSDGSS YFADSVKGRFTISRDYGKDMLYLQMNSLRAEDT TRGGTYDPKYYGMDY 15 CAAS D VAY T AIYYC EVKLVESGGDLVKPGGSLRLS GFTLSNY MNWVRQAPGKGLQW ISSGGSS FYEDSVKGRFTISRDNGKDMLYLQMNSLRAEDT TRGGTYDPKYYTMDY 15 CAAS D VAY T AMYYC

EVILVESGGDLVKPGGSLRLSC INWVRQAPGKGLQWV INSDGSS YYADSVKGRFAISRDNDKDILYLQMNSLRAEDTA 43 GFIFSSYD ARAGTISPHYYAMGY 15 AAS AS T MYYC

237 EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVSGRFTISRDNDNDMLYLLMNSLRAEDT ARALSLDAPYYAMDY 15 CAAS D VAY T ALYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVSGRFTISRDNDNDMLYLLMNSLRAEDT ARALSLDAPYYAMDY 15 CAAS D VAY T ALYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YSSDSVKGRFTISRDNGKDILYLQMNRLRAEDTA ARALSLDAPYYPMDY 15 CAAS D VAY T MYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YSSDSVKGRFTISRDNGKDILYLQMNRLRAEDTA ARALSLDAPYYPMDY 15 CAAS D VAY T MYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDDDKDMLYLQMNSLRAEDT ARAMSLAPPYYPMDY 15 CAAS D VAY T AIYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDDDKDMLYLQMNSLRAEDT ARAMSLAPPYYPMDY 15 CAAS D VAY T AIYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLPW ISSGGST YYADSVKGRFTISRDDDKDMLYLQMNSLRAEDT ARAVTLAPPYYPMDY 15 CAAS D VAY T AIYYC EVKLVESGGDLVKPGGSLRLS GFTFNNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDYGKDMLYLHMNSLRTEDT ARGGTIDPLYYALDY 15 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFNNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDYGKDMLYLQMNSLRTEDT ARGGTVDPLYYALDY 15 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFNNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRIEDTA ARGGTVDPLYYAMDS 15 CAAS D VAY T MYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY IDWVRQAPGKGLQWV ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRAEDT ARGGTYDPKYYAMDY 15 CAAS D AY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW INSDGSS YFADSVKGRFTISRDYGKDMLYLQMNSLRAEDT ARGGTYDPKYYGMDY 15 CAAS D VAY T AMYYC

EVKLVESGGDLVKPGGSLRLS GFTFNTY MNWVRQAPGKRLQW ISSGGGS YYADAVIGPVTISRDNGKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARAPGVSPNYYAMDY EVKLVESGGDLVKPGGSLRLS EFTFSNY MNWVRQAPGKGLQW ISSGGSS YYVDSVKGRFTISRDNGKDMVYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARDPGSYPAYYAMDY EVKLVESGGDLVKPGGSLRLS GFTFSNY INWVRQAPGKGLQWV ISSGGSS YYADAVKGRFTISRDNGKDILYLQMNSLRAEDTA 15 CAAS D AY T MYYC ARDPGTYPAYYAMDY EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 15 CAAS D VAY ISSGGIST AMYYC ARGETVAPGYYAMGY EVKFVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADPVKGRFTISRDDGKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGGSFDPVYYAMDY EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ITSGGSS YYADAVKGRFTISRDNGKDMMYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGGTIAPAYYAMGY 44 EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGGTISPYYYAMGY EVKLVESGGDLVKPGGSLRLS GFTFSHY MNWVRQAPGKGLQW ISSGGSS YYADAVKGRFTISRDNGKDMLHLQMNSLRTEDT 15 CAAS D VAY T AMYYC ARGGTRSPYYYAMGY EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGGTTSPLYYAMAY EVKLVESGGDLVKPGGSLRLS GFTFSDY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDDDRDMLHLQMNSLRAEDT 15 CAAS D VAY T AMYHC ARGGTTSPVYYAMGY EMKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGGTVAPHYYAMGY EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGGTVAPNYYAMDY

238 EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGGTVAPTYYAMGY EVMLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGGTVAPTYYAMGY EVKLVESGGDLVKPGGSLRLS GFTFSNF MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGGTVAPYYYAMGY EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRAEDT 15 CAAS D VAY T ATYYC ARGGTVSTVYYAMDY EVKLVESGGDLVKPGGSLRLS GFTFSNF MNWVRQAPGKGLQW ISSGGSS YYADSVRGRFTISRDNGKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYHC ARGGTVSTVYYAMDY EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW YYADSVKGRFTISRDNGKDMLYLQMNSLRAEDT 15 CAAS D VAY ISSGGIST AMYYC ARGGTYDPYYYAMDY EVKLVESGGDLVKPGGSLRLS GFTFSDY MNWVRQAPGKGLQW ISSGGSS YYTDSVKGRFTISRDNGKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGSTYDPEYYAMGY EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADAVNGPFTISRDNGKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGSTYDPGYYAMDY EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADAVVGPFTISRDNSKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARGSTYDPGYYAMGY EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNVKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARSNTVAPTYYAMGY EVKVVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 15 CAAS D VAY T AMYYC ARSNTVAPTYYAMGY

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISGGGSS YYADSVKGRFTISRDDDKDMLYLQMNSLRAEDT ANNIGGYEDYYYAMDY 16 CAAS D VAY T AIYYC 45 EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISGGGSS YYADSVKGRFTISRDDDKDMLYLQMNSLRAEDT ANNIGGYEDYYYAMDY 16 CAAS D VAY T AIYYC

EVKLVESGGDLVKPGGSLRLS EFTFSNY MNWVRQAPGKGLQW ISSGGSD YFADSVQGRFTISRDNDKDMLYLQMNSLRAEDT TRGVGGDHDDYYAMGY 16 CAAS D VAY T ATYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT TRGVGGDRDDYYAMVY 16 CAAS D VAY T AMYHC EVQLAESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSDESS YSADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 46 TRGVGGDRDDYYGMAY 16 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW YYADSVQGRFTISRDNDKDMLYLQMNSLRAEDT ISIGESST TRGVGGDRDDYYTMAY 16 CAAS D VAY AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW YYADSVQGRFTISRDNDKDMLYLQMNSLRAEDT ISIGESST TRGVGGDRDDYYTMAY 16 CAAS D VAY AMYYC

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ITSGGSS YSADSVKGRFTISRDNDKDMLYLQMNSLRAEDT ASNIGGIEDYYYAMDY 16 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YSADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 47 TNNIGGDEDYYYAMGY 16 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLA GFTFSNY MNWVRQAPGKGLQW ISSDGSS YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT TNNIGGYEDYYYAMGY 16 CAAS D VAY T FQYGC

239 EVKLVESGGDLVKPGGSLRLS GFTFSSF MNWVRQAPGKGLQW ITSGGSS YYADSVRGRFTISRDNDKDMLYLQMNSLRVEDT ARGGGGDYDDYYAMDY 16 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSSF MNWVRQAPGKGLQW ITSGGSS YYADSVRGRFTISRDNDKDMLYLQMNSLRVEDT ARGGGGDYDDYYAMDY 16 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDKVSAWLGFQMNSLRSRD ARGLGGTGWGGYDMDY 16 CAAS D VAY T MALCYC EVKLVESGGDLVKPGGSLRLS GFTFNNY MNWVRQAPGKGLQW YYADSVQGRFTISRDTDKDMLYLQMNSLKTVDT ITSSGNDI ARGVGGDNDEYYAMDY 16 CAAS D VAY AMYYC EVKLVESGGDLVKPGGSLRLS GFTFNNY MNWVRQAPGKGLQW YYADSVQGRFTISRDTDKDMLYLQMNSLKTVDT 48 ITSSGNDI ARGVGGDNDEYYAMDY 16 CAAS D VAY AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ITSDRSS YSADSVKGRFTISRDNDKDMLYLQMNSLRAEDT ARGVGGDRDDYYAMAY 16 CAAS D VAY T AMYYC EVRLLESGGDLVKPGGSLRLS GFTFSNS MNWVRLAPGKGLQW ISSGDIG YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT ARGVGGDRDDYYAMGY 16 CAAS D VAY T AMYYC EVRLLESGGDLVKPGGSLRLS GFTFSNS MNWVRLAPGKGLQW ISSGDIG YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT ARGVGGDRDDYYAMGY 16 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFSNH MNWVRQAPGKGLQW IYSGGSD YYADSVKGRFTISRDNDKDMLYLQMKSLRAEDT ARGVGGDRDDYYGMGC 16 CAAS D VAS T ATYYC

EVNLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YSADSVQGRFTISRDNGKDELYLQMNSLRAEDT ARALGGDSDDYYAIDY 16 CAAS D VAY T AIYYC 49 EVKLVESGGDLVKPGGSLRLS GFTFSKY MNWVRQAPGKGLQW ISSDGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRAEDR ARALGGDSDDYYAMDY 16 CAAS D VAY T AMYYC

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW IGSDWS YYADSVKGRFTISRDNDKDMLYLQINSLRAEDTA ARGGDSSLSESYYAMAY 17 CAAS D VAY NI MYYC 50 EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW IGSDWS YYADSVKGRFTISRDNDKDMLYLQINSLRAEDTA ARGGDSSLSESYYAMAY 17 CAAS D VAY NI MYYC

EVNLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW IYSDGSS YYADSVKGRFTTSRDNDKDMLYLQMNGLRAED ARRPYGSSSEDYYAMGY 17 CAAS D VAY T TAMYYC 51 EVNLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW IYSDGSS YYADSVKGRFTTSRDNDKDMLYLQMNGLRAED ARRPYGSSSEDYYAMGY 17 CAAS D VAY T TAMYYC

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISGGGSG YYADSVRGRFTISRDNDKDMLYLQMNSLRAEDT ARGRDDNFSDAYYAMGY 17 CAAS D VAY T AMYYC 52 EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISGGGSG YYADSVRGRFTISRDNDKDMLYLQMNSLRAEDT ARGRDDNFSDAYYAMGY 17 CAAS D VAY T AMYYC

EVKLVESGGDLVKPGGSLRLS GFTFSNH MNWVRLAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRTEDT ARGAHYDSSEESYYAMNY 18 CAAS D VAY T AMYHC EVKLVESGGDLVKPGGSLRLS GFTFSNH MNWVRLAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRTEDT 53 ARGAHYDSSEESYYAMNY 18 CAAS D VAY T AMYHC EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDDGKDMLYLQMNSLRAEDT ARGAHYDSSEGSYYAMVY 18 CAAS D VAY T AMYYC

240 EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDDGKDMLYLQMNSLRAEDT ARGAHYDSSEGSYYAMVY 18 CAAS D VAY T AMYYC EVKLVESGGDLVKPGGSLRLS GFTFNNY MNWVRLAPGKGLQW ISTGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRTEDT ARGSHYDSSEESYYAMNN 18 CAAS D VAY T AMYHC EVKLVESGGDLVKPGGSLRLS GFTFNNY MNWVRLAPGKGLQW ISTGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRTEDT ARGSHYDSSEESYYAMNN 18 CAAS D VAY T AMYHC

EVKFVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRAEDT ARGGYWNSPSEDYYAMAY 18 CAAS D VAY T AIYYC 54 ELKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADYLKGRFTISRDDDKDMLYLQMNSLRAEDT ARGSVYWNSMGDYYAMGY 18 CAAS D VAY T AMYYC

EVQLVESGGGLVKPGASLRLS GFTFSNF MHWVRQAPGKGLQW TFTDESG FYADSVKGRFTISRDDGKNTLYLQMNSLRTEDT ARNADCDSYGYCLSAMDY 18 CAAS G VAI T AVYYC 55 EVQLVESGGGLVKPGASLRLS GFTFSNF MHWVRQAPGKGLQW TFTDESG FYADSVKGRFTISRDDGKNTLYLQMNSLRTEDT ARNADCDSYGYCLSAMDY 18 CAAS G VAI T AVYYC

EVHLVDSGGDLVKPGGSLRLS GFTFSSY MSWVRQAPGKGLQW ISNSGSD YYTDSVKGRFTISTDNGKNTLYLQMNTLRAEDM ARSDVNFDGYGYQVFYYVM 21 CAAS W VAS T AVYYC DY 56 EVHLVDSGGDLVKPGGSLRLS GFTFSSY MSWVRQAPGKGLQW ISNSGSD YYTDSVKGRFTISTDNGKNTLYLQMNTLRAEDM ARSDVNFDGYGYQVFYYVM 21 CAAS W VAS T AVYYC DY

EVKLVESGGDLVKPGGSLRVS GFTFSNY MKWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRAEDT ARDRTDCDGFGNCFVGEYY 23 CAAS D VAY T AMYYC AMDY 57 EVKLVESGGDLVKPGGSLRVS GFTFSNY MKWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRAEDT ARDRTDCDGFGNCFVGEYY 23 CAAS D VAY T AMYYC AMDY

EVQLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGEGLQW YYADSVKGRFTVSRDNGKNTLYLQMNSLRAEDT 58 CAAS Y VAF INTGGSIT ACYYC AALDRIYAIDY 11

EVHLVESGGDLVKPGASLRLS GFTFSSY MYWVRQAPGKGLQW TSTDGS YYADSLKGRFTISRDNGKNTLYLQMNSLRTEDTA 59 CAAS G VAF DT VYYC ARVTEEWEMGY 11

EVQLVESGGDLVKPGGSLRLS GFTFSSY MYWVRQAPGKGLQC INTGESS SYAGSVKGRFTISRDDGWNTLYLQMNSLRVEDT 60 CAAS D VAW T AMYYC ATGGSDNRIEY 11

EVQLVESGGDLVKPGASLRLS GFTFSNY MHWVRQAPGKGLQW TNTDESD YYADSVKGRFTFSRDNGKNTLYLQMNSLRTEDT 61 CAAS G VAI T AVYYC ARASHYGSEFEY 12

EVQLVESGGDLVKPGASLRLS GFTFSYY MHWIRQAPGKGLQWV TSTDESG YYADSVKGRFTISRDNGKNTLYLQMNSLKAEDT 62 CAAS V AV T AVYFC AREGGGDTAFEY 12

241 EVQLVESGGDLVKPGGSLRLS GFTFSNY MHWVRQAPGKGLQW GSYDAS SYADSVKGRFTISRDNGKNTLYLQMSSLRTEDT 63 CAAS E VGL ST AVYYC AREGYDNSDFEF 12

EVRLVESGGDLVKPGGSLRLS GFTFSNY MHWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRTEDT 64 CAAS D VAY I AMYYC ARGEEVDYVMGY 12

EVKLVASGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGESS FYAASVKGRFTLSRDDDKDMLYLHMNSLRPGDT 65 CAAS A VAY T AMYYC ARGGLEGYDMDY 12

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDDDKDMLYLQMNSLRAEDT 66 CAAS D VTY T AMYYC ARGSYSDYAMAY 12

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ILSGGSS YYADSVKGRFTISGDSDKDMLYLQMNSLRTEDT 67 CAAS D VAS T AMYYC AAGIKVEDYAMDC 13

EVQLVESGGDLVKPGGSLRLS GFTFSNN MSWVRQAPGKGLQW ISSYESS SYADSVKGRFTISRDDGKNTLYLQMNSLRAEDT 68 CEAS F VAY T AMYYC ATEGDTYDNSFEY 13

EVKLVESGGDLVKPGGSLRLS GFTFSNY INWVRQAPGKGLQWV ILSGGSS YYADSVKGRFTISGDNDKDMLYLQMNSLRTEDT 69 CAAS D AS T AMYYC VAGIKVEDYAMDC 13

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYVDSVKGRFTISRDNDQDMLYLQMNSLKTEDT 70 CAAS D VAY T AMYYC ARDLNWEPYYAMDY 14

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISGGGSS YYADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 71 CAAS D VAY T AMYYC ARDYNYDPYYAMDY 14

EVQLVESGGDLVKPGGSLRLS GFTFSSY MQWVRQAPGKGLQW IRYNGGS TYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT 72 CTAS S VAY T ALYYC ARVEDALYYYAMDY 14

EVQLVESGGDLVKPGGSLRLS GFTFSDY MSWVRQAPGKGLQW INTGGSD YYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT 73 CAAS F VAW T AMYYC ARVKLTGTVYAMDY 14

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGEGLQW ISSDATS YSADSVKGRFTISRDNDKDMLYLQMNSLRAEDT 74 CAAS D VAY T AKYYC ATRDGSYEGYYFEY 14

EVILVESGGDLVKPGGSLRLSC GFTFSNY MNWVRQAPGKGLQW INSDGSS YYADSVKGRFAISRDNGKDMLYLQMNSLRAEDT 75 AAS D VAY A AMYYC VRAETISPHYYGMGY 15

242 EVKLVESGGDLVKPGGSLRLS GFTFSNY INWVRQAPGKGLQWV ISSGGSS YYADSVKGRFTISRDNDKDMLFLQMNSLRAEDT 76 CAAS D AY T AMYYC ARAGSTEATAYYAMDY 16

EVQLVESGGDLVKPGGSLRLS GFTFSNY MSWVRQAPGKGLQW IDTGGST SYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT 77 CAAS F VAW T AMYYC ARGDAGIVELGYYFES 16

EVKLVESGGDLVKPGGSLRLS GFTFTNY MNWVRQAPGKGLQW ITRDGNN YYADSVKGRFTISRDNDKDILYLQMNSLRAEDTA 78 CAAS D VAY T MYSC ARTCGSDNDYYYAMDY 16

EVQLVESGGDLVKPGGSLRLS GFTFSSY MYWVRQAPGKGLQC INTGGSS SYADSVKGRFTISRDNGKNTLYLQMNSLRVEDT 79 CAAS D VAW T AMYYC ATAGTGIVSLYYYAMDY 17

EVKLVESGGDLVKPGGSLRLS MNWVRQAPGKGLQW ISSGGYS YYADSLKGRFTISRDDGEDMLYLQMNSLRAEDT 80 CAAS EFTFSYYD VAY T AMYYC ARGAHFGSSDGSYYAMAF 18

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISGYGSS YYADSVKGRFTISRDDGKDMLYLQMNSLRAEDT 81 CAAS D VAY T AMYYC SRGGETINGGPDYYAMGY 18

EVKLVESGGDLVKPGGSLRLS GFTFSNY MNWVRQAPGKGLQW ISSGGSS YYADSVKGRFTISRDNGKDMLYLQMNSLRVEDT ARADCDSYGDCFLSAYYAM 82 CAAS D VAY T AIYYC GY 21

EVQLVESGGDLVKPGGSLRLS GFTFSSY MYWVRQAPGKGLQC INTGGSS SYADSVKGRFTISRDNGKNTLYLQMNSLRAEDT AKITQSQYCDSYGYCNLHDY 83 CAAS D VAW T AMYYC LDV 23

EVQLVESGGDLVKPGGSLRLS GFTFSDY MSWVRQTPGKGLQW ITYDGRS SYADSVKGRFTVSRDNGKNTLYLQMNSLRAEDT ARGPTTYCHSSACFGEYYYP 84 CAAS Y VAY A AMYYC MAY 23

243 Appendix 4.2 Ferret immunoglobulin lambda chain clonal families recovered from influenza infected ferrets

Clonal CDR1- CDR2- CDR3 family FR1-IMGT IMGT FR2-IMGT IMGT FR3-IMGT CDR3-IMGT length SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSENTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QSYDSSGNV 9 SSELTQTPFVSVAPGQTARITCS VQWYQQNPGQAPLM ERASGIPERFSGSKSENTATLTISSVQAEDEAD GE ELGNSY VIY LDN YFC HSYDSSGHV 9 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QSYDSSGPV 9 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERYSGSKSGNTATLTISSIQAEDEADY 1 GE EVDYGY VIY LDN FC HSYDSSGKV 9 SSELTQTPFVSVAPGQTARITCS VQWYQQNPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELGNSY VIY LDN YFC QSYDSSGHV 9 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLMII ERASGIPERFSGSKSGNTATLTISSVEAEDEAD GE EVDYGY Y LDN YVC QSYDSGGNV 9 SSELTQTPFVSVAPGQTATITCS IQWYQQKPGQAPLMII ERASGIPERFSGSKSGNTATLTISSVEAEDEAD GE EVDYGY Y LDN YFC QSYDSGGNV 9

QPVLSPRFSLSASPGTTARLTC RDISVGST MHWYQQKPGTPPRYL QLGPGIPSRFSGSKDTSANAGILLISGLQPEDEA TLS Y LY YYSDSST DYYC AVWHSGAYV 9 QPVLLSSSSLSASPGTSARLTCT RDISVGSD MHWYQQKPGTPPRYL QLGPGIPSRFSGSKDTSANAGILLISGLQPEDEA LN Y LY YYSDSTT DYYC AVWHSGAYV 9 QPVLLPRVSLSASPGTTARLTCT RDISVGSK MHWYQQKPGTPPRYL QLGPGIPSRFSGSKDTSANAGILLISGLQPEDEA LS Y LY YYSDSST DYYC AVWHSGAYV 9 QPVLLSSFLLSASPGTTARLTCT RDISVGTK MHWYQQKPGTPPRYL QLGPGIPSRFYGSEDTSANAGILHISGLQPEDE LS Y LY YYSDSTT ADYYC AVWHSGDYV 9 QPVVLFLASLSASPGTTAGLTCT RDFSVAG MHWYQRKPGTPPRYL QLGPGIPSRFSGSEDTSANAGTLLISGLQPEDE LS NY LY YYSDSTT ADYYC AVWRSGAYV 9 QPVLLLPGSLSASPGTTARLTCT RDISVIGK IHWYQQKPGTPPRYLL QLGPGIPSRFSGSEDTSANAGILLISGLQPEDEA LS Y Y YYSDSTT DYYC AVWHSGVYV 9 SLCFSPSVLLSASPGTTAGLTCT RDFSVAG MHWYQKKPGTPPRYL QLGPGITSRFSGSEDTSANAGILLISGLQPEDEA 2 LS NY LY YYSDSTT DYYC AVWHSGAYV 9 QPVLLYPVPLSASPGTTAGLTCT RDFSVAG IHWYQQKPGTPPRYLL QLGPGIPSRFSGSEDTSANAGILLISGLQPEDEA LS NY Y YYSDSTT DYYC AVWHSGAYV 9 QPVLLLRVSLSASPGRTARLTCT RDISVDSK MHWYQQKTGTPPRYL QLGPGIPSRFSGSEDTSGNAGILLISGLQPEDE LS Y LY HYSDSTT ADYYC AVWHSGVYV 9 ACASLLGSSLSASPGTTARLTCT RDISVGSK MHWYQQKPGTPPRYL QLGPGIPSRFSGSKDTSANAGILLISGLQPEDEA LS Y LY YYSDSST DYYC AVWHSGAIV 9 SLCFSPRSSLSASPGTTARLTCT RDISVDSK MHWYQQKPGTPPRYL QLGPGIPSRFSGSEDTSGNAGILLISGLQPEDE LS Y LY HYSDSTT ADYYC ALWHSGVYV 9 SLCFSPSFSLSASPGTTARLTCT RDISVGSK MHWYQQKPGTPPRYL QLGPGIPSRFYGSEDTSANAGILHISGLQPENE LS Y LY YYSDSTT ADYYC AVWHSGDYV 9 QPVLLSSSSLSASPGTSARLTCT RDISVGSD MHWYQQKPGTPPRYL QLGPGIPSRFSGSKDTSANAGILLISGLQPDDEA LS Y LY YYSDSTT DYYC AVWHSDTYV 9

244 QPVLLSSFSLSASPGTSARLTCT RDISVGSD MHWYQQKPGTPPRYL QLGPGIPSRFSGSKDTSANAGLLLISGLQPEDE LS Y LY YYSDSTT ADYYC AVWHSNTYV 9 QPVLLSSSSLSASPGTTARLTCT RDIIVGSK MHWYQQKPGTPPRYL QLGPGIPSRFSGSEDTSANAGILLISGLQPEDEA LS Y LY YYSDSTT DYYC AVWHSRLYV 9

SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGH VIY LDN YFC QSQDSSGDAV 10 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QSYDSSGNPV 10 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLIISSVQAEDEADY GE ELDYGY VIY LDD FC QSYDSSGNPV 10 SSELTQTPFVSVAPGQTATITCS LQWYQQKPGQAPLMV ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE EVDYGF IY LDN YFC QSYDNSGNPV 10 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGKMATLTISSVQAEDEAD GE QLDYGY VIY LDN YFC QSYDSSGNPM 10 SGELTQTPFVSVAPGQTATIACS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISRVQAEDEAD GE EVDNGY VIY LDN YFC QSYDTSDNAV 10 SSEPTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSRSGNTATLTISSVQAEDEAD GE QLDYGY VIY LDN FFC HSYDSSNNPV 10 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE DLDYGY VIY LDN YFC QSYDSSGNVV 10 SSEVTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QSYDSIGNVV 10 SSEPTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSRSGNTATLTISSVQAEDEAD GE QLDYGY VIY LDN YFC QSYDSSNNPV 10 SSELTQTPFVSVAPGQTATITCS VQWYRLKPGQAPLMV ERASGIPERFSGSKSGNTATLTISSVQAEDEAD 3 GE ELDYGY IY LDN YFC QSYDSSGNPV 10 SGELTQTPFVSVAPGQTATITCS VHWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIF LDN YFC HSYDSSGNPV 10 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSENTATLTISNVQAEDEAD GE ELDYGY VIY LDN YFC QSYDSSGNPV 10 SSEPTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSRSGNTATLTISSVQAEDEAD GE QLDYGY VIY LDN YFC QSYDSSNNPV 10 SSALTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QSSDSSGNAV 10 SSELTQTPLVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QSYDSSGNPV 10 SSELTQTPFVAVAPGQTATITCS VQWYQQKQGQAPLM ERASGIPERFCGYKSGNTATLGISSVQAEDEAD GE ELDYGY VIY LDN FLS QSYDSSGTPV 10 SSELTQTPFVSVAPGQTATITCS VQWYQRKPGQAPLM ERASGIPERFSGSKSGNTATLTISTVQAEDEAD GE ELDYGY VIY LDN YFC QSYDSSGNPV 10 SSELTQTPFVSVAPGQTATITCS VQWYRQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY MDD YFC QSYDSSGNPV 10 SSEPTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QSYDSSGNPV 10 SGELTQTPFVSVAPGQTATIACS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTVSRVQAEDEA GE EVDNGY VIY LDN DYFC QSHDSSGNAV 10

245 SSELAQTPIVSVAPGQTATITCS VQWYKQKPGQAPLMV ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDHGY IY LDN YFC QSYDSSGNPV 10 SSELTQTPFVSVAPGQTATITCS VQWYQQKSGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QTSDSSSKAV 10 SSEPTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSRSGNTATFTISSVQAEDEAD GE QLDYGY VIY LDN YFC QSYDSSNNPV 10 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGHAPLM ERASGIPERFSGSKSGNMATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QSYDTSVNPV 10 SSELTQTPFVSVAPGQTATIPCS VQWYQLRPGQAPLMV ERASGIPERFSGSKSGNTATLTITSVQAEDEAD GE ELDYGY IY LDN YFC QSCDSSGYPV 10 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE RLDYGY VIY LDN YFC QSYDSSGNPV 10

SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTINSVQAEDEAD GE ELDYGY VIY LDN YFC QSYDSSGNAV 10 4 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QSYDSSGNAV 10

SSELTQAPFVSVAPGQTAIIICSG VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLIISSVQAEDEADY E ELDYGY VIY LDN FC QSYDNRGNVYV 11 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSENTATLTITSVQAEDEAD GE ELDSGY VIY LDN YFC QSYDSIGNTVV 11 SSELTQTSFVSVAPGQTATITCS VQWYQRKPGQAPLM ERASGIPDRFSGSKSGNTATLTISSVQAEDEAD GE ELDYGF VIY LDN YFC QSYDSSGHTYV 11 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELENGY VIY LDN YFC QSYDITTDPYV 11 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC HSYDSSGNAYV 11 SSELTQTPFVSVAPGQTATITCS VQWYRQKPGQAPLM ERASGIPERFSGSKSGNTATLTISNIQAEDEADY GE ELDYGY VIY LDN FC QSYDSSGNAIV 11 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIF LDN YFC QSYDNSDNMII 11 5 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLMII ERASGIPERFSGSESGNTATLTISSVQAEDEAD GE EVDFGY Y LDD YFC HSYANSGDVYV 11 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSIQAEDEADY GE ELDYGY VMY LDD FC QSYDSSGHAPV 11 SSDLTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QSYDNSGNAYV 11 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQVPLM ERASGIPERFSAFKSGNTATLTISSVQAEDEAD GE ELDYGY MVY LDN YFC QSYDSSDDIIL 11 SSELTQTPFVSVAPGQTATITCS VQWYQQKSGQAPLM ERASGIPERFSGSKSGNTATLTINGVQAEDEAD GG ELDHGY VIY LDD YFC QSYDSTGDTPV 11 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYDY VIF LDN YFC QSYDNSDDAIM 11 SSELTQTPFVSVAPGQTATITCS VQWYRQKPGQAPLM ERASGIPERFSGSKSENTATVTITSVQAEDEAD GE DLDSNH VIY LDN YFC QSYDSIGNAVV 11

