DEVELOPMENT AND CHARACTERIZATION OF LASSA VIRUS VACCINES AND MOUSE MODEL BY GAËLLE CAMUS A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements of the degree of Doctor of Philosophy Department of Immunology University of Manitoba Winnipeg Copyright © 2012 by Gaëlle Camus ACKNOWLEDGEMENTS I am extremely thankful to my advisory committee, Dr. Heinz Feldmann, Dr. Redwan Moqbel, my external examiner Dr. Lisa Hensley and especially my supervisor, Dr. Jude Uzonna and my co-supervisor, Dr. Judie Alimonti for their support, guidance and encouragement throughout the years of my PhD. I would like to thank Dr. Steven Jones for giving me the opportunity to work on Lassa fever, support throughout the years and believing in me. I want to acknowledge Dr. Ute Ströher for sharing her enthusiasm for Lassa virus with me and helpful discussions. I would also like to thank Dr. Paul Hazelton for the electron microscopy studies. I am grateful to the Department of Immunology and all its members at the University of Manitoba for their help and support over the years. Thanks to everyone in Special Pathogens, especially Kinola Williams, Alex Silaghi, Heidi Breitling, Lisa Fernando, Xiangguo Qiu and Salipa Sinkala. I appreciate your support, help and friendship. I am also grateful to the Manitoba Health Research Council for the “Graduate Student Fellowship” I received in 2006-2010. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................................... ii TABLE OF CONTENTS ...................................................................................................................... iii LIST OF TABLES .............................................................................................................................. vii LIST OF FIGURES ........................................................................................................................... viii LIST OF ABBREVIATIONS ................................................................................................................. x ABSTRACT ...................................................................................................................................... xv 1. Introduction ............................................................................................................................. 1 1.1. Arenaviruses ........................................................................................................................ 1 1.1.1. Taxonomy and classification ........................................................................................ 1 1.1.2. Genome and virion structure ....................................................................................... 5 1.1.3. Lassa virus life cycle ..................................................................................................... 8 1.2. Lassa fever ......................................................................................................................... 10 1.2.1. Discovery of Lassa fever ............................................................................................. 10 1.2.2. Clinical presentation of Lassa fever ........................................................................... 10 1.2.2.1. Clinical and virological laboratory parameters ...................................................... 11 1.2.2.2. Severe cases and complications............................................................................. 12 1.2.2.3. Pathology of Lassa fever ........................................................................................ 15 1.2.3. Epidemiology .............................................................................................................. 15 1.2.4. Transmission .............................................................................................................. 16 1.2.5. Laboratory diagnosis .................................................................................................. 16 1.2.6. Animal models of LASV infection ............................................................................... 17 1.2.6.1. Non-human primates ............................................................................................. 17 1.2.6.2. Guinea pigs ............................................................................................................. 21 1.2.6.3. Mice ....................................................................................................................... 22 1.2.7. Pathogenesis and immunology of LASV infection ..................................................... 24 1.2.7.1. Infection ................................................................................................................. 24 1.2.7.2. Host immune response .......................................................................................... 24 1.2.8. Therapy ...................................................................................................................... 33 1.2.9. Vaccine design strategies ........................................................................................... 37 1.2.9.1. Vaccines against Lassa fever virus ......................................................................... 40 iii 1.2.9.2. Mopeia virus .......................................................................................................... 40 1.2.9.3. Vaccinia recombinant constructs ........................................................................... 43 1.2.9.4. Alphavirus replicons ............................................................................................... 45 1.2.9.5. Recombinant Salmonella ....................................................................................... 46 1.2.9.6. Inactivated Lassa virus ........................................................................................... 46 1.2.9.7. Mopeia/Lassa reassortant virus ............................................................................. 47 1.2.9.8. DNA vaccine ........................................................................................................... 50 1.2.9.9. Yellow Fever recombinant vaccine ........................................................................ 50 1.2.9.10. Recombinant VSV vaccine ...................................................................................... 52 1.3. Global objectives and hypotheses .................................................................................... 54 1.3.1. Global rationale .......................................................................................................... 54 1.3.2. Hypotheses ................................................................................................................ 56 1.3.2.1. Development and characterization of vaccines against Lassa fever ..................... 56 1.3.2.2. Establishment of a novel mouse model of Lassa fever .......................................... 57 1.3.3. Objectives................................................................................................................... 57 2. Materials and Methods ......................................................................................................... 58 2.1. Cell lines ............................................................................................................................. 58 2.2. Viruses ............................................................................................................................... 58 2.2.1. Recombinant VSV virus stock preparation ................................................................ 59 2.2.2. Plaque assay ............................................................................................................... 59 2.3. SDS-PAGE and Western blotting (WB) .............................................................................. 60 2.3.1. Membrane stripping .................................................................................................. 62 2.4. Mouse studies ................................................................................................................... 63 2.4.1. Animal ethics .......................................................................................................... 63 2.5. Quantitative ELISA ............................................................................................................. 63 2.5.1. LASV antigen preparation .......................................................................................... 63 2.5.2. Mouse serum samples ............................................................................................... 64 2.5.3. ELISA protocol ............................................................................................................ 64 2.6. ELISPOT assay .................................................................................................................... 65 2.6.1. Preparation of mouse splenocytes ............................................................................ 65 2.6.2. ELISPOT protocol ........................................................................................................ 66 2.6.2.1. LASV peptides ........................................................................................................ 67 iv 2.7. Biosafety ...........................................................................................................................