Development of Novel Anti-Respiratory Syncytial Virus Therapies by Michael J Norris A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto © Copyright by Michael J Norris 2017 Development of Novel Anti-Respiratory Syncytial Virus Therapies Michael J Norris Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto 2017 Abstract Respiratory syncytial virus (RSV) is a leading cause of mortality in infants and young children. Despite the RSV disease burden, no vaccine is available and treatment remains non-specific. New drug candidates are needed to combat RSV. Towards this goal, we investigated two broad strategies to control RSV infection. First, we examined the utility of delivering an anti-4-1BB agonist antibody to enhance CD8 T cell costimulation in a mouse model of RSV infection. This strategy enhanced CD8 T cell numbers, however failed to reduce RSV titers in the lung. We found that this was likely due to reduced RSV specific CD8 T cells in the lung. Overall, this strategy did not attenuate RSV associated disease outcomes and was associated with increased morbidity. Second, we screened over 2000 FDA approved compounds to identify approved drugs with novel anti-RSV activity. Cardiac glycosides, inhibitors of the membrane bound Na+/K+-ATPase, were identified to have anti-RSV activity. Cardiac glycosides diminished RSV infection in HEp-2 cells and in primary human airway epithelial cells grown at an air liquid interface. Digoxin, an FDA approved cardiac glycoside, was further able to inhibit RSV infection of community isolates of RSV in primary nasal epithelial cells. Our results suggest that the antiviral effects of cardiac glycosides are primarily dependent on changes in the intracellular Na+ and K+ composition. Consistent with this mechanism, we subsequently demonstrated that ii the ionophoric antibiotics salinomycin, valinomycin, and monensin inhibited RSV in HEp-2 cells and primary nasal epithelial cells. Our data indicate that the K+/Na+ sensitive steps in the RSV lifecycle occur within the initial 4 hours of virus infection, but do not include virus binding/entry. We employed an RSV mini-replicon assay allowing the independent assessment of RSV-specific RNA synthesis. Our findings demonstrated that digoxin does not alter the RSV RNA polymerase. Future studies will focus on the possibility that digoxin and other ion-modulating drugs may impact viral uncoating (i.e. release of viral nucleic acid from RNP complex). iii Acknowledgments I would like to thank everyone that has made this work possible, beginning with my family and friends who have supported me through all my educational pursuits. I am eternally grateful to my parents, Karen and Bob, for their endless love and support throughout my life; without you, none of this could have ever been possible. Thank you for helping me reach my dreams. To my sister Terri, thank you for always being there for me, your support has meant the world. To my beloved aunt and uncle, Matt and Tania, your faith in me has always been an inspiration, and I will always strive to never let you down! To my adopted aunt Sarah, thank you for always believing in me and helping me see my potential. To my best friends, Tracy, Jco, and Jamie, you all have seen me through my darkest times; thank you for your unwavering support. Last and certainly not least, a special note to my amazing husband Justin, thank you for your love, support, and patience that carried me through all the long nights in the lab, failed experiments, and the never ending hours of writing. Thank you especially for listening to all my crazy science talk! You are not only my soul mate and life partner, you are my rock and my best friend. Without your encouragement, I would not be here today. Thank you for believing in me. I would also like to thank the members of the Moraes lab. Thank you to both Wenming and Hong, your willingness to help has been extraordinary and you both have been instrumental in making this work possible. Additionally, thank you to May (an honorary Moraes lab member); you kept me sane through these five years! Thank you for always being there for me. Also, I would like to thank my committee members Dr. Tania Watts, Dr. Brian Kavanaugh, and Dr. Nades Palaniyar for providing positive feedback and different perspectives within and outside of committee meetings. Special thanks to iv my co-supervisor Dr. Richard Hegele, for his continuous optimism and guidance, which has been essential for this work. Lastly I would like to thank my supervisor Dr. Theo Moraes, it has been an absolute honor to work with you these past five years. Words cannot express how thankful I am to have been given the opportunity to learn from you. Thank you for all your encouragement, advice, and support over the years! I was lucky to have such a wonderful supervisor. v Table of Contents Abstract ........................................................................................................... ii Acknowledgments ......................................................................................... iv Table of Contents ........................................................................................... vi List of Tables ............................................................................................... viii List of Figures ................................................................................................ ix List of Abbreviations ..................................................................................... xi Submitted Work ........................................................................................... xiii Chapter I ......................................................................................................... 1 1. Review of the Literature ............................................................................. 1 1.1 Respiratory Syncytial Virus ........................................................................... 1 Classification .............................................................................................................. 1 Virion .......................................................................................................................... 1 RNA ............................................................................................................................ 2 Proteins ....................................................................................................................... 4 1.2 Replicative Cycle of RSV .............................................................................. 5 1.3 RSV Pathogenesis and Pathology .................................................................. 8 1.4 Host Immune Response to RSV ................................................................... 13 The Innate Immune Response and Inflammation ..................................................... 13 The Adaptive Immune Response .............................................................................. 17 1.5 Burden of RSV Infection .............................................................................. 20 1.6 Strategies and Design of Antiviral Drugs .................................................... 23 Virus-Targeting Antivirals ........................................................................................ 24 Host-Targeting Antivirals ......................................................................................... 28 Immune Modulating Antivirals ................................................................................ 30 1.7 Management of RSV Infection .................................................................... 31 Current Therapies ..................................................................................................... 31 Emerging Therapies .................................................................................................. 36 Need for Additional RSV Therapies ......................................................................... 39 1.8 Opportunities for Novel Anti-RSV Therapies ............................................. 39 Viral Targets ............................................................................................................. 40 vi Host Targets .............................................................................................................. 41 1.9 Proposed Research ....................................................................................... 43 Chapter II ...................................................................................................... 45 2. Agonistic 4-1BB antibody fails to reduce disease burden during acute respiratory syncytial virus (RSV) infection .................................................. 45 2.1 Abstract ........................................................................................................ 45 2.2 Introduction .................................................................................................. 45 2.3 Methods ........................................................................................................ 46 2.4 Results .......................................................................................................... 48 2.5 Discussion .................................................................................................... 58 Chapter III ....................................................................................................