246 SSELTQTPFVSVAPGQTATITCS IQWYRQKPGQAPLMVI ERASRIPERFSGSKSGNTATLTISSVQAEDEAD GE QLGYGY Y LDN YFC QSYDSSGDAIV 11 SSELTQTPFVSVAPGQTATITCS IQWYRQKPGQAPLMVI ERASRIPERFSGSKSGNTATLTISSVQAEDEAD GE QLGYGY Y LDN YFC QSYDSSGDAIV 11

SSELTQTPFVSVAPGQTATISCS VQWYQQKPGQAPLM ERASGFPERFSASKSGNTATLTISSIQAEDEAD GE DLDYAY VIY LDN YFC QSYDSSGNAPV 11 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLM ERASGIPERFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIF LDN YFC QSYDNSDNAII 11 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQAPLMII ERASGIPERFSGSKSGNTATLTISSVQAEDEAD 6 GE ELDYGY Y LDN YFC QSYANSGNVYV 11 SSELTQTPFVSVAPGQTATITCS VQWYQQKPGQVPLM ERASGIPERFSGSKSGNTATLIISNLQAEDEADY GE ELDYGY VIY LDD FC QSYDSSSDAIV 11 SSELTQTSFVSVAPGQTATITCS VQWYQRKPGQAPLM ERASGIPDRFSGSKSGNTATLTISSVQAEDEAD GE ELDYGY VIY LDN YFC QSYDSNNHAYV 11

SYVLTQPPSISVKLEQTAKITCG AYWYQQKPGRVPVLII KRPSGIPDQFSGTNSGNTATLTISGARAEDEAD GD NIGNRY Y EDS YYC QVWDISTFAPV 11 7 SYVLTQPPSISVKLGQTARITCG AYWYQQKPGRVPVLII KRPSGIPDQFSGTNSGNTATLTISGARAEDEAD GD NIGNKY Y EDS YYC QVWDSSTNAYV 11

AYELTQDSSVSVNLGQTARITC AYWYQQKPSQAPLLII NRPSGIPERFSGTNSGNTATLTISGAQAEDEAD 8 RGD DIGIKF Y GDS YYC QVGTVVM 7

QLLVTQPPFLSASLETTARLTCT IFWCQQKPGSPPRYLL HQGSGVPSRFSGSKDASASAGILLISGLQAEDE 9 LS SDISIDIYP T YVTDSNK ADYYC SHLMLVMV 8

SSELTQTPFVSVAPGQTATIICS VQWFQQKPGQAPLM ERASGIPERLSGSKSGNTATLTISSVQAEDEAD 10 GE ELDSGY VIY LDN YFC QSYDSSGNPV 10

SYVLTQPPSVSVALGQTAQVTC VHWYQQKPGQAPVVII KRPSGIPGRFSGTKSGNTATLTISRSRAEDEAD 11 GGN NIGNKS Y NNN YYC QVWDRSSDYV 10

SSELTQLPSVSVALGQTATITCT VQWYQKKPGQAPLTII ERPSGIPERFSGSRSGNTATLTISGVQAKDEAD 12 GE ELDYT N DES YYC QSYDSNADAV 10

QSVLTQPSSLSGSLGQRVTISC VYWYQQHQGKAPKTII SRPSGVPERFSGSRSGNTGSLTITGLKTEDEG 13 TGG STNIGDGY N GSD DYYC SSWDKTLSGPV 11

SSELTQLPSVSVALGQTATITCT VQWYQKKPGQAPLTII ERPSGIPERFSGSRSGNTATLTISGVQAEDEAD 14 GE ELDYT Y DES YYC QSYDSNADAYV 11

247

SFVLTQPPSMSVNLGQTVRMTC VPWYQQKPGLAPVMII SRPSGIPDRFSGTNSGNTATLTISGVRAEDEAD 15 GGN NIGRKS Y GDS YYC QVWDNSADAWV 11

AYELTQPPLMSVNLGRTASITCS VSWYQQKLGKAPMMII NRPSGIPDRFSGSNSGNTATLTISGAQAEDEAD 16 RD NIGNTY Y SGS YYC LSYKSGSGDYV 11

LPVLSHLSSESASLPPSHTVSCT CRWKQQRPCNAPLC TSEDAMPNRFSSSPSECDRYCSISNIHLTDDPE GDDYRVGGQSG 17 LI SERRIHM MMT LYKLASY DDC YV 13

248

Appendix 4.3 Ferret immunoglobulin kappa chain clonal families recovered from influenza infected ferrets

Clonal CDR2- CDR3 family FR1-IMGT CDR1-IMGT FR2-IMGT IMGT FR3-IMGT CDR3-IMGT length EVVLTQTPLSLSVTPGEPVSISC QSLVHSDGN LHWYLQKPGQSPQA NRFTGVPDRFSGSGSGTDFTLRISRVEVEDMGV RAS TY LIY KVS YFC EQGTQWS 7 1 EFVLTQNQFSLSVTRESRSPFPA QSLVHSQGN LHWYLQKPGQAPQG NRFTGVPDRFSGSGSGTDFTLRISRVEVKDMGV GP TY LIY KVS YFC EQGTQWS 7

EVVLTQTPLSLSVTPGEPVSISC QSLVHSNGN LHWNLQKPGQSPQG HRFTGVPDRFSGSGSGTDFTLRMSRVEADDMG RAS TF LIY KVS VYFC EQGKQWT 7 EVVLTQTPLSLSVTPGEPVSISC QSLVHSNGN LHWNLQKPGQSPQG HRFTGVPDRFSGSGSGTDFTLRMSRVEADDMG RAS TY LIY KVS VYFC EQGKQWT 7 EVVLTQSPLSLSVTRGEPLSISG QSLVHSNGN LHWNLQKPGQSPQA PRFRGVPDRFSGSGSGSDFPLRMSRVEEEDMG RPS TN LIY KVS VYF EQGKQWS 7 EVVLTQNQLSLSVTPGEPVSISC QSLVHSHGN LHWYLQKPGQAPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDMGV RAS TY LIY KVS YFC DQVTQWS 7 EVVLTQTPLSLSVTPGEPVSISC QSLVHSNGN LHWNLQKPGQSPQG HRFTGVPDRFSGSGSGTDFTLRMSRVEADDMG RAS TY LIY KVS VYFC EQGKQWS 7 EIVLTQTPLSLSVTPGEPVSISCR QSLVHSNGN LHWNLQKPGQSPQG HRFTGVPDRFSGSGSGTDFTLRMSRVEADDMG AG TY LIY KVS VYFC EQDKQWS 7 EVVLTQTPLSLSVTPGEPVSISC QSLVHSNGN LHWNLQKPGQSPQG HRFTGVPDRFSGSGSGTDFTLRMSRVEADDMG RAS TY LIY KVS VYFC EQGKQWS 7 EVVLTQTPLSLSVTPGEPVSISC QSLVHSNGN LHWNLQKPGQSPQG HRFTGVPDRFSGSGSGTDFTLRMSRVEADDMG RAS TY LIY KVS VYFC EQGKQWT 7 EVVLTQTPLSLSVTPGEPVSISC QSLVHSHGN LHWYLQKPGQAPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDMGV 2 RAS TY LIY KVS YFC EQDTQWT 7 EVVLTQTPLSLSVTPGEPVSISC QSLVHSNGN LHWNLQKPGQSPQG HRFTGVPDRFSGSGSGTDFTLRMSRVEADDMG RAS TY LIY KVS VYFC EQGEQWT 7 EVVLTQTPLSLSVTPGEPVSISC QSLVHSYGNT LHWYLQKPGQSPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDMGV RAS Y LIY KVS SFC DQGTQWT 7 EVVLTQTPLSLSVTPGEPVPISC QSLVHSYGNT LHWYLQKPGQSPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDMGV RAS Y LIY KVS YFC EQGTQWT 7 EVVLTQTPLSLSVTPGEPVSISY QSLVHSHGN LHWYLQKPGQAPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDMGV RAS TY LIY KVS YFC EQGTQWT 7 EVVLTQTPLSLSVTPGEPVSISC QSLVHSHGN LHWYLQKPGQAPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDMGV RAS TY LIY KVS YFC EQGTQWT 7 EVVLTQTTLSLSVTPGEPVSISC QSLVHSYGNT LHWYLQKPGQSPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDMGV RAS Y LIY KVS YFC EQGTQWT 7 EVVLTQTPLSLSVTPGEPVSISC QSLVHSYGNT LHWYLQKPGQSPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDMGV RAS Y LIY KVS FFC EQGTQWS 7 EVVLTQTPLSLSVTPGEPVSISC QSLVHSYGNT LHWYLQKPGQSPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDMGV RAS Y LIY KVS YFC EQGTQWT 7

249

EVVLTQTPLSLSVTPGEPVSISC QSLLHSNGNT LHWYLQKPGQSPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDMGV DQGTHAQW RAS Y LIY KVS YFC T 9 3 EVVLTQTPLSLSVTPGEPASISC QSLVHSDGN LSWYLQKPGQSPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDVGV FQGTHFPW RAS TY LIY KVS YYC T 9

EVVLTQTPLSLSVTPGESASISC QSLVHSDGN LNWYLQMPGQSPQL NRFTGVPDRFTGSGSGTDFTLRISRVEADDVGV 4 RAS TY LIY RVS YYC YQDTHAPLS 9

EVVLTQTPLSLSVTPGEPVSISC QSLVHSDGN LDWYLQKPGQSPQG NRFTGVPDRFSGSGSGTDFTLRISRVEADDMGV 5 RAS TY LIY KVS YAC EQGTQWP 7

EIVLTQTPLSLSVSSREPASISCR QSLLHSNGN LHWYLQKPGQSPQLL NRFSGVPDRFSGSGSGTDFTLRISRVEADDVGV GQSLHVPQ 6 AS NF IF FAT YYC YF 10

250 Appendix 4.4 HA sequences from viral escape mapping of 4A06 mAb. K163E antibody induced escape mutation is highlighted.

251 Chapter 5 appendix

Appendix 5.1 IgK+ transcripts recovered from NKp46, CD138 and LAMP specific murine memory B-cells

NKp46 J-GENE and CDR1- CDR2- CDR3- CDR3 V-GENE and allele allele FR1-IMGT IMGT FR2-IMGT IMGT FR3-IMGT IMGT JUNCTION length Musmus IGKV14- Musmus DIKMTQSPSSMYASLGE LSWFQQKPGK RLVDGVPSRFSGSGSGQDYSLTIS LQYDEF CLQYDEF 111*01 F IGKJ2*01 F RVTITCKAS QDINSY SPKSLIY RAN SLEYADMGIYYC PYT PYTF 9 Musmus IGKV14- Musmus DIKMTQSPSSMYASLGE LSWFQQKPGK RLVDGVPSRFSGSGSGQDYSLTIS LQYDEF CLQYDEF 111*01 F IGKJ2*01 F RVTITCKAS QDINSY SPKTLIY RAN SLEYEDMGIYYC PYT PYTF 9 Musmus IGKV14- Musmus DIKMTQSPSSMYASLGE LSWFQQKPGK RLVDGVPSRFSGSGSGQDYSLTIS LQYDEF CLQYDEF 111*01 F IGKJ1*01 F RVTITCKAS QDINSY SPKTLIY RAN SLEYEDMGIYYC PST PSTF 9 Musmus IGKV14- Musmus DIKMTQSPSSMYASLGE LSWFQQKPGK RLVDGVPSRFSGSGSGQDYSLTIS LQYDEF CLQYDEF 111*01 F IGKJ5*01 F RVTITCKAS QDINSY SPKTLIY RAN SLEYEDMGIYYC PLT PLTF 9 Musmus IGKV14- Musmus DIKMTQSPSSMYASLGE LSWFQQKPGK RLVDGVPSRFSGSGSGQDYSLTIS LQYDEF CLQYDEF 111*01 F IGKJ5*01 F RVTITCKAS QDINSY SPKTLIY RAN SLEYEDMGIYYC PLT PLTF 9 Musmus IGKV14- Musmus DIKMTQSPSSMYASLGE LSWFQQKPGK RLVDGVPSRFSGSGSGQDYSLTIS LQYDEF CLQYDEF 111*01 F IGKJ1*01 F RVTITCKAS QDINSY SPKTLIY RAN SLEYEDMGIYYC PWT PWTF 9 Musmus IGKV14- Musmus DIKMTQSPSSMYASLGE LSWYQQKPWK SLADGVPSRFSGSGSGQDYSLTIS LQHGES CLQHGES 126*01 F IGKJ5*01 F RVTITCKAS QDIKSY SPKTLIY YAT SLESDDTATYYC PPT PPTF 9 Musmus IGKV14- Musmus DIKMTQSPSSMYASLGE LSWYQQKPWK SLADGVPSRFSGSGSGQDYSLTIS LQHGES CLQHGES 126*01 F IGKJ4*01 F RVTITCKAS QDFKSY SPKSLIY YAT SLESDDTATYYC PFT PFTF 9

CD138 J-GENE and CDR1- CDR2- CDR3- CDR3 V-GENE and allele allele FR1-IMGT IMGT FR2-IMGT IMGT FR3-IMGT IMGT JUNCTION length Musmus IGKV19- Musmus DIQMTQSPSSLSASLGG IAWYQHKPGKG TLQPGIPSRFSGSGSGRDYSFSIS LQYDNL CLQYDNL 93*01 F IGKJ2*01 F KVTITCKASQ PNINKY PRLLIH YTS NLEPEDIATYYC YT YTF 8 Musmus IGKV4- Musmus QIVLTQSPAIMSASLGEEI MHWYQQKSGT NLASGVPSRFSGSGSGTFYSLTIS HQWSS CHQWSSY 80*01 F IGKJ1*01 F TLTCSAS SSVSY SPKLLIY STS SVEAEDAADYYC YPT PTF 8 Musmus IGKV8- Musmus DIVMSQSPSSLAVSAGE QSLLNSRT LAWYQQKPGQ TRESGVPDRFTGSGSGTDFTLTIS KQSYNL CKQSYNL 21*01 F IGKJ4*01 F KVTMSCKSS RKNY SPKLLIY WAS SVQAEDLAVYYC FT FTF 8 Musmus IGKV8- Musmus NIMMTQSPSSLAVSAGE QSVLYSSN LAWYQQKPGQ TRESGVPDRFTGSGSGTDFTLTIS HQYLSS CHQYLSS 27*01 F IGKJ5*01 F KVTMSCKSS QKNY SPKLLIY WAS SVQAEDLAVYYC LT LTF 8

