Immunoprophylaxis of Influenza using AAV Vector Delivery of Cross-Subtype Neutralizing Single Domain Antibodies JOANNE MARIE M. DEL ROSARIO Thesis submitted for the degree of Doctor of Philosophy Infection and Immunity University College London 2020 To Chris, as fate would have it. To Teki, thank you for everything. 2 DECLARATION I, Joanne Marie M. Del Rosario, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. __________________________ 3 ABSTRACT Cross-subtype neutralizing single domain antibodies against influenza present new opportunities for immunoprophylaxis and pandemic preparedness. Their simple modular structure and single open reading frame format are highly amenable to gene therapy-mediated delivery. R1a-B6, an alpaca-derived single domain antibody (nanobody), that is capable of potent cross-subtype neutralization in vitro of H1N1, H5N1, H2N2, and H9N2 influenza viruses, through binding to a highly conserved epitope in the influenza hemagglutinin stem region, was previously described. To evaluate the potential of R1a-B6 for immunoprophylaxis via adeno-associated viral (AAV) vector delivery, it was reformatted as Fc fusions of mouse IgG1 (ADCC-) and IgG2a (ADCC+) isotypes. This is also to extend R1a-B6’s half-life and to assess the requirement for ADCC for efficacy of R1a-B6 in vitro and in vivo. It was found that reformatted R1a-B6 of either mouse IgG isotype retained its potent binding and neutralization activity against different Group I influenza A subtypes in vitro. The findings in this study also demonstrate that a single intramuscular injection in mice of AAV encoding R1a-B6-Fc was able to drive sustained high-level expression (0.5–1.1 mg/mL) of the nanobody-Fc in sera with no evidence of reduction for up to 6 months. R1a-B6-Fc fusions of both isotypes gave complete protection against lethal challenge with both pandemic A/California/07/2009 (H1N1)pdm09 and mouse-adapted avian influenza A/Vietnam/1194/2004 (H5N1). These data suggest that R1a-B6-Fc delivered via AAV is capable of cross-subtype protection and ADCC was not essential for protection. These findings reveal that AAV delivery of cross-subtype neutralizing nanobodies may be an effective strategy to prevent influenza infection and provide long-term protection independent of a host induced immune response. 4 IMPACT STATEMENT Although vaccines are the main countermeasure against pandemic influenza, their timely implementation and ineffectiveness in vulnerable patient groups present challenges. We need a pro-active and pre-emptive approach in tackling influenza especially in the case of a pandemic emergency. Novel interventions using broadly neutralizing monoclonal antibodies (mAbs) are under clinical development, however their structural complexity, expensive production and requirement for repeated injections are limitations to widespread use. Adeno associated virus (AAV) vectors were used to deliver a much simpler broadly neutralizing single domain antibody (nanobody R1a-B6) specific for a highly conserved epitope in the influenza hemagglutinin stem region. A one-time intramuscular delivery route aiming for longer expression and stricter dosing control in preference to intranasal delivery that may be obstructed due to congestion and a reflex sniffing response of the recipient leading to reduced efficacy of delivery was evaluated. Findings demonstrate high level, long- term expression in vivo, which protected mice from lethal challenge by both pandemic H1N1 and highly pathogenic avian influenza H5N1. Surprisingly, Fc-mediated antibody dependent cellular cytotoxicity (ADCC) was not essential for R1a-B6 efficacy when expressed at high levels in vivo which contrasts with that described previously for similar stem binding human mAbs when injected as purified protein. These findings have implications in developing the optimum antibody format and delivery option to provide safe, long-term protection, independent of the patient’s immune response and prior exposure to influenza, that can be utilized in the instance of a pandemic. 