The Creation of a Hantavirus Vaccine Using Reverse Genetics and Its Evaluation in the Lethal Syrian Hamster Model by Kyle Brown B.Sc. (Microbiology), University of Manitoba 2003 A thesis submitted to the Faculty of Graduate Studies of the University of Manitoba in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Medical Microbiology University of Manitoba Winnipeg, Manitoba © 2012 by Kyle Brown TABLE OF CONTENTS ACKNOWLEDGEMENTS vi DEDICATION vii ABSTRACT viii LIST OF TABLES x LIST OF FIGURES xi LIST OF COPYRIGHTED MATERIAL xiii LIST OF ABBREVIATIONS xiv 1.0 INTRODUCTION 1 1.1 Emergence and Epidemics of Hantaviruses 1 1.1.1 Emergence and Viral Transmission of Old World Hantaviruses 1 1.1.2 Emergence and Viral Transmission of New World Hantaviruses 3 1.2 Natural Reservoir of Hantaviruses 4 1.3 Biology of Hantaviruses 8 1.4 Viral Life Cycle 9 1.4.1 Virus Entry 9 1.4.2 Viral Transcription 12 1.4.3 Translation and Processing of Viral Proteins 15 1.4.4 Viral Replication 16 1.4.5 Assembly and Egress 17 1.5 Hantavirus Infection of Humans 19 1.5.1 Clinical Signs of Disease 19 1.5.2 Pathogenesis and Pathology 21 1.5.3 Immune Evasion 23 1.5.4 Diagnosis 24 1.5.5 Treatment and Prevention 25 1.5.5.1 Old World Hantavirus Vaccines 26 1.5.5.2 New World Hantavirus Vaccines 27 1.6 Rationale: Reverse Genetics Systems 28 1.7 Rationale: Use of Recombinant VSV as a Vaccine Platform 35 1.8 Hypothesis and Objectives 36 ii 2.0 MATERIALS AND METHODS 40 2.1 Cells and Viruses 40 2.2 Antibodies and Primers 41 2.3 Molecular Biology Techniques 42 2.3.1 RNA Extractions 42 2.3.2 Polymerase Chain Reaction (PCR) 43 2.3.3 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 45 2.3.4 Amplicon Analysis 46 2.3.5 Quantitative Real-Time RT-PCR (qRT-PCR) 47 2.4 Molecular Cloning Techniques 49 2.4.1 DNA Digestion 49 2.4.2 DNA Ligation 50 2.4.3 DNA Plasmid Transformation 50 2.4.4 Screening and Verification of the Constructs by Restriction Digest 51 2.5 Cloning Strategy – Creation of ANDV N Expression Constructs 52 2.6 Cloning Strategy – Creation of ANDV Glycoprotein Expression Constructs 52 2.7 Cloning Strategy – Creation of ANDV L Expression Constructs 56 2.8 Cloning Strategy – Creation of Minigenome Constructs 58 2.9 Cloning Strategy – Creation of Recombinant VSV Constructs 59 2.10 Transfection 64 2.11 Detection of Protein Expression by Immunostaining Techniques 65 2.11.1 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS- PAGE) Gels and Wet Transfer 65 2.11.2 Western Blot 66 2.11.3 Immunofluorescent Assay (IFA) 67 2.12 Minigenome Rescue 68 2.13 Detection of Minigenome Reporter Signal 68 2.13.1 Chloramphenicol Acetyl Transferase (CAT) Assay and Thin-Layer Chromatography (TLC) 69 2.13.2 Luciferase (LUC) Assay 70 2.14 Rescue of Recombinant VSV 70 2.15 Infectivity Assays 74 2.15.1 Plaque Forming Assay 74 2.15.2 Focus Forming Assay 75 2.15.3 50% Tissue Culture Infectious Dose 76 2.16 Transmission Electron Microscopy (TEM) of VSV 78 2.17 Growth Kinetic Analysis of VSV 80 2.18 Immunization and Challenge of Syrian Hamster Lethal Disease Model 80 2.19 Hamster Serological Assays 81 2.19.1 Neutralization Assays 82 2.19.2 N-Specific Indirect Enzyme Linked Immunosorbent Assay (ELISA) 83 2.20 qRT-PCR Hamster Cytokine Assay 84 iii 3.0 RESULTS 85 3.1 Cloning and Expression of ANDV Proteins in Mammalian Expression Constructs 85 3.1.1 ANDV N Constructs 85 3.1.2 ANDV Glycoprotein Constructs 86 3.1.3 ANDV L Constructs 86 3.1.4 Confirmation of Protein Expression 92 3.2 Cloning and Rescue of Minigenome Constructs 95 3.3 Cloning and Rescue of Recombinant VSV 98 3.4 In Vitro Characterization of Recombinant VSV Vaccine 104 3.4.1 TEM Imaging of VSV 104 3.4.2 Growth Kinetic Analysis of VSV 108 3.5 In Vivo Protection Studies 110 3.5.1 Vaccine Efficacy Against Lethal ANDV Challenge 110 3.5.2 Detection and Quantification of ANDV Replication 113 3.5.2.1 ANDV RNA 113 3.5.2.2 N Protein-Specific Seroconversion 113 3.5.3 Measurement of Immune Response 115 3.5.3.1 Humoral Response – Neutralizing Antibodies 115 3.5.3.2 Validation of Assay 118 3.6 Time to Protection and Post-Exposure Vaccine Efficacy 120 3.6.1 Time to Protection Survival Study 120 3.6.2 Post-Exposure Survival Study 122 3.6.3 Time to Protection – Measurement of Immune Response 124 3.6.3.1 Time to Protection – ANDV Replication 124 3.6.3.2 Time to Protection – ANDV Glycoprotein Neutralizing Antibodies 126 3.6.3.3 Time to Protection – Stimulation of Innate Immune System 126 4.0 DISCUSSION 131 4.1 Cloning and Expression