LAMP J-GENE and CDR1- CDR2- CDR3- CDR3 V-GENE and allele allele FR1-IMGT IMGT FR2-IMGT IMGT FR3-IMGT IMGT JUNCTION length Musmus IGKV15- Musmus DIQMNQSPSSLSASLGD LNWYQQKPGNI NLYTGVPSRFSGSGSGTGFTFTIS QQGQS CQQGQSY 103*01 ORF IGKJ4*01 F TITITCHAS QNINVW PKLLIY KAS SLQPEDIATYYC YPFT PFTF 9 Musmus IGKV15- Musmus DIQMNQSPSSLSASLGD LNWYQQKPGNI NLYTGVPSRFSGSGSGTGFTLTIS QQGQS CQQGQSY 103*01 ORF IGKJ4*01 F TITITCHAS QNINVW PKLLIY KAS SLQPEDIATYYC YPFT PFTF 9 Musmus IGKV15- Musmus DIQMNQSPSSLSASLGD LNWYQQRPGS NLHTGVPSRFSGSGSRTGFTLTIS QQGQS SQQGQSY 103*01 ORF IGKJ2*01 F TITITCHAS QNNNVW FPKLLIY KAS SLQPEDIATYYS YPYT PYTF 9

252 Musmus IGKV15- Musmus DIQMNQSPSSLSASLGD LNRYQQKPGNI NLHTGVPSRFSGSGSGTGFTLTIS QQGQS CQQGQSY 103*01 ORF IGKJ2*01 F TITITCHAS HNVNVW PKLLIY KAS SLQPEDIATYYC YPYT PYTF 9

253 Appendix 5.2 Murine immunoglobulin IgG clonal families recovered from ferret CD19 specific B-cells

Clonal V-GENE and J-GENE and CDR1- CDR2- CDR3 FR1-IMGT FR2-IMGT FR3-IMGT CDR3-IMGT family allele allele IMGT IMGT length Musmus Musmus EVKLEESGGGLVRPGGS GFTFS MNWVRQSPEKG IRLKSE HYAESVKGRFTISRDDSKSSVYLEMN TDYDV 5 IGHV6-3*01 F IGHJ1*03 F MKLSCIAS KYW LDWVTQ NYAT NLRAEDTGIYYC Musmus Musmus EVKLEESGGGLVRPGGS GFTFS MNWVRQSPEKG IRLKSE HYAESVKGRFTISRDDSKSSVYLEMN TDYDV 5 IGHV6-3*01 F IGHJ1*03 F MKLSCIAS KYW LDWVTQ NYAT NLRAEDTGIYYC Musmus Musmus EVKLEESGGGLVQPGGS GFTFS MNWVRQSPEKG IRLKSE HYAESVKGRFTISRDDSKSSVYLQM TDYDV 5 IGHV6-3*01 F IGHJ1*03 F MKLSCVAS KFW LEWVAQ NYAT NNLRTEDTGIYYC Musmus Musmus EVKLEESGGGLVRPGGS GFTFS MNWVRQSPEKG IRLKSE HYAESVKGRFTISRDDSKSSVYLEMN TDYDV 5 IGHV6-3*01 F IGHJ1*03 F MKLSCIAS EYW LDWVTQ NYAT NLRAEDTGIYYC 1 Musmus Musmus EVKLEESGGGLVRPGGS GFTFS MNWVRQSPEKG IRLKSE HYAESVKGRFTISRDDSKSSVYLEMN TDYDV 5 IGHV6-3*01 F IGHJ1*03 F MKLSCIAS KYW LDWVTQ NYAT NLRAEDTGIYYC Musmus Musmus EVKLEESGGGLVRPGGS GFTFS MNWVRQSPEKG IRLKSE HYAESVKGRFTISRDDSKSSVYLEMD TDYDV 5 IGHV6-3*01 F IGHJ1*03 F MKLSCIAS KYW LDWVTQ NYAT NLRAEDTGIYYC Musmus Musmus EVKLEESGGGLVRPGGS GFTFS MNWVRQSPEKG IRLKSE HYAESVKGRFTISRDDSKSSVYLEMN TDYDV 5 IGHV6-3*01 F IGHJ1*03 F MKLSCIAS KYW LDWVTQ NYAT NLRAEDTGIYYC Musmus Musmus EVKLEESGGGLVRPGGS GFTFS MNWVRQSPEKG IRLKSE HYAESVKGRFTISRDDSKSSVYLEMN TDYDV 5 IGHV6-3*01 F IGHJ1*03 F MKLSCIAS KYW LDWVTQ NYAT NLRAEDTGIYYC

Musmus Musmus QAYVQQSGAELVRPGA GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F SVNMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFRGKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VKMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QDYLQQFGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKGKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VKMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus LAYLQQFGGELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYLQLS ARIGSGRAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGPGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDSAVYFC 2 Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SHN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDCAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIEA DT SLTSEDSAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDSTVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDSAVYFC

254 Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDCAVYFC Musmus Musmus QAYLQQFGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QAYLQQFGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKNKATLTVDKSSITAYMQVS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VNMSCKAS SYN LEWIGA DT SLTSEDFAVYFC Musmus Musmus QAYLQQSGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKGKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VKMSCKAS SYN LEWIGA DT SLTSEDSAVYFC Musmus Musmus QADLYQFGAELVRPGAS GNTFT MHWVKQTPRQG IYPGNG SYNQKFKGKATLTVDKSSITAYMQLS ARIGSGFAY 9 IGHV1-12*01 F IGHJ3*01 F VKMSCKAS SYN LEWIGA DT SLTSEDCAVYFC

Musmus Musmus EVKLEESGGGLVQPGGS GLTFS MNWVRQSPEKG IRLKSD HYAESVKGQFIISRDDSKNSVYLQMN TGGRDVEDY 9 IGHV6-3*01 F IGHJ2*01 F MKLSCVAS NYW LEWVAQ NYAT NLRTEDTGIYYC Musmus Musmus EVKLEESGGGLVQPGGS GLTFS MNWVRQSPEKG IRLKSD HYAESVKGQFIISRDDSKNSVYLQMN 3 TGGRDVEDY 9 IGHV6-3*01 F IGHJ2*01 F MKLSCVAS NYW LEWVAQ NYAT NLRTEDTGIYHC Musmus Musmus EVKLEESGGGLVQPGGS GLTFS MNWVRQSPEKG IRLKSD HYAESVKGQFIISRDDSKNSVYLQMN TGGRDVEDY 9 IGHV6-3*01 F IGHJ2*01 F MKLSCVAS NYW LEWVAQ NYAT NLRTEDTGIYYC

Musmus Musmus EVKLEESGGGLVQPGGS GLTFS MNWVRQSPEKG IRLKSD HYAESVKGQFIISRDDSKNSVYLQMN TGGRDVEDY 9 IGHV6-3*01 F IGHJ2*01 F MKLSCVAS NYW LEWVAQ NYAT NLRTEDTGIYYC Musmus Musmus EVKLEESGGGLVQPGGS GLTFS MNWVRQSPEKG IRLKFD LYAEFVKGQFIISREDFKNSVYLQMN 4 TGGGDVEDL 9 IGHV6-3*01 F IGHJ2*01 F MKLSCVAS NYW IEWVAQ NYAT KLRIEDIGIYYC Musmus Musmus EVKLEESGGGLVQPGGS GLTFS MNWVRQSPEKG IRLKSD HYAESVKGQFIISRDDSKNSVYLQMN TGGRDVEDY 9 IGHV6-3*01 F IGHJ2*01 F MKLSCVAS NYW LEWVAQ NYAT NLRTEDTGIYYC

Musmus Musmus EVQLQQSGPELVKPGAS GYSFT LNWVKQSPEKTL INPSSD TYNQKFKAKATLTVDRSSSTAYMQL SRSDGGGFA 10 IGHV1-42*01 F IGHJ3*01 F VKISCKTS GNY EWIGE DT RSLTSEDSAIYYC Y Musmus Musmus EVQLQQSGPELVKPGAS GYSFT LNWVKQSPEKTL INPSSD TYNQKFKAKATLTVDRSSSTAYMQL SRSDGGGFA 10 IGHV1-42*01 F IGHJ3*01 F VKISCKTS GNY EWIGE DT RSLTSEDSAIYYC Y 5 Musmus Musmus EVQLQQSGPELVKPGAS GYSFT LNWVKQSPEKTL INPSSD TYNQKFKAKATLTVDRSSSTAYMQL SRSDGGGFA 10 IGHV1-42*01 F IGHJ3*01 F VKISCKTS GNY EWIGE DT RSLTSEDSAIYYC Y Musmus Musmus EVQLQQSGPELVKPGAS GYSFT LNWVKQSPEKTL INPSTD TYNQKFKAKATLTVDRSSSTAYMHLR SRSDGGGFA 10 IGHV1-42*01 F IGHJ3*01 F VKISCKTS GKY EWIGE DT SLTSEDSAIYYC Y

Musmus Musmus QAYLQQSGAELVRPGAS DYTFT IHWVKQTLRQGL IYPGNG SYNQNFKGKATLTIDKSSSTAYMQLS ARLTTVVATN 6 11 IGHV1-12*01 F IGHJ2*01 F VKMSCKAS SYN EWIGA AS SLTSEDSAVYFC Y

255 Musmus Musmus QAYLQQSGAELVRPGAS DYTFT IHWVKQTLRQGL IYPGNG SYNQNFKGKATLTIDKSSSTAYMQLS ARLTTVVATN 11 IGHV1-12*01 F IGHJ2*01 F VKMSCKAS SYN EWIGA AS SLTSEDSAVYFC Y Musmus Musmus QAYLQQSGAELVRPGAS DYTFT IHWVKQTLRQGL IYPGNG SYNQNFKGKATLTIDKSSSTAYMQLS ARLTTVVATN 11 IGHV1-12*01 F IGHJ2*01 F VKMSCKAP SYN EWIGA AS SLTSEDSAVYFC Y Musmus Musmus QAYLQQSGAELVRPGAS DYTFT IHWVKQTLRQGL IYPGNG SYNQNFKGKATLTIDKSSSTAYMQLS ARLTTVVATN 11 IGHV1-12*01 F IGHJ2*01 F VKMSCKAS SYN EWIGA AS SLTSEDSAVYFC Y Musmus Musmus QAYLQQSGAELVRPGAS DYTFT IHWVKQTLRQGL IYPGNG SYNQNFKGKATLTIDKSSSTAYMQLS ARLTTVVATN 11 IGHV1-12*01 F IGHJ2*01 F VKMSCKAS SYN EWIGA AS SLTSEDSAVYFC Y Musmus Musmus QAYLQQSGAELVRPGAS DYTFT IHWVKQTLRQGL IYPGNG SYNQNFKGKVTLTIDKSSSTAYMQLS ARLTTVVATN 11 IGHV1-12*01 F IGHJ2*01 F VKMSCKAS SYN EWIGA AS SLTSEDSAVYFC Y Musmus Musmus QAYLQQSGAELVRPGAS DYTFT IHWVKQTLRQGL IYPGNG SYNQNFKGKATLTIDKSSSTAYMQLS ARLTTVVATN 11 IGHV1-12*01 F IGHJ2*01 F VKMSCKAS SYN EWIGA AS SLTSEDSAVYFC Y Musmus Musmus LANLQQSGGELGRPGAS AYTFT IHWVKQTFRQGL IYPGNV SYNQNFKGKATPRIDKSSSTAYMQLS ARLTTLVATN 11 IGHV1-12*01 F IGHJ2*01 F VKISCKGS SYY EGIGA VS SMTFEEYAVYLC Y Musmus Musmus QAYLQQSGAELVRPGAS DYTFT IHWVKQTLRQGL IYPGNG SYNQNFKGKATLTIDKSSSTAYMQLS ARLTTVVATN 11 IGHV1-12*01 F IGHJ2*01 F VKMSCKAS SYN EWIGA AS SLTSEDSAVYFC Y

Musmus Musmus EVQLQQSGPELVKPGAS GYTFT MNWVKQSHGKS INPNNG TYNQKFQGTATLTVDKSSNTASMEL ARSYYSASY 12 IGHV1-26*01 F IGHJ1*03 F VKISCKAS DYY LEWIGN GT RSLTSEDSAVYYC FDV 7 Musmus Musmus EVQLQQSGPELVKPGAS GYTFT MNWVKQSHGKS INPNNG SYKQNFTGKAILTVDKSSSTAYMELR ARSYYESGY 12 IGHV1-26*01 F IGHJ1*03 F VKISCKAS DYY LEWIGD GT SLTSEDSAVYYC FDV

Musmus Musmus QAYLQQSGAELVRPGAS GYTFT MHWVKQTPGQG IYPGNG SYNQKFKGKATLTVDKSSSTAYMQV ARSGDRLGY 13 IGHV1-12*01 F IGHJ3*01 F VKMSCKAS SYN LEWIGA DT SSLTSEDSAVYFC PFAY Musmus Musmus QAYLQQSGAELVRPGAS GYTFT MHWVKQTPGQG IYPGNG SYNQKFKGKATLTVDKSSSTAYMQV ARSGDRLGY 13 IGHV1-12*01 F IGHJ3*01 F VKMSCKAS SYN LEWIGA DT SSLTSEDSAVYFC PFAY Musmus Musmus QAYLQQSGAELVRPGAS GYTFT MHWVKQTPGQG IYPGNG SYNQKFKGKATLTVDKSSSTAYMQV ARSGDRLGY 13 IGHV1-12*01 F IGHJ3*01 F VKMSCKAS SYN LEWIGA DT SSLTSEDSAVYFC PFAY Musmus Musmus QAYLQQSGAELVRPGAS GYTFT MHWVKQTPGQG IYPGNG SYNQKFKGKATLTVDKSSSTAYMQV ARSGDRLGY 13 IGHV1-12*01 F IGHJ3*01 F VKMSCKAS SYN LEWIGA DT SSLTSEDSAVYFC PFAY 8 Musmus Musmus QAYLQQSGAELVRPGAS GYTFT MHWVKQTPGQG IYPGNG SYNQKFKGKATLTVDKSSSTAYMQV ARSGDRLGY 13 IGHV1-12*01 F IGHJ3*01 F VKMSCKAS SYN LEWIGA DT SSLTSEDFAVYFC PFAY Musmus Musmus QAYLQQSGAELVRPGAS GYTFT MHWVKQTPGQG IYPGNG SYNQKFKGKATLTVDKSSSTAYMQV ARSGDRLGY 13 IGHV1-12*01 F IGHJ3*01 F VKMSCKAS SYN LEWIGA DT SSLTSEDSAVYFC PFAY Musmus Musmus QAYLQQSGAELVRPGAS GYTFT MHWVKQTPGQG IYPGNG SYNQKFKGKATLTVDKSSSTAYMQV ARSGDRLGY 13 IGHV1-12*01 F IGHJ3*01 F VKMSCKAS SYN LEWIGA DT SSLTSEDSAVYFC PFAY Musmus Musmus LDYVQQYVDEVVCQGA GNTFT THWVKQTLRQAL IYPGND SYYQKYNGKFTGTVDKSSSTSYIQVN ARSGARLGS 13 IGHV1-12*01 F IGHJ3*01 F SLRTSCKIS NTN EQIGD AR SQICEESSVDLC PFAY

Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 9 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY

256 Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL PYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F PVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F TVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGSSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQL ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI GSLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SCW EWIGE RI SLTSEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSPSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTFSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY Musmus Musmus QVQLQQPGAEVVKPGA GYTFT ITWVNQRPGQGL LYPGSG NYNEKFKSKATLTVDTSSSTGYMQLS ARGNGNSQS 14 IGHV1-55*01 F IGHJ2*01 F SVKMSCETS SYW EWIGE RI SLTAEDSAVYYC YYFDY

257 Musmus Musmus QAYLQQCGAELVRPGAS DYTFT IHRVKHSFFPFFK SSFYYT QRGGVSISRDKATNTPFSQKTTVRC 10 QATSVDY 7 IGHV1-12*01 F IGHJ4*01 F VKMSCKAS SYN KIQV ET EDTATYYCARGTT Musmus Musmus EVKLEESGGGLVQPGGS GFTFS MNWVRQSPEKG IRLKSD HYAESVKGRFTISRDDSKSSVYLQM 11 TDPGQGF 7 IGHV6-3*01 F IGHJ2*01 F MKLSCVAS NYW LEWVAQ NYVT NNLRAEDTGIYYC Musmus Musmus QVQLQQSGAELVKPGAS GYAFS MNWVKQRPGKG IYPGDG NYNGKFKGKATLTADKSSSTAYMQL 12 ARGSPSYY 8 IGHV1-80*01 F IGHJ2*01 F VKISCKAS SYW LEWIGQ DT SSLTSEDSAVYFC Musmus Musmus QVQLQQPGAELVKPGAS GYSFIS IHWIKQRPGQGL ILPSDS NYNQKFKGKATLTVDKSSSTAYMQL 13 ALWGWLGY 8 IGHV1-74*01 F IGHJ2*01 F VKMSCKAS YW EWIGR DT TSLTSEDSAVYFC Musmus Musmus QVQLQQPGAELVRPGS GYTFT MHWVKQRPIEGL IDPCDS HYNQKFKDKATLTVDKSSNTAYMQL 14 ARNYYGADY 9 IGHV1-52*01 F IGHJ2*01 F SVKLSCKAS SYR EGIGN GA SSLTYEDSAVYYS Musmus Musmus QVQLQQSGAELVKPGAS GYTFT IEWMKQNHGKSL FHPYND RYTENFKGKATLTVEKSSSTVYLELS ARSTVGGFD 15 10 IGHV1-47*01 F IGHJ2*01 F VKMSCKAS NYP EWIGN DT RLTSDDSAVYYC Y Musmus Musmus QVQLQQSGPELVKPGAS GYTFT INWVKQRPGQGL IYPRDG KYNEKFKGKATLTVDTSSSTAYMELH ARGDYGGFF 16 11 IGHV1-85*01 F IGHJ3*01 F VKLSCKAS SYD EWIGW ST SLTSEDSAVYFC AY Musmus Musmus QVQLQQPGTELVKPGAS GYTFT MHWVKQRPGQG INPSNG NYNEKFKSKATLTVDKSSSTAYMQLS ARGSLRQYF 17 11 IGHV1-53*01 F IGHJ1*03 F VKLSCKAS SYW LEWIGN GT SLTSEDSAVHYC DV Musmus Musmus QVQLQQPGAELVKPGAS GYIFIS MHWVKQRPGQG IHPSDS DYNQNFKGKATLTVDKSSSTAYMQL TTRAGRAWF 18 11 IGHV1-74*01 F IGHJ3*01 F VKVSCKAS YW LEWIGR DT SSLTSDDSAVYYC AY Musmus Musmus QVQLQQPGAELVKPGAS GYTFT MHWVKQRPGQG IHPSDS NYNQMFKGKATLTVDKSSSTAYMQL AIKGRVAWF 19 11 IGHV1-74*01 F IGHJ3*01 F VKVSCKAS SYW LEWIGR DT SSLTSEDSAVYHC AY Musmus Musmus QVQLQQPGAELVRPGA GYTFT ITWVKQRPGQGL IYPGSGI KYNEKFNSKATLIVDTSSSTAYMQLS ARWDYDNYL 20 12 IGHV1-55*01 F IGHJ4*01 F SVKMSCKAS NYW EWIGD T SLTSEDSAVYYC PGY Musmus Musmus EVKLLQSGGGLVQPEGS GIDFSR MSWVRRAPGKG INPESN NYAPSLKDKFIISRDNAKNTLYLQMSK ARTGNWAYA 21 12 IGHV4-1*01 F IGHJ4*01 F LKLSCAAS FW LEWIGE TI VRSEDTALYYC MDY Musmus Musmus QVQLQQPGAELVKPGAS GYTFT ITWVKQRPGQGL IYSGSG NFNEKFKSKATLTVDTSSSTAYMQLS ARSGYSNPS 22 12 IGHV1-55*01 F IGHJ2*01 F VKMSCKAS SYW EWIGD SS SLTSEDSAVYYC FDY Musmus Musmus QVQLQQSGAELMKPGA AYTFT IEWVKQRPGHGL ILPGRG NYNEQFKDKATFTADTSSNTAYMQL ARETAQARG 23 13 IGHV1-9*01 F IGHJ2*01 F SVKLSCKAT GYW EWIGE KI SSLTTEDSAIYFC SFDY

258 Appendix 5.3 Murine immunoglobulin IgK clonal families recovered from ferret CD19 specific B-cells

Clonal V-GENE and CDR1- CDR2- CDR3- CDR3 J-GENE and allele FR1-IMGT FR2-IMGT FR3-IMGT family allele IMGT IMGT IMGT length Musmus IGKV1- DIGGTQSPLSLPVSLGD QSLVHSN LHWYLQKPGQS NRFSGVPDRFSGSGSGTDFTLKIS SQSTHV Musmus IGKJ1*01 F KVS 9 110*01 F QASISCRSS GNTY PKLLIY RVEAEDLGVYFC PWT 1 Musmus IGKV1- DIGGTQSPLSLPVSLGD QSLVYSNG LHWYLQKPGQS NRFSGVPDRFSGSGSGTDFTLKIS SQSTHV Musmus IGKJ4*01 F KVS 9 110*01 F QASISCRSS NIH PKLLIY RVEAEDLGVYFC PFT

Musmus IGKV1- DIVATQSPLALSVTIGQS QSLLDSDG LNWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT Musmus IGKV1- DIVATQSPLALSVTIGQS QSLLDSDG LSWLFQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT Musmus IGKV1- DIVATQSPLALSVTIGQS QSLLDSDG LSWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLEIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT Musmus IGKV1- DIVATQSPLALSVTIGQS QSLLDSDG LSWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT Musmus IGKV1- DIGATQSPLTLSVTIGQP QSLLDSDG LSWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLEIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT Musmus IGKV1- DIVATQSPLALSVTIGQS QSLLDSDG LNWLLQRPGQS KLDSGVPDRFIGSGSGTDFTLKISR RQGTHD Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY VEAEDLGVYYC PWT Musmus IGKV1- DIGPTQSPLTLSVTIGQP QSLLDSDG LSWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT Musmus IGKV1- DIVATQSPLALSVTIGQS QSLLDSDG LNWFLQRPGQS VLDSGVPDRFTGSGSGTDFTLKIS WQGKH Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLMY RVEAEDLGVYYC FPWT Musmus IGKV1- DIGATQSPLTLSVTIGQP QSLLDSDG LNWLLQRPDQS KLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT 2 Musmus IGKV1- DIVATQSPLTLSVTIGQP QSLLDSDG LNWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT Musmus IGKV1- DIGGTQSPLTLSVTIGQP QSLLDSDG LNWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT Musmus IGKV1- DIGATQSPLTLSVTIGQP QSLLDSDG LSWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT Musmus IGKV1- DIVATQSPLALSVTIGQS QSLLDSDG LNWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PRT Musmus IGKV1- DIVATQSPLTLSVTIGQP QSLLDSDG LNWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLIISR WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PRRLIY VEAEDLGVYYC PWT Musmus IGKV1- DIVPTQTPLTLSVTIGQP QSLLDSDG LNWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT Musmus IGKV1- DIGAPQTPLTLSVTIGQP QSLLDSDG LSWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLEIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGIYYC PWT Musmus IGKV1- DIVATQSPLTLSVTIGQP QSLLDSDG LSWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT Musmus IGKV1- DIVATQSPLTLSVTIGQP QSLLDSDG LNWFLQRPGQS VLDSGVPDRFTGSGSGTDFTLKIS WQGTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS KTY PKRLIY RVEAEDLGVYYC PWT

Musmus IGKV1- DIGATQSPLTLSVAIGQP QSLLDRG LNWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS CQSTHF 3 Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS GKTY PKRLIY RVEAEDLGVYYC PWT

259 Musmus IGKV1- DIGGTQSPLTLSVAIGQP QSLLDRG LNWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS CQSTHF Musmus IGKJ1*01 F LVS 9 135*01 F ASISCKSS GKTY PKRLIY RVEAEDLGVYYC PWT

Musmus IGKV10- DIVEPQTTSSLSASLGD LDWYQQKPDGT RLHSGVPSRFSGSGSGTDYSLTIS QQGNTL Musmus IGKJ5*01 F QDISNY YTS 9 96*01 F RVTISCRAS VKLLIY NLEQEDIATYFC PLT 4 Musmus IGKV10- DIVATQTTSSLSASLGDR LNWYQQKPDGT RLHSGVPSRFSGSGSGTDYSLTIS QQGNTL Musmus IGKJ1*01 F QDINNY YTS 9 96*01 F VTINCRAS VRLLIY NLEQEDIAIYFC PWT

Musmus IGKV12- DIVATQSPASLSVSVGE LAWYQQKQGK NLADGVPSRFSGSGSGTQYSLKIN QHFWGT Musmus IGKJ1*01 F ENIYSN AAT 9 46*01 F TVTITCRAS SPQLLVY SLQSEDFGSYYC PRT 5 Musmus IGKV12- DIVATQSPASLSVSVGE LAWYQQKQGK NLADGVPSRFSGSGSGTQYSLKIN QHFWGT Musmus IGKJ5*01 F ENIYSN AAT 9 46*01 F TVTITCRAS SPQLLVY SLQSEDFGSYYC PLT

Musmus IGKV3- DIGVTQSTDSLGVSLGQ ESVDSYG MHWYQQKPGQ NLEFGVPARFSGSGSRTEFTLTID QQNNED Musmus IGKJ1*01 F LAS 9 10*01 F RATISCRAS NSF PPKLLI PVEGDDVATYYC PRT 6 Musmus IGKV3- DIGGTQSPASLAVSLGQ ESVDSYG MHWYQQKPGQ NLESGVPARFSGSGSRTDFTLTID QQNNED Musmus IGKJ1*01 F LAS 9 10*01 F RATISCRAS NSF PPKLLIY PVEADDAATYYC PRT

Musmus IGKV3- DIGETQTPTSLAVSLGQ QSVHYDG LNWYQQKPGQ NLESGIPARFSGSGSGTDFTLNIHP QQSNED Musmus IGKJ1*01 F AAS 9 4*01 F RATISCKAS DSY PPKLLIY VEEEDAATYYC PRT 7 Musmus IGKV3- DIGVTQSPASWAVSLGQ QSVDYDG MNWYQQKPGQ NLESGIPARFSGSGSGTDFTLNIHP QQSNED Musmus IGKJ2*01 F AAS 9 4*01 F RATISCKAS DSY PPKLLIY VEEEDAATYYC PYT

Musmus IGKV6- DIVATQSPKFMSTSVGD VAWYQQKPGQ YRYSGVPDRFTGSGSGTDFTLTIS QQYNSY Musmus IGKJ5*01 F QNVGTN SAS 9 15*01 F RVSVTCKAS SPKALIY NVQSEDLAEYFC PLT 8 Musmus IGKV6- DIVATQSPKFMSTSVGD VAWYQQKPGQ YRYSGVPDRFTGSGSGTDFTLTIS QQYNSY Musmus IGKJ2*01 F QNVGTN SAS 9 15*01 F RVSVTCKAS SPKALIY NVQSEDLAEYFC PYT

Musmus IGKV8- DIGATQSPSSLTVTAGE QSLLHSGT LTWHQQKPGQ TRESGVPDRFTGSGSGTDFTLTIS QNDYSY Musmus IGKJ5*01 F WAS 9 19*01 F RVTMSCTSS QKNY PPKLLIY SVQAEDRAVYT PLT 9 Musmus IGKV8- DIGATQSPSSLTVTAGE QSLLNSRT LIWYQQKPGQP TRESGVPERFTGRGSGTDFTLTIS QNDSSH Musmus IGKJ5*01 F WAS 9 19*01 F RVTMSCTSS QKNY PKLLIY SVQAEDLAVYYC PLT

Musmus IGKV1- DIGETQTPLSLPVSLGD QSIVHSNG LEWYLQKPGQS NRFFGVPDRFSGSGSGTDFTLKIS FQGSHV 10 Musmus IGKJ5*01 F KVS 9 117*01 F QASISCRSS NTY PKLLIY RVEAEDLGVYYC PLT Musmus IGKV1- DIVATQSPLTLSVTIGQP QSLLYSNG LNWLLQRPGQS KLDSGVPDRFTGSGSGTDFTLKIS VQGTHF 11 Musmus IGKJ2*01 F LVS 9 133*01 F ASISCKSS NTY PKRLIY RVEAEDLGLYYC PYT Musmus IGKV10- DIGVAHSPSSPCASLGE LKRYQRKPEGD RLHSGVSPRVSGRGCGKDYSLST QHGIMR 13 Musmus IGKJ2*01 F LDTSTY YTS 9 96*01 F RVSITCRAS VKISIY SNTEQEDIATYFC PYT Musmus IGKV12- DIDATQSPASLSASVGE LAWYQQKQGK TLADGVPSRFSGSGSGTQYSLKIN QHFWST 14 Musmus IGKJ2*01 F GNIHNY NAK 9 41*01 F TVTITCRAS SPQLLVY SLQPEDFGSYYC PYT Musmus IGKV12- Musmus IGKJ1*01 F, or DIVAPQTPASLSASVGE LAWYQQKQGK TLAEGVPSRFSGSGSGTQFSLKIN QHHYGT 15 ENIYSY NAK 9 44*01 F Musmus IGKJ1*02 F TVTITCRAS SPQLLVY SLQPEDFGSYYC PPT