5 ACKNOWLEDGEMENTS I would like to extend my gratitude to the following people who have made this PhD a unique and positive learning experience. The support of these amazing people has been vital in the most challenging and humbling of times. My supervisors, Dr. Yasuhiro Takeuchi and Dr. Simon Hufton, for really taking on the job and teaching, correcting, and guiding me every step of the way. I finish this PhD, hopefully a better scientist, and definitely a better person, than I was before, because of your training, encouragement, patience, kindness and friendship. I have been very blessed to have you as my advisers. The DPSM faculty and team, and the Chemistry unit of the University of the Philippines Manila for their unwavering support from start to finish of my PhD. None of this would have been possible without you. Professor Mary Collins for helpful insight and advice. I would not have been here if you and Yasu did not take a chance on me. Professor Paul Digard and Dr. Laura McCoy, whose extensive examination have helped make this piece of work better. I have learned so much in those 4 hours. Dr. Matthew Smith for all the exciting influenza work that you taught me. It has been a pleasure sharing the bench with such an awesome scientist like you. Christina Ball, Paul Risley, Dr. Kam Zaki and Dr. Ilaria Nisoli for sharing your expertise and time with me. Dr. Othmar Engelhardt and Dr. Nigel Temperton for being supportive and critical collaborators, who have enriched this body of work. Rose, Alan, Shaun, Christine, Vicky and all the wonderful people in the Biological Services Division at NIBSC for going over and beyond to help me with the animal work. Our colleagues at the Biotherapeutics Division and the Influenza Resource Center at NIBSC for help and advice with this project. 6 My former students, for always cheering me on. Your belief in me has made me become more of what I already am. The friends that I have made here in London especially Bea, Jasmine and Pedro, and Chris’s family who have made being in another country an incredibly fun, uplifting and a most rewarding experience. And all my friends back in the Philippines and scattered all around the world who have always cheered me on and believed in me even at times when I doubt myself. Gabby, Ciara, Andrea (x2) and Diana, for listening to all my ranting, and for always calming me down when things don’t go as planned. Jules, Kevin, Dem, Jireh and Hermie, eternal friends, rambunctious supporters, together we are untouchable. Arlou, it is finally done! And I share this victory with you, one of many in our bright future. Tom, Ryan and Martin, NIBSC became home because you were there. Work did not feel like work, and we fostered a happy, productive, supportive environment, that I would always cherish. Teki, for being my rock. I have borrowed from your strength so much and you have kept me standing through the best and worst of it. My family and my Bicho for your unconditional love and faith in me. Being away from you has not been easy but I thank you for sharing my dreams and helping me in making them happen, and always, always, praying for me. This is the product of all our sacrifices. Chris, my clever boy, for all the late nights, stimulating discussions and inspiring banter. For suffering and rejoicing with me and holding my hand through our inflatable bed journey. Everything will be worth it in the end. Smart ka talaga for sharing this incredible adventure with me. And finally, infinite thanks to HIM, the Giver of all good things. 7 TABLE OF CONTENTS DECLARATION . 3 ABSTRACT . 4 IMPACT STATEMENT . 5 ACKNOWLEDGEMENTS . 6 TABLE OF CONTENTS . 8 LIST OF FIGURES . 12 LIST OF TABLES . 14 ABBREVIATIONS . 15 CHAPTER 1. INTRODUCTION . 19 1.1 Thesis Overview . 19 1.2 Influenza . 19 1.2.1 Influenza A Biology . 19 1.2.2 Replication cycle . 22 1.2.3 Pathogenesis . 24 1.2.4 Burden and Epidemiology . 27 1.2.5 Pandemic threats . 29 1.2.6 Prevention and Treatment . 31 1.3 Innate Immune Response to Influenza . 37 1.4 Antibody Immune Response to Influenza . 38 1.5 Passive Immunotherapy against Influenza . 42 1.5.1 Broadly neutralizing Antibodies (bnAb) . 44 1.5.2 Nanobodies (Nb) . 48 1.5.2.1 Nanobody Re-Formatting to increase Half-Life . 52 1.5.2.2 Nanobodies with Effector functions . 56 1.5.2.3 Nanobodies against Influenza . 59 1.6 Gene Therapy and Viral Vectors . 62 1.6.1 Recombinant Adeno-Associated Virus (rAAV) . 64 1.6.2 rAAV in clinical trials . 68 1.6.3 rAAV and influenza . 71 8 1.7 Universal Influenza Vaccine . 73 1.8 Thesis Objectives . 75 CHAPTER 2. MATERIALS AND METHODS . 77 2.1 General . 77 2.1.1 Propagation and maintenance of cell cultures . 77 2.1.1.1 ExpiCHO-S™ cells . 77 2.1.1.2 Human embryonic kidney (HEK) 293T cells . 77 2.1.1.3 Madin-Darby canine kidney (MDCK) cells . 77 2.1.2 Restriction Digestion . 77 2.1.3 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis . 78 2.1.4 Western Blotting . 79 2.1.5 Enzyme-Linked Immunosorbent Assay (ELISA) . 79 2.1.6 Statistical Analysis . 80 2.2 Production and purification of nanobody-Fc fusion proteins . 80 2.2.1 Cloning of nanobody-Fc in mammalian expression vector . 80 2.2.2 Transfection in ExpiCHO-S™ cells . 81 2.2.3 Protein A Affinity Chromatography . 82 2.2.4 Dialysis . 82 2.3 In vitro assays for nanobody-Fc specificity and neutralization activity . 83 2.3.1 Influenza ELISA panel . 83 2.3.2 Antibody Dependent Cell Cytotoxicity (ADCC) reporter assay .