An Inhalational Antiviral Strategy for the Potential Use of Nitric Oxide during Influenza Infection by Bevin Brent McMullin RT, Cariboo College, 1992 MAppSc, Charles Sturt University, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (VANCOUVER) September 2012 ©Bevin Brent McMullin, 2012 Abstract Since the discovery in the late 1980’s that the endothelium relaxing factor is nitric oxide (NO) there has been intensive scientific pursuit to understand the many roles of NO in biological systems. NO is a messenger molecule with both paracrine and autocrine functions. NO is produced by phagocytes as part of the immune system as a non-specific antimicrobial which may be effective against Influenza. Influenza is a virus that infects millions of people each year resulting in hundreds of thousands of deaths. While vaccination and antivirals have helped reduce the death toll, their effectiveness is limited because of the rapidly evolving nature of the influenza virus and the development of resistance. The recent 2009 influenza pandemic has highlighted the need for new and novel antivirals. We hypothesize that the direct exposure of influenza viruses to gaseous nitric oxide (gNO) will have an antiviral effect. We also show that it is feasible and safe to deliver inhaled gNO to humans at antiviral concentrations using an intermittent high dose regimen. ii Preface In Chapter 1, an independent search of the literature was done in collaboration with Tom Oliver, manager of Clinical Epidemiology and Biostatistics, McMaster University. I then reviewed and synthesized the papers for inclusion in Chapter 1 and throughout the remaining chapters. A version of Chapter 2 has been submitted for publication. I was responsible for the in-vitro testing and proof of concept. Gilly Regev-Shoshani was responsible for the pH and nitrite portion of the study. Gilly Regev-Shoshani, Chris Miller and I were responsible for manuscript preparation. A version of Chapter 4 has been published as: Gaseous Nitric Oxide Bactericidal Activity Retained During Intermittent High-Dose Short Duration Exposure, Miller C, McMullin B, Ghaffari A, Stenzler A, Pick N, Road J, Av-Gay Y, Nitric Oxide 20 (2009) 16-23. Copyrighted material used by permission from Elsevier. The initial concept was developed and patented by Chris Miller, Alex Stenzler and myself. I was responsible for all NO exposure and testing of bacteria, virus and cell lines. Evaluation of cells was done by Abdi Ghaffari and Neora Pick. Manuscript preparation was done by Chris Miller and myself. Funding for the project was provided by Pulmonox Medical Inc. and Cardinal Health Care. A version of Chapter 5 has been published as: A Phase I Clinical Study of Inhaled Nitric Oxide in Healthy Adults, Chris Miller, Minna Miller, Bevin McMullin, Gilly Regev, Lena Serghides, Kevin Kain, Jeremy Road, Yossef Av-Gay. Journal of Cystic Fibrosis, available online 18 April 2012. The study had UBC CREB Approval #H09-00872 and Health Canada approval TPD CTA #129958. Under the supervision of Chris Miller, I took part in the study design, review and implementation, including space allocation, patient recruitment, evaluation, set-up, monitoring of iii treatments and lung function testing. I was responsible for data collection, collation and review. Gilly Regev-Shoshani and Lena Serghides performed nitrite, cytokine/chemokines and angiopoietin assays and provided interpretation of results. Chris Miller and I were responsible for manuscript preparation. iv Table of Contents Abstract ....................................................................................................................................... ii Preface........................................................................................................................................ iii Table of Contents ........................................................................................................................ v List of Tables ............................................................................................................................. ix List of Figures ............................................................................................................................. x Acknowledgements .................................................................................................................... xi Dedication ................................................................................................................................ xiii Chapter I: Introduction to Nitric Oxide and Influenza............................................................... 1 1.1 Nitric Oxide .......................................................................................................................... 1 1.1.1 Nitric Oxide Biochemistry ............................................................................................. 1 1.1.2 Nitric Oxide in the Immune System .............................................................................. 3 1.1.3 Nitric Oxide Delivery .................................................................................................... 4 1.1.4 Gaseous Nitric Oxide as an Antibacterial ...................................................................... 5 1.1.5 Inhalation of Nitric Oxide .............................................................................................. 6 1.2 Nitric Oxide as an Antiviral .................................................................................................. 7 1.2.1 Literature Search ............................................................................................................ 8 1.2.2 Baltimore Classification............................................................................................... 11 1.2.3 Viruses Susceptible to the NO Donor, SNAP .............................................................. 14 1.3 Influenza ............................................................................................................................. 15 1.3.1 Nitric Oxide and Influenza ........................................................................................... 16 1.4 Hypothesis........................................................................................................................... 18 Chapter 2: Gaseous Nitric Oxide and Influenza: An in-vitro model ........................................ 20 2.1 Introduction ......................................................................................................................... 20 2.2 Materials and Methods ........................................................................................................ 20 2.2.1 Viruses and Cell Lines ................................................................................................. 20 2.2.2 Gaseous Nitric Oxide Delivery .................................................................................... 21 2.2.3 Post Infection Effect of Gaseous Nitric Oxide ............................................................ 22 2.2.4 Virucidal (Cell-free) Effect of Gaseous Nitric Oxide .................................................. 22 2.2.5 Nitrites and pH Content ............................................................................................... 23 v 2.2.6 Effect of Nitrites, pH and Combination on H1N1 Virion (Cell-free) .......................... 23 2.2.7 Nitric Oxide Effect on Dried H1N1 ............................................................................. 23 2.2.8 Statistical Analysis ....................................................................................................... 24 2.3 Results ................................................................................................................................. 24 2.3.1 Post Infection Effect of Gaseous Nitric Oxide in Cells ............................................... 24 2.3.2 Virucidal Effect of Gaseous Nitric Oxide on Cell-free Virions .................................. 27 2.3.3 Nitrite Concentrations, pH and Their Effect on Cell-free H1N1 Virions .................... 30 2.4 Summary ............................................................................................................................. 32 Chapter 3: A Mechanism by which Nitric Oxide Inhibits the Infectivity of Influenza ............ 34 3.1 Introduction ......................................................................................................................... 34 3.2 Materials and Methods ........................................................................................................ 34 3.2.1 Influenza Viruses ......................................................................................................... 34 3.2.2 Gaseous Nitric Oxide Delivery .................................................................................... 35 3.2.3 Hemagglutination Inhibition Assay ............................................................................. 36 3.2.4 Nitrosylation Assay ...................................................................................................... 36 3.2.5 Nitric Oxide Effect on Dried H1N1 ............................................................................. 37 3.2.6 Hemagglutinin.............................................................................................................. 37 3.3