260 Musmus IGKV14- DIGATQSPSSMYASLGE LSWFQQKPGKS RLVDGVPSRFSGSGSGQDYSLTIS LQYDEF 16 Musmus IGKJ5*01 F QDINSY RAN 9 111*01 F RVTITCKAS PKTLIY SLEYEDMGIYYC PLT Musmus IGKV14- DIGAPQSPSSMYASLGE LSWFQQKPGKS KLVDGVPPRFSGSGSGQDYSLTIS LQYDDF 17 Musmus IGKJ2*01 F QDINSY RAN 10 111*01 F RVTITCKAS PKTLIY SLEYEDMGIYYC PPYT Musmus IGKV3- DIVATQSPASLAVSLGQ ESVEYYGT MQWYQQKPGQ NVESGVPARFSGSGSGTDFSLNIH QQSRKV 18 Musmus IGKJ1*01 F AAS 9 1*01 F RATISCRAS SL PPKLLIY PVEEDDIAMYFC PWT Musmus IGKV3- DIVVTQSPASLAVSLGQ ESVDSYG MHWYQQKPGQ NLESGIPARFSGSGSRTDFTLTINP QQSNED 19 Musmus IGKJ1*01 F RAS 9 5*01 F RATISCRAS NSF PPKLLIY VEADDVATYYC PRT Musmus IGKV3- DIGATQSPASLAVSLGQ ESVSFAGT MHWYQQKPGQ NLESGVPARFSGSGSESDFTLTID MQSME 20 Musmus IGKJ5*01 F RAS 9 9*01 F RATISCQAS NL PPKLLIY PVEEDDAAMYYC DPLT Musmus IGKV4- DIGATQSPAIMSASPGE LHWYQQKSGAS NLASGVPARFSGSGSGTSYSLTIS QQYSGY 21 Musmus IGKJ5*01 F SSVSSSY STS 9 57-1*01 F KVTMTCRAS PKLWIY SVEAEDAATYYC PLT Musmus IGKV6- Musmus IGKJ1*01 F, or DIGTNPFHKFMSTSVGD VAWYQQKPGQ YRYTGVPDRFTGSGSGTDFTFTIS QQHYST 22 QDVSTA SAS 9 17*01 F Musmus IGKJ1*02 F RVSITCKAS SPKLLIY SVQAEDLAVYYC PPT Musmus IGKV6- DIGMNPFHKFMSTSVGD VAWYRQKPGQ ARHTGVPDRFTGSGSGTDFTLTIT QQYNTY 23 Musmus IGKJ5*01 F QDVGTA WSS 9 23*01 F GVSITCKAS SPKLLIF NVQSEDLADYFC PLT Musmus IGKV8- DIVMTQSPSSLAVSVGE QSLLYSSN LAWFSQVIWRC STESGVPARFSGSGSRTDFTLTID QQNNED 24 Musmus IGKJ1*01 F VPG 9 30*01 F KVTMSCKSS QKNY LVLLLW PVEADDAATYYC PRT Musmus IGKV8- DIVLTQSPSSLTVSAGEK QSLLASGN LAWHQQKPGR TRVSGVPDRFIGSGSGTDFTLTINS QQSYSA 25 Musmus IGKJ1*01 F WAS 9 34*01 F VTMSCKSS QNNY SPKMLII VQAEDLAVYYC PWT

261

Appendix 5.4 Murine immunoglobulin IgG clonal families recovered from ferret IgD specific B-cells

Clonal V-GENE and CDR1- CDR2- CDR3 J-GENE and allele FR1-IMGT FR2-IMGT FR3-IMGT CDR3-IMGT family allele IMGT IMGT length Musmus EVKLEESGGGLVRPG GFTFS MNWVRQSPEK IRLKSE HYAESVKGRFTISRDDSKSSVYL Musmus IGHJ1*03 F TDYDV 5 IGHV6-3*01 F GSMKLSCIAS KYW GLDWVTQ NYAT EMNNLRAEDTGIYYC Musmus EVKLEESGGGLVRPG GFTFS MNWVRQSPEK IRLKSE HYAESVKGRFTISRDDSKSSVYL Musmus IGHJ1*03 F TDYDV 5 IGHV6-3*01 F GSMKLSCIAS KYW GLDWVTQ NYAT EMNNLRAEDTGIYYC 1 Musmus EVKLEESGGGLVRPG GFTFS MNWVRQSPEK IRLKSE HYAESVKGRLTISRDDSKSSVYL Musmus IGHJ1*03 F TDYDV 5 IGHV6-3*01 F GSMKLSCIAS KYW GLDWVTQ NYAT EMNNLRAEDTGIYYC Musmus EVKLEESGGGLVRPG GFTFS MNWVRQSPEK IRLKSE HYAESVKGRFTISRDDSKSSVYL Musmus IGHJ1*03 F TDYDV 5 IGHV6-3*01 F GSMKLPCIAS KYW GLDWVTQ NYAT EMNNLRAEDTGIYYC

Musmus QVQLQQPGAELVKP GYTFI MHWVKQRPG IHPSDS NYNQKFKGKATLTVDKSSSTAY IGHV1-74*01 Musmus IGHJ2*01 F AIGFDY 6 GASVKVSCKAS SFW QGLEWIGR DT MQLSSLTSEDSAVYYC F Musmus QVQLQQPGAELVKP GYIFV MHWVKQRPG IHPSDS YYNQKFKGRATLTVDTSSSTAY IGHV1-74*01 Musmus IGHJ2*01 F AIGFDY 6 GASVKVSCKAS SFW QGLEWIGR ET MQLSSLTSEDSAVYYC F Musmus QVQLQQPGAELVKP GYTFI MHWVKQRPG IHPSDS NYNQKFKGKATLTVDKSSSTAY IGHV1-74*01 Musmus IGHJ2*01 F AIGFDN 6 GASVKVSCKAS SFW QGLEWIGR ET MQLSSLTSEDSAVYYC F Musmus QVQLQQPGAELVKP GYTFI MQWVKLRPGQ IHPCDS HYNQQFKGKATLTVDKSSSTAY 2 IGHV1-74*01 Musmus IGHJ1*03 F AMGFDV 6 GASVKVSCEAS SFW GLDWVGR ET MQFSSLTSEDCAVYFC F Musmus QVQLQQPGAELVKP GYTFI MHWVKLRPGQ IHPSDS HYNQQFKGKATLTVDKSSSTAY IGHV1-74*01 Musmus IGHJ1*03 F AMGFDV 6 GASVKVSCEAS SFW GLDWVGR ET MQLSSLTSEDSAVYFC F Musmus Musmus IGHJ1*01 F, or QVQLQQPGAELVRP GYTFI MHWMKQRPG IHPSDS YYNQNFNDKATLTVDKSSSTAH IGHV1-74*01 AIGFDV 6 Musmus IGHJ1*03 F GASVRVSCKAS SFW QGLEWIGR ET MQLSGLTSEDSAVYFC F Musmus QVQLQQPGAELVKP GYTFI MQWVKQRPG IHPYDS KYNQKFKDKATLAVDKSSSTAY IGHV1-74*01 Musmus IGHJ2*01 F AIGFDN 6 GASVKVSCRAC SFR QGLEWIGR ET MQFSSLTCEDCAVYFR F

Musmus EVQLQQSGPELVTPG GYTFT IDWVKQSRGK IDPNN IYNQKFEGKATLTVDKSSNTAYM IGHV1-18*01 Musmus IGHJ3*01 F VRSWFAY 7 SSVKIPCKAS DYT SLEWIGH GGT ELRSLTSEDTAVFFC F Musmus EVQLQQSGPELVKPG GYTFT MDWLKQSHGK INPNN IYNQKFKGKATLTVDKSSSTAYM 3 IGHV1-18*01 Musmus IGHJ3*01 F ARSWFAY 7 ASVKIPCQAS DYN SLEWIGH GGT DLRSLTSEDTGVYYC F Musmus EVQLQQSGPDLVKP GYTFT MDWVKQSHGK INPND IYNQKFKGKATLTVDKSSSTAYM IGHV1-18*01 Musmus IGHJ3*01 F ARSWFAY 7 GASVKIPCKAS DYN SLEWIGH GDT ELRSLTSEDTAVYYC F

262

Musmus QAYLQQSGAELVRP GYTFI MHWVKQTPRQ IYPGN SYNQNFKGKATLTVDKSSSTAY IGHV1-12*01 Musmus IGHJ3*01 F ASNWDRFTY 9 GASVKISCKAS SYS GLEWIGS GDT MQLNSLTSDDSAVYFC F 4 Musmus Musmus IGHJ3*01 F, or QAYLQQSGAELVRP GYTFI MHWVKQTPRQ IYPGN SYNQLFKAKATLTVDKSSSTAYI IGHV1-12*01 ANNWDRFDY 9 Musmus IGHJ3*02 P GASVKISCKAS NYS GLEWIGS GET QLNSLTSEDSAVYFC F

Musmus EVKLEESGGGLVQPG GLTFS MNWVRQSPEK IRLKSD HYAESVKGQFIISRDDSKNSVYL Musmus IGHJ2*01 F TGGRDVEDY 9 IGHV6-3*01 F GSMKLSCVAS NYW GLEWVAQ NYAT QMNNLRTEDTGIYYC 5 Musmus EVKLEESGGGLVQPG GLTFS MNWVRQSPEK IRLKSD HYAESVEGQFIISRDDSKNSVYL Musmus IGHJ2*01 F TGGRDVEDY 9 IGHV6-3*01 F GSMKLSCVAS NYW GLEWVAQ NYAT QMNNLRTEDTGIYYC

Musmus QVQVQQSGAELVKP GYAFS MNWVKQRPGK IYPGD RYNGQFKDRVTLTVDKSSSTAY IGHV1-80*01 Musmus IGHJ2*01 F ARGTGYYFDY 10 GASVKIPCKAS SYW GLEWIGQ GET MQFTSLTSEDSAVYFC F Musmus QVQVQQSGAELVKP GYAFS MNWVKQRPGK IYPGD RYNGKFKDKATLTVDRSSSTAY IGHV1-80*01 Musmus IGHJ2*01 F ARGTGYYFDY 10 GASVKIPCKAS SYW GLEWIGQ GET MQFSSLTSEDSAVYFC F 6 Musmus QVQLQQSGAEVVKP GYAFN MDWVKQRPGK IYPGD NYNGNFKGKATVTADKSSSKAY IGHV1-80*01 Musmus IGHJ2*01 F ARGTGYYFDY 10 GASVKISCKAS NYW GLEWIGQ GET MQLSSLTSEDSAVYFC F Musmus QVQVQQSGAELVKP GYAFS MNWVKQRPGK IYPGD RYNGKFKDKATLTVDRSSSTAY IGHV1-80*01 Musmus IGHJ2*01 F ARGTGYYFDY 10 GASVKIPCKAS SYW GLEWIGQ GET MQFSGLTSEDSAVYFC F

Musmus QVQLQQSGAELVKP GYIFS MNWVKQRPGK IYPGD NYNGKFRGKAALTADKSSNTAS AREGDYSYTM IGHV1-80*01 Musmus IGHJ4*01 F 12 GASVKVSCTPS DYW GLEWIGQ GDT MQLSSLTSEDSAVYFC DY F 7 Musmus QVQLQQSGAELVKP GYAFT MNWVKQRPG IYPGD NYNGKFKGKATLTADKSSSTAY ARGDYASIYVD IGHV1-80*01 Musmus IGHJ2*01 F 12 GASVKISCKAS SYW RGLEWIGQ GET MQLSSLTSEDSAVYFC Y F

Musmus RVHFQQYGEELGRH GNTFT TSWVKQTLKQ KDPGN KNNQKFKGKFTMKVDKSSSTSYI GRSGARLGYG IGHV1-12*01 Musmus IGHJ3*01 F 13 GASLKTSGKTS SSN ALEQIG GD QVSSQTCEDSSVYFC FAY F 8 Musmus QAYLQQSGAELVRP GYTFT MHWVKQTPGQ IYPGN SYNQKFKGKATLTVDKSSSTAY ARSGDRLGYP IGHV1-12*01 Musmus IGHJ3*01 F 13 GASVKMSCKAS SYN GLEWIGA GDT MQVSSLTSEDSAVYFC FAY F

Musmus QVQLQQPGAEVVKP GYTFT ITWVNQRPGQ LYPGS NYNEKFKSKATLTVDTSSSTGY ARGNGNSQSY 9 IGHV1-55*01 Musmus IGHJ2*01 F 14 GASVKMSCETS SYW GLEWIGE GRI MQLSSLTAEDSAVYYC YFDY F

263 Musmus QVQLQQPGAEVVKP GYTFT ITWVNQRPGQ LYPGS NYNEKFKSKATLTVDTSSSTGY ARGNGNSQSY IGHV1-55*01 Musmus IGHJ2*01 F 14 GASVKMSCETS SYW GLEWIGE GRI MQLSSLTAEDSAVYYC YFDY F Musmus QVQLQQPGAEVVKP GYTFT ITWVNQRPGQ LYPGS NYNEKFKSKATLTVDTSSSTGY ARGNGNSQSY IGHV1-55*01 Musmus IGHJ2*01 F 14 GASVKMSCETS SYW GLEWIGE GRI MQLSSLTAEDSAVYYC YFDY F Musmus QVQLQQPGAEVVKP GYTFT ITWVNQRPGQ LYPGS NYNEKFKSKATLTVDTSSSTGY ARGNGNSQSY IGHV1-55*01 Musmus IGHJ2*01 F 14 GASVKMSCETS SYW GLEWIGE GRI MQLSSLTAEDSAVYYC YFDY F Musmus QVQLQQPGAEVVKP GYTFT ITWVNQRPGQ LYPGS NYNEKFKSKATLTVDTSSSTGY ARGNGNSQSY IGHV1-55*01 Musmus IGHJ2*01 F 14 GASVKMSCETS SYW GLEWIGE GRI MQLSSLTAEDSAVYYC YFDY F