of ANDV Proteins In Vitro 131 4.2 Cloning and Rescue of Minigenome Constructs 134 4.3 Cloning and Rescue of Recombinant VSV Constructs 136 4.4 In Vivo Protection Studies 139 4.4.1 Efficacy of VSVΔG/ANDVGPC as a Vaccine Platform 139 4.4.2 Immune Response 141 4.5 Time to Protection and Post-Exposure Vaccine Efficacy 144 4.5.1 Time to Protection Efficacy 144 4.5.2 Post-Exposure Efficacy 145 4.5.3 Time to Protection – Adaptive Immune Response 147 4.5.4 Time to Protection – Innate Immune Response 149 iv 5.0 CONCLUSIONS 152 6.0 REFERENCES 155 7.0 APPENDICES 177 Appendix 1 – Buffer Recipes 177 Appendix 2 – List of Primary and Secondary Antibodies 180 Appendix 3 – List of Primers 181 Appendix 4 – DNA and Protein Ladders 184 Appendix 5 – Minigenome Rescue Conditions 185 v ACKNOWLEDGEMENTS First of all, I would like to thank my supervisor Heinz Feldmann for all of the support that he’s given me throughout the years. Despite all of the difficulties that were encountered throughout this study, Heinz has ensured that I would be able to come to Montana on many occasions to complete this work, and would continue to be financially supported while I remained in Winnipeg. His belief in my work as a student has provided the ultimate encouragement for me throughout the years. Additionally, I would like to thank Gary Kobinger for supporting me in Winnipeg through funding and the provision of lab space so that this work could continue in Heinz’s absence. Thank you to the Department of Medical Microbiology for the support over the years, and especially to Angela Nelson who was always quick to answer any necessary questions. Thank you also to my committee members Steven Pind and Kevin Coombs for all of the helpful suggestions they’ve provided for the project and ultimately for reading my thesis. I would also like to thank all of the various members of the Special Pathogens program at the National Microbiology Laboratory that I have shared lab space with, become close friends with, and who have helped to improve my techniques, scientific understanding, and helped provide fun outside the lab. Without them, these years would have been much less eventful. I also have to thank the members of the Rocky Mountain Laboratory in Montana who have proved to be absolutely essential in completing this work. Specifically, thank you to David Safronetz, Andrea Marzi, Marko Zivcec, Hideki Ebihara, Yoshimi Tsuda and Jessica Levine Spengler for all of their direct involvement in the different experiments throughout this study. In addition, I would like to acknowledge the people involved in animal care for the hamster experiments, as well as Beth Fisher and David Dorward in the EM department for their help in imaging the VSV particles. Finally thank you so much to Kay Menk and Joyce Walczynski who did so much of the behind the scenes work in getting me down to Montana every time, and letting me in and out of the building on a daily basis! I must also thank the people that have provided reagents towards this work. Thank you to Jack Rose for the VSV reverse genetics system and rescue plasmids, Connie Schmaljohn for the ANDV stock, and to Robbin Lindsay and Antonia Dibernardo for the recombinant SNV N antigen and ELISA plates. Finally, thank you to the Canadian Institutes of Health Research (CIHR) for funding towards portions of this project. vi DEDICATION This thesis is dedicated to my parents who have been a constant encouragement to me throughout all of my years. They have pushed me to achieve, to be successful and have supported me when I’ve stumbled. It is also dedicated to my loving wife Alanna, who has been by my side throughout this entire lengthy process and never questioned why I was doing something so ridiculous. She also provided me with our wonderful son Nathaniel, who has served as a good distraction during my thesis writing and with whom I hope to spend much more time with now that my work is finally complete. Finally, it is also dedicated to my close friends and family members that have always had time to go out and have a good time when the work has become overwhelming. vii ABSTRACT Andes virus (ANDV) is a highly pathogenic New World hantavirus found in Chile and Argentina that causes Hantavirus Pulmonary Syndrome (HPS). A significantly high case fatality rate, the potential for human-to-human transmission, and the lack of licensed vaccines or effective treatments for the disease suggest an urgent need for the development of such measures.