Musmus QVQLQQSGAELARP GYTFR ITWMRERTGQ IYPTSD YYNEKFKGKATLTADKSSSTAY ARSTYGSNYD IGHV1-81*01 Musmus IGHJ4*01 F 15 GASVKLSCKAS NYG GLEWIGE NT MEFRSLTSEDSAVYFC DALDH F 10 Musmus QAQLQQSGAELARP GYTFT IHWVKQRTGQ IYPRSG YYNEKFKGKATLTADKSSSTAY ARSSYGSYYD IGHV1-81*01 Musmus IGHJ4*01 F 15 GASVKLSCKTS SFG GLEWIGE NT MELRSLTSEASAVYFC DAVDY F

Musmus QVQLQQSGAELVKP GYAFS MNWVKQRPGK IYPGH NYNGKFKGKATLTANKSSNTAY 11 IGHV1-80*01 Musmus IGHJ2*01 F ARGDY 5 GASVKISCKAS SFW GLEWIGQ GDT MQLSSLTSEDSAVYFC F Musmus QVQLQQPGAELVRP GYTFT MHWVKQRPIQ IDPSDS HYNQKFKDKATLTVDKSSSTAY 12 IGHV1-52*01 Musmus IGHJ3*01 F ASSFTY 6 GSSVKLSCKAS TYW GLEWIGN ET MQLSSLTSEDSAVYYC F Musmus QVQLQQPGAELVRP GYTFT LHWVIQRPIQG IDPSDS HYNQKFKNKATLTVDKSSSTAY 13 IGHV1-52*01 Musmus IGHJ2*01 F ARDQEH 6 GSSVKLSCKAS NFW LEWIGN ET MQLSSLTSEDSAVYYC F Musmus QVQLQQPGAELVRP GYTFT MHWVKQRPG IAPSDN NYNQKFKGKATLTVDTSSNTAY 14 IGHV1-59*01 Musmus IGHJ3*01 F TLGFPY 6 GTSVKLSCKAS SYW QGLEWIGV YT MQLTSLTSEDSAVYYC F Musmus QVQLQQPGAELVMP GYTFI MHWMKQRPG IDPSDS NYNQKFKGKSTLTVDKSSSTAY 15 IGHV1-69*01 Musmus IGHJ2*01 F AVSFDY 6 GASVKLSCKAS SYW QGLEWIGE YT MQLSSLTSEDSAVYYC F Musmus QVQLQQPGADLVKP SSAVP MHWVKQRPG IDPNSG EYNEKFKNKATLTVDKSSSTAY 16 IGHV1-72*01 Musmus IGHJ3*01 F AVGFAS 6 GASVKLSCEAW TFW RGLEWIGR DS MQLSSLTSEDSAIYFC F Musmus QAYLQQSGAELVRP GNTFT MHWVKQTPRQ IYPGN SYNQKFKNKATLTVDKSSITAYM 17 IGHV1-12*01 Musmus IGHJ3*01 F ARIGSGFAY 9 GASVNMSCKAS SYN GLEWIGA GDT QLSSLTSEDSAVYFC F Musmus QAYLQQSGAEVVRP GYTFT MHWVKQTPRQ IYPGN SYNQKFKGKAILSVDKTSSTAYM 18 IGHV1-12*01 Musmus IGHJ1*03 F ARGYWYFDV 9 GASVKMSCKAS SYN GLEWIGA GDT QLSSLTSEDSAVYFC F

264 Musmus QAYLQQSGAELVRP GNTFT MHWVKQTPRQ IYPGN SYNQKFKNKATLTVDKSSITAYM 19 IGHV1-12*01 Musmus IGHJ3*01 F ARIGSGFAY 9 GASVNMSCKAS SYN GLEWIGA GDT QLSSLTSEDSAVYFC F Musmus QVQLQQPGAELVKP GYTFT MHWVKQRPG IHPNSG NYNEKFKSKATLTVDKSSSTAY 20 IGHV1-64*01 Musmus IGHJ2*01 F ARFGTTPLDY 10 GASVKLSCKAS SYW QGLEWIGM ST MQLSSLTSEDSAVYYC F Musmus QVQLQQSGAELVRP GYTFT MHWVKQTPVH IDPETG AYNQKFKGKAILTADKSSSTAYM TRSHDYDWFA 21 IGHV1-15*01 Musmus IGHJ3*01 F 11 GGSVTLSCKAS DYE GLEWIGA GT ELRSLTSEDSAVYYC Y F Musmus EVQLQQSGPELVKPG GYTFT MHWVKQSHGK INPNN SYNQKFKGKATLTVNKSSSTAY ARWENWGRFA 22 IGHV1-22*01 Musmus IGHJ3*01 F 11 ASVKMSCKAS DYN SLEWIGY GGT MELRSLTSEDSAVYFC Y F Musmus QVQLQQPGTELVKP GYTFT IHWVKQRPGQ INPSNG HYNEKFKIKATLTVDKSSSTAYM ARSDDYDWFA 23 IGHV1-53*01 Musmus IGHJ3*01 F 11 GASVKLSCKAS SYY GLEWIGN GT QLSSLTSEDSAVYYC Y F Musmus QVQLQQSGAELMKP GYSFT IEWVKQRPGH IFPGSD NYSEKFKGKATFTADTSSNTAY 24 Musmus IGHJ2*01 F ARSYYSNYFDY 11 IGHV1-9*01 F GASVKLSCKAT GYW GLEWIGE ST MQLSSLTTEDSAIYYC Musmus QAQLQQSGTELMKP GYRFT IEWVKERPGH ILPESG NYNGNFKDKATFTVDTSSNTAY ARLGNNHPFA 25 Musmus IGHJ3*01 F 11 IGHV1-9*01 F GASVKLSCKAS GYW GLEWIGE ST MQLSRLTTEDSAIYYC Y Musmus QVQLQQSGAELVRP GYTFS IHWVKQTPVH IDPETG AYNQKFKGKAIVTADKSSSTAYM TRHDYDYDWF 26 IGHV1-15*01 Musmus IGHJ3*01 F 12 GASVTLSCKAS DYV GLEWIGA GT EFRSLTSEDSAVYYC AY F Musmus EVQLQQSGPEVMKP GYTFT MNWVKQSHGK INPNKG SYNQKFKGKATLTVDKSSSTAY ARSLLQSYYFD 27 IGHV1-26*01 Musmus IGHJ2*01 F 12 GASVNISCKAS DYY SLEWIGD GT MELRSLTSEDSAVYYC Y F Musmus EVKLEESGGGLVQPG GFTFS MDWVRQSPEK IRNKAN YYAESVKGRFTISRDDSKSSVYL TRPLYGSSGSF 28 Musmus IGHJ3*01 F 13 IGHV6-6*01 F GSMKLSCTAS DAW GLEWVAE NHAT QMNSLRAEDTGIYYC AY Musmus EVQLQQSGPELVKPG GYTFT MNWVKQSHGK INPNN SYNQKFKGKATLTVDKSSSTAY ARKGGYYSSS 29 IGHV1-26*01 Musmus IGHJ2*01 F 14 ASVKISCKAS DYF SLEWIGD GGT MELRSLTSEDSAVYYC YFDY F Musmus Musmus IGHJ2*01 F, or QVQLQQSGPELVKP GYAFS MNWVKQRPGK IYPGD NYNGKFKGKATLTADKSSSTAY ARGSYDAYRY 30 IGHV1-82*01 14 Musmus IGHJ2*02 F GASVKISCKAS SSW GLEWIGR GDT MQLSSLTSEDSAVYFC YFDY F Musmus DVKLVESGGGLVKPG GFTFS MSWIRQTPEK LVSSG FYADAVKGRFTISRDNAGSTLFL TRENWDNYGR 31 IGHV5-9-1*02 Musmus IGHJ2*01 F 18 GSLKLSCTAS SYA RLEWLAY VHT QMSSLQSEDTAMYYC SFSYYFDY F

265 Appendix 5.5 Murine immunoglobulin IgK clonal families recovered from ferret IgD specific B-cells

Clonal V-GENE and J-GENE and allele FR1-IMGT CDR1- FR2-IMGT CDR2- FR3-IMGT CDR3- CDR3 family allele IMGT IMGT IMGT length 1 Musmus Musmus IGKJ1*01 F DIVLTQTTSSLSASLG QDISNY LNWYQQKPD YTS RLHSGVPSRFSGSGSGTDYS QQGNT 9 IGKV10-96*01 DRVTISCRAS GTVKLLIY LTISNLEQEDIATYFC LPWT F Musmus Musmus IGKJ5*01 F DIQMTQTTSSLSASL QDISNY LNWYQQKPD YTS RLHSGVPSRFSGSGSGTDYS QQGNT 9 IGKV10-96*01 GDRVTISCRAS GTVKLLIY LTISNLEQEDTATYFC LPLT F Musmus Musmus IGKJ1*01 F DIQMTQTTSSLSASL QDISNY LNWYQQKPD YTS RLHSGVPLRFSGGGSGTDYS QQVNT 9 IGKV10-96*01 GDRVTISCRAS GTVKLLIY LTISNLEQEDIATYFC LPWT F Musmus Musmus IGKJ5*01 F DIQETQTTSSLSASL QDISNY LNWYQQKPD YTS RLHSGVPSRFSGSGSGTDYS QQGNT 9 IGKV10-96*01 GDRVTISCRAS GTVKLLIY PTISNLEQEDIATYFC LPLT F Musmus Musmus IGKJ5*01 F DIVLTQTTSSLSASLG QDISNY LNWYQQKPD YTS RLHSGVPSRFSGSGSGTDYS QQGNT 9 IGKV10-96*01 DRVTISCRAS GTVKLLIY LTISNLEQEDIATYFC LPLT F Musmus Musmus IGKJ5*01 F DIQMTQTTSSLSASL QDISNY LNWYQQKPD YTS RLHSGVPSRFSGSGSGTDYS QQGNT 9 IGKV10-96*01 GDRVTISCRAS GTVKLLIY LTISNLEQEDIATYFC LPLT F

2 Musmus Musmus IGKJ1*01 F DIQCPQSPASLSVSV ENIYSN LAWYQQKQG AAT NLADGVPSRFSGSGSGTQYS QHFWG 9 IGKV12-46*01 GETVTITCRAS KSPQLLVY LKINSLQSEDFGSYYC TPWT F Musmus Musmus IGKJ1*01 F DIQATQSPASLSVSV ENIFSN LAWYQQKQG AAT KLADGVPSRFSGSGSGTQYS QHFWG 9 IGKV12-46*01 GETVTITCRAS KFFQLLVY LKINSLQSEDFGSYYC TPRT F Musmus Musmus IGKJ1*01 F DIQCPQSPASLSVSV ENIYSN LAWYQQKQG AAT NLADGVPSRFSGSGSGTQYS QHFWG 9 IGKV12-46*01 GETVTITCRAS KSPQLLVY LKINSLQSEDFGSYYC TPRT F

3 Musmus Musmus IGKJ5*01 F DIVLTQSPSSTYASL QDINSY LSWFQQKPG RAN RLVDGVPSRFSGSGSGQDYS LQYDE 9 IGKV14- GERVTITCKAS KSPKTLIY LTISSLEYEDMGIYYC FPLT 111*01 F Musmus Musmus IGKJ5*01 F DIVLTQSPSSTYASL QDINSY LSWFQQKPG RAN RLVDGVPSRFSGSGSGQDYS LQYDE 9 IGKV14- GERVTITCKAS KSPKTLIY LTISSLEYEDMGIYYC FPLT 111*01 F Musmus Musmus IGKJ2*01 F DIVLTQSPSSTYASL QDINSY LSWFQQKPG GAN RPQNGVPSRFSGSGSGQDY TQDDE 9 IGKV14- GERVTITCKAS KYIKTLIY SFTISSLECEDMGKHYC HPHT 111*01 F

266 4 Musmus Musmus IGKJ5*01 F DIKMTQSPSSMYASL QDINNY LTWFQQKPG HTN RLLNGVPSKFSGSGFGQDYS YKYYEF 9 IGKV14- EERVTITCKAS KFPRTLIY LTIISREYEDMGIYYV PLT 111*01 F Musmus Musmus IGKJ2*01 F, or Musmus DIKMTQSPSSSYASL QDISSY LNWFQQKPE YTN RLVDGVPSRFSGSGSGQDYS IQCNEF 9 IGKV14- IGKJ5*01 F GERVTISCRAS KSLKTLIY LTISKPEQEDMEFIM PLT 111*01 F

5 Musmus Musmus IGKJ5*01 F DIKMTQSPSSMYASL QDINSY LSWFQQKPG RAN RLVDGVPSRFSGSGSGQDYS LQYDE 9 IGKV14- GERVTITCKAS KSPKTLIY LTISSLEYEDMGIYYC FPLT 111*01 F Musmus Musmus IGKJ1*01 F DIKMTQSPSSMYASL QDIVKN LNWYQQKPG YAT ELAEGVPSRFSGSGSGSDYS LQFYEF 9 IGKV14- GERVTITCKAS KPPSFLIY LTISNLESEDFADYYC PRT 130*01 F

6 Musmus Musmus IGKJ2*01 F ETTVTQSPASLSVAT TDIDDD MNWYQQKP EGN SPRPGVPSRFSSSGYGTDFV LQTDT 9 IGKV17- G EKVTIRCITS GEPPKLLIS FTIENTLSEDVADYYC MPYT 127*01 F Musmus Musmus IGKJ2*01 F, or Musmus ETTVTQSPASLSVAT TDIDDD MNWYQQKP EGN TLRPGVPSRFSSSGYGTDFV LQSDN 9 IGKV17- IGKJ5*01 F G EKVTIRCITS GEPPKLLIS FTIENTLSEDVADYYC MPLT 127*01 F Musmus Musmus IGKJ2*01 F, or Musmus ETTVTQSPASLSVAT TDIDDD MNWYQQKP EGN TLRPGVPSRFSSSGYGTDFV LQSDN 9 IGKV17- IGKJ5*01 F G EKVTIRCITS GEPPKLLIS FTIENTLSEDVADYYC MPLT 127*01 F

7 Musmus Musmus IGKJ4*01 F DIGATQSPASLAVSL ESVDSY MHWYQQKP LAS NLESGVPARFSGSGSRTDFT QQNNE 9 IGKV3-10*01 F GQRATISCRAS GNSF GQPPKLLIY LTIDPVEADDAATYYC DPFT Musmus Musmus IGKJ1*01 F DIGATQSPASLAVSL ESVDSY MHWYQQKP LAS NLESGVPARFSGSGSRTDFT QQNNE 9 IGKV3-10*01 F GQRATISCRAS GNSF GQPPKLLIY LTIDPVEADDAATYYC DPRT

8 Musmus Musmus IGKJ1*01 F DIGPTQSPASLAVSL ESVDNY MNWFQQKPG AAS NQGSGVPARFSGSGSGTDFS QQSKE 9 IGKV3-2*01 F GQRATISCRAS GISF QPPKLLIY LNIHPMEEDDTAMYFC VPWT Musmus Musmus IGKJ2*01 F DIVLTQSPASLAVSLG ESVDNY MNWFQQKPG AAS NQGSGVPARFSGSGSGTDFS QQSKE 9 IGKV3-2*01 F QRATISCRAS GISF QPPKLLIY LNIHPMEEDDTAMYFC VPYT Musmus Musmus IGKJ5*01 F DIVLTQSPASLAVSLG ESVDNY MNWFQQKPG AAS NQGSGVPARFSGSGSGTDFS QQSKE 9 IGKV3-2*01 F QRATISCRAS GISF QPLKLLIY LNIHPMEEDDTAMYFC VPLT Musmus Musmus IGKJ5*01 F DIVLTQSPASLAVSLG ESVDNY MNWFQQKPG VAS NQGSGVPARFSGSGSGTDFS QQSKE 9 IGKV3-2*01 F QRATISCRAS GISF QPPKLLIC LNIHPMEEDDTAMYFC VPLT Musmus Musmus IGKJ5*01 F DIAQTQSPASLAVSL ESVDNY MNWFQQKPG AAS NQGSGVPARFSGSGSGTDFS QQSKE 9 IGKV3-2*01 F GQRATISCRAS GISF QPPKLLIY LNIHPMEEDDTAMYFC VPLT Musmus Musmus IGKJ1*01 F DIVLPQTPASLAVSLG ESVDNY MNWFQQKPG AAS NQGSGVPARFSGSGSGTDFS QQSKE 9 IGKV3-2*01 F QRATISCRAS GISF QPPKLLIY LNIHPMEEDDTAMYFC VPWT Musmus Musmus IGKJ1*01 F DIVLTQSPASLAVSLG ESVDNY MNWFQQKPG AAS NQGSGVPARFSGSGSGTDFS QQSKE 9 IGKV3-2*01 F QRATISSRAS GISF QPPKLLIY LNIHPMEEDDTAMYFC VPWT

267 9 Musmus Musmus IGKJ2*01 F DIGATQSPAIMSASL SSVNY IYWYQQKSD YTS NLALDVPTRFSGSGCGNYYS QQSTS 9 IGKV4-50*01 F GEKVTMSCRAS ASPKLWIY LTISSMEGEDGATYYC SPYT Musmus Musmus IGKJ2*01 F DIGATQSPAIMSASL SSVNY MYWYQQKSD YTS NLAPGVPARFSGSGSGISYSL QQFTN 9 IGKV4-50*01 F GEKVTMSCRAS ASPKLWIY TISSLETEDTATYYC FPYT Musmus Musmus IGKJ2*01 F DIGATQSPAIMSASL SSVNY MYWYQQKSD YTS NLAPGVPARFSGSGSGISYSL QQFTS 9 IGKV4-50*01 F GEKVTMSCRAS ASPKLWIY TISSMEGEDAATYYC FPYT

10 Musmus Musmus IGKJ2*01 F, or Musmus ENVLTQSPAIMSASP SSVSY MHWFQQKPG STS NLASGVPARFSGSGSGTSYS QQRSS 9 IGKV4-57*01 F IGKJ2*03 F or Musmus IGKJ5*01 F GEKVTITCSAS TSPKLWIY LTISRMEAEDAATYYC YPLT Musmus Musmus IGKJ5*01 F ENVLTQSPAIMSASP SSVSY MHWFQQKPG STS NLASGVPARFSGSGSGTSYS QQRSS 9 IGKV4-57*01 F GEKVTITCSAS TSPKLWIY LTISRMEAEDAATYYC YPLT Musmus Musmus IGKJ5*01 F DIVATQSPAIMSASP SSVSY MHWFQQKPG STS NLASGVPARFSGSGSGTSYS QQRSS 9 IGKV4-57*01 F GEKVTITCSAS TSPKLWIY LTISRMEAEDAATYYC YPLT Musmus Musmus IGKJ5*01 F DIVQTQTPAIMSASP SSVSY MHWFQQKPG STS NLASGVPARFSGSGSGTSYS QQRSS 9 IGKV4-57*01 F GEKVTITCSAS TSPKLWIY LTIGRMEAEDAATYYC YPLT

11 Musmus Musmus IGKJ2*01 F DILLTQSPAILSVSPG QSIGTS IHWYQQRTN YAS ESISGIPSRFSGSGSGTDFTL QQSNS 9 IGKV5-48*01 F ERVSFSCRAS GSPRLLIK SINSVESEDIADYYC WPYT Musmus Musmus IGKJ5*01 F DILLTQSPAILSVSPG QSIGTT IHWYQQRTN YAS VSISGIPSRFSGSGSGTDFTL QQSNS 9 IGKV5-48*01 F ERVSFSCRAS GSPRLLIK SINSVESEDIADYYC WPLT Musmus Musmus IGKJ5*01 F DILLTQSPAILSVSPG QSIGTS IHWYQQRTN YTS ESISGIPSRFSGSGSGTDFTL QQSNS 9 IGKV5-48*01 F ERVSFSCRAS GSPRLLIE SINSVESEDIADYYC WPLT

12 Musmus Musmus IGKJ1*01 F DIVCPQSPSSPAMSV QSLLNSS LAWYQQKPG FAS TRESGVPDRFIGSGSGTDFTL QQHYS 9 IGKV8-24*01 F GQKVTMSCKSS NQKNY QSPKLLVY TISSVQAEDLADYFC TPRT Musmus Musmus IGKJ4*01 F DIVCPQSPSSPAMSV QSLLNSN LAWYQQKPG FAS TRESGVPDRFIGSGSGTDFTL QQHYS 9 IGKV8-24*01 F GQKVTMSCKSS NQKNY QSPKLLVY TISSVQAEDLADYFC TPFT

13 Musmus Musmus IGKJ2*01 F DIGATQSPSSMYASL QDINSY LSWFQQKPG RAN KLVDGVPPRFSGSGSGQDYS LQYDD 10 IGKV14- GERVTITCKAS KSPKTLIY LTITSLEYEDMGIYYC FPPHF 111*01 F Musmus Musmus IGKJ2*01 F DIVLTQSPSSMYASL QDINGY LSWFQQKPG RAN RLVDGVPSRFSGSGSGQDYS LQYDD 10 IGKV14- GERVTITCKAS KPPKTLIY LTISSLEYEDMGIYYC FPPYT 111*01 F Musmus Musmus IGKJ2*01 F DIVLTQSPSSMYASL QDINGY LSWFQQKPG RAN RLVDGVPSRFSGSGSGQDYS LQYDD 10 IGKV14- GERVTITCKAS KPPKTLIY LTISSLEYEDMGIYYC FPPYT 111*01 F Musmus Musmus IGKJ2*01 F DIVLTQSPSSMYASL QDINSY LSWFQQKPG RAN KLVDGVPSRFSGSGSGQDYS LQYDD 10 IGKV14- GERVTITCKAS KSPKTLIY LTISSLEYEDMGIYYC FPPYT 111*01 F Musmus Musmus IGKJ2*01 F DIGATQSPSSMYASL QDINSY LSWFQQKPG RAN RLVDGVPSRFSGSGSGQDYS LQYDD 10 IGKV14- GERVTITCKAS KSPKTLIY LTISSLEYEDMGIYYC FPPYT 111*01 F

268 14 Musmus Musmus IGKJ1*02 F DIQLTQTTSSLSASLG QDISNY LNWYQQKPD YTS RLHSGVPSRFSGSGSGTDYS QQGNT 8 IGKV10-96*01 DRVTISCRAS GTVKLLIY LTISNLEQEDIATYFC LPT F 15 Musmus Musmus IGKJ1*01 F NIMMTQSPSS QSVLYSS LAWYQQKPG WAS TRESGVPDRFTGSGSGTDFT HQYLS 8 IGKV8-27*01 F LAVSAENVTMSCKSS NQKNY QSPKLLIY LTISSVQAEDLAVYYC SRT 16 Musmus Musmus IGKJ1*01 F DIGGPQSPLSLPVSL QSLVHS LHWYLQKPG KVS NRFSGVPDRFSGSGSGTDFT SQSTH 9 IGKV1-110*01 GDQASISCRSS NGNTY QSPKLLIY LKISRVEAEDLGVYFC VPWT F 17 Musmus Musmus IGKJ2*01 F DIGATQSPLSLPVSL QSIVHSN LEWYLQKPG KVS NRFSGVPDRFSGSGSGTDFT FQGSH 9 IGKV1-117*01 GDQASISCRSS GNTY QSPKLLIY LKISRVEAEDLGVYYC VPYT F 18 Musmus Musmus IGKJ1*01 F DIVATQTPLSLPVSLG QSLENIN LNWYLQKPG RVS NRFSGVLDRFSGSGSGTDFT LQITHV 9 IGKV1-122*01 DQASISCRSG GNTY QSPQLLIY LKISRVEAEDLGVYFC PWT F 19 Musmus Musmus IGKJ1*01 F DIQMTQTTSSLSASL QGISNY LNWYQQKPD YTS SLHSGVPSRFSGSGSGTDYS QQYSK 9 IGKV10-94*01 GDRVTISCSAS GTVKLLIY LTISNLEPEDIATYYC LPWT F 20 Musmus Musmus IGKJ2*01 F DIQMTQSPASLFASA GNIHNY LAWYQQKQG NAK TLADGVPSRFSGSGSGTQYS QHVWS 9 IGKV12-41*01 GETVTITCRAS KSPQLLVY FKINSLQPKDFGSYY TPYT F 21 Musmus Musmus IGKJ1*01 F DIQMTQSPASLSASV ENIYSY LAWYQQKQG NAR TLPEGVPSRFSGSGSGTQFS QHHYG 9 IGKV12-44*01 GETVTITCRAS KSPQLVVY LKINSLQPEDFGSYYC TPRT F 22 Musmus Musmus IGKJ2*01 F DIKMTQSPSSMYASL QDINGY LSWFQQKPG RAN RLVDGVPSRFSGSGSGQDYS LQLMTF 9 IGKV14- GERVTITCKAS KPPKTLIY LTISSLEYEDMGIYY LRT 111*01 F 23 Musmus Musmus IGKJ1*01 F RHCATQSPSYLAASP KSISKY LAWYQEKPG SGS TLQSGIPSRFSGSGSGTDFTL QQHNE 9 IGKV16- GETITINCRAS KTNKLLIY TISSLEPEDFAMYYC YPWT 104*01 F 25 Musmus Musmus IGKJ1*01 F DIVLTQSPASLAVSLG ESVDSY MHWYQQKP RAS NLESGIPARFSGSGSGTDFTL HQSNE 9 IGKV3-5*01 F QRATISCRAS GNSF GQPPKLLIY TINPVEADDVATYYC DPRT 26 Musmus Musmus IGKJ2*03 F QIVLTQSPAIMSASP SSVSF MYWYQQKPG DTS NLASGVPVRFSGSGSGTSYS QQWSS 9 IGKV4-55*01 F GEK VTMTCSAS SSPRLLIY LTISRMEAEDTATYYC YPIT 27 Musmus Musmus IGKJ1*01 F DIVLTQSPAIMSASPG SSVSSSY LHWYQQKSG STS NLASGVPARFSGSGSGTSYS QQYSG 9 IGKV4-57- EKVTMTCRAS ASPKLWIY LTISSVEAEDAATYYC YPWT 1*01 F 28 Musmus Musmus IGKJ2*01 F, or Musmus QIVLTQSPAIMSA SSVSY MYWYQQKPG RTS NLASGVPARFSGSGSGTSYS QQYHS 9 IGKV4-61*01 F IGKJ2*03 F or Musmus IGKJ5*01 F SHGEVTISCSAS SSPKPWIY LTISSMEAEDAATYYC YPLT 29 Musmus Musmus IGKJ1*01 F, or Musmus DIGPTQSPALMYASP SSVSY MYWYQQKPR LTS NLASGVPARFSGSGSGTSYS QQWSS 9 IGKV4-68*01 F IGKJ1*02 F GEKVTRTCSAS SSPKPWIY LTISSMEAEDAATYYC NPPT 30 Musmus Musmus IGKJ5*01 F DIVMTQSQKFMSTSV QNVGTA VAWYQQKPG SAS NRYTGVPDRFTGSGSGTDFT QQYSS 9 IGKV6-13*01 G DRVSITCKAS QSPKLLIY LTISNMQSEDLADYFC YPLT (F) 31 Musmus Musmus IGKJ4*01 F DIVMTQSQKFMSTSV QNVRTA VAWYQQKPG LAS NRHTGVPDRFTGSGSGTDFT LQHWN 9 IGKV6-14*01 F G DRVSITCKAS QSPKALIY LTICNVQSEDLADYFC YPFT 32 Musmus Musmus IGKJ1*01 F DIGGPQSPSSPAVSA QSLLNSR LAWYQQKPG WAS TRESGVPDRFTGSGSGTDFT KQSYN 9 IGKV8-21*01 F GEKVTMSCKSS TRKNY QSPKLLIY LTISSVQAEDLAVYYC LPWT

269 33 Musmus Musmus IGKJ5*01 F DMGAPQSPSSLSVS QSLLNS LAWYQQKPG GAS TRESGVPDRFTGSGSGTDFT QNDHS 9 IGKV8-28*01 F AGEKVTMSCKSS GNQKNY QPPKLLIY LTISSVQAEDLAVYYC YPLT 34 Musmus Musmus IGKJ4*01 F DIQMTQSPSSLSASL QEIGGF LSWFQQKPD AAS TLDSGVPKGFSGSRSGSDYS LQYAS 9 IGKV9-124*01 G ERVSLTCRAS GTIKRLIY LTISSLESEDFADYYC YPFT F 35 Musmus Musmus IGKJ1*01 F DVVMTQTPLS QSLVHS LHWYLQKPG KVS NRFSGVPDRFSGSGSGTDFT SQSTH 10 IGKV1-110*01 LPVSLGDQASISCRS NGNTY QSPKLLIY LKISRVEAEDLGVHFC VPPWT F S

270 Publications Review paper : Improving Immunological insights into the ferret model of human viral infectious disease

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282 Research Paper : Sequencing B cell receptors from ferrets (Mustela putorius furo)

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Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Julius, Wong Jin Liang

Title: Advancing the ferret as an immunological model to study B-cell responses

Date: 2020

Persistent Link: http://hdl.handle.net/11343/247854

File Description: Final thesis file

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