DEVELOPMENT OF BORDETELLA PERTUSSIS AS A LIVE VEHICLE FOR HETEROLOGOUS ANTIGEN DELIVERY, AND ITS APPLICATION AS A UNIVERSAL INFLUENZA A VACCINE LI RUI NATIONAL UNIVERSITY OF SINGAPORE 2010 DEVELOPMENT OF BORDETELLA PERTUSSIS AS A LIVE VEHICLE FOR HETEROLOGOUS ANTIGEN DELIVERY, AND ITS APPLICATION AS A UNIVERSAL INFLUENZA A VACCINE LI RUI B.Sc.; M.Sc. A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE ACKNOWLEDGEMENTS If you ask me which part of the thesis I have spent most of the time to write, I would say it is the acknowledgements. In the past five years of my PHD study, there have been too many people who have given me so much help that without them all these work never have been possible. So, from the beginning to the end of my study, I am always thinking how I would write the acknowledgements to express my sincere appreciation and thanks to them. First of all, I would like to express my deepest gratitude to my supervisor Assistant Professor Dr Sylvie Alonso whose invaluable guidance as well as remarkable patience have made this project possible. Your constant advice, encouragement and support has inspired me and propelled my passion for research. You has been a most inspiring and considerate supervisor throughout these five years of my research here. Besides research, you have also showered me endless care and help in my life throughout these years. I cannot thank you enough. The experience and knowledge gained from you will always benefit my future and will always be embedded in my mind. I am also very grateful to Associate Professor Vincent Chow and Sim Meng Kwoon for their helpful suggestions and generous support in this project. I would also like to express my special thanks to Annabelle, whose help has been extremely valuable especially during the days when I was pregnant and away to give birth to my baby. Special thanks also to Mrs Phoon Meng Chee, Dr Raju, Jowin and Wee Peng, for their constant and great support and help to this project. i I would also thank Mr Joe Tong and Mr Goh for their selfless help and support in handling the administrative matters, which had helped a lot in the progress of this project. I would also like to thank my friends in the lab, Siying, Wenwei, Lili, Stephanie, Adrian, Grace, Wei Xin, Damian, Emily, Aakanksha, Regina, Weizhen, Jian Hang, Zarina, Michelle, Vanessa, for the help they gave in various aspects. I have shared five cherished years with you in a cozy environment. This wonderful time will always be a beautiful memory in my life. Special thanks are also addressed to Professor Camille Locht for providing the BPZE1 strain; Lew Fei Chuin and Weiqiang for their invaluable assist in FACS analysis; friends in Professor Kemeny‟s lab, especially Richard, Benson, Kenneth and Yafang; friends in Associate Professor Fred Wang‟s lab and Associate Professor Herbert Schwarz‟s lab; as well as all those who have helped me in one way or another. I really appreciate it. I am immensely grateful to my parents, my sister and brother, as well as my uncles for their endless love, encouragement, support and belief in me all these years. It is your love and support that has made me who I am today. Last but not least, I would like to dedicate this thesis to My husband Xu Yan and my dear baby Chenyu. Chenyu, my little angel, thank you for accompanying me since last year, your lovely smile has made my life colorful and joyful and let me forgot all the not so good times. Xu Yan, thank you so much for your endless love, understanding and support throughout this project. Thank you for waiting there for me. This piece of work is the fruit of our years of separation. I will cherish it forever. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS iii LIST OF FIGURES . xiv LIST OF TABLES xvii ABBREVIATIONS xviii SUMMARY xxiii CHAPTER INTRODUCTION . CHAPTER SURVEY OF LITERATURE . (I) INFLUENZA VIRUSES . 2.1 INFLUENZA MORBIDITY, MORTALITY AND HISTORY OF INFLUENZA PANDEMICS . 2.2 INFLUENZA VIRUSES CLASSIFICATION . 2.3 INFLUENZA A VIRUS: STRUCTURE AND REPLICATION . 2.3.1 Influenza A Virus Genome and Its Major Protein Products 2.3.2 Antigenic Shift and Drift . 11 2.3.3 Determinants of Tissue Tropism and virulence . 11 2.4 IMMNUE RESPONSE TO INFLUENZA A VIRUS INFECTION 16 2.4.1 Innate Immunity . 16 2.4.2 Effector Mechanisms of the Adaptive Immunity . 17 iii 2.4.3 Immune Correlates of Protection . 20 2.4.3.1 Role of HA, NA and M2 specific antibodies in the protection of influenza virus infection . 20 2.4.3.2 Role of influenza specific cell mediated immunity in the protection of influenza virus infection . 24 2.5 INFLUENZA PATHOGENESIS . 26 2.5.1 Clinical Presentations of Influenza and Links with Immune Dysregulation . 26 2.5.2 The “Cytokine Storm Theory”- Cytokines and Chemokines in Influenza Immunopathology . 27 2.5.3 CD4+ and CD8+ in Influenza Immunopathology . 29 2.5.4 Alveolar Macrophages and Neutrophils in Influenza Immunopathology 31 2.6 OPTIONS FOR PANDEMIC CONTROL . 34 2.6.1 Antivirals Treatment: Effectiveness and Limitations 34 2.6.2 Licensed and Trial Vaccines 36 2.6.2.1 Current licensed vaccines . 37 2.6.2.1.1 Inactivated virus vaccines 37 2.6.2.1.2 Live attenuated virus vaccine- Cold attenuated vaccine (CAV) 38 2.6.2.1.3 Limitations of Current Licensed Vaccines 39 2.6.2.2 Alternative approaches to pandemic influenza vaccine development 40 2.6.2.2.1 Virosome-based influenza vaccines . 40 iv 2.6.2.2.2 DNA Vaccines . 41 2.6.2.2.3 Recombinant Vectored Subunit Vaccines . 42 2.6.3 Universal influenza virus vaccines 45 2.6.3.1 Ecto-domain of Matrix protein (M2e) as a Universal Vaccine Candidate 45 2.6.3.2 Nucleocapsid Protein (NP) as a Universal Vaccine Candidate 47 2.6.3.3 Conserved Neutralizing Epitopes of HA protein as Universal Vaccine Candidates . 48 (II) BORDETELLA PERTUSSIS AS A LIVE VEHICLE FOR HETEROLOGOUS VACCINE ANTIGENS DELIVERY THROUGH THE NASAL ROUTE . 50 2.7 MUCOSAL VACCINATION 50 2.7.1 Mucosal Immunity . 50 2.7.2 Vaccination via the Mucosal Route . 51 2.7.3 Intranasal Vaccination . 52 2.8 BORDETELLA PERTUSSIS MICROBIOLOGY . 53 2.8.1 Bordetella pertussis Pathogenesis and Whooping Cough . 53 2.8.2 Treatment and Pertussis Vaccines . 54 2.8.3 Virulence Determinants of B. pertussis 55 2.9 IMMUNITY TO B. PERTUSSIS 61 2.9.1 Humoral and Cell-mediated Immunity . 61 v 2.9.2 Immune Subversion and Immunomodulatory Effects of B. pertussis . 64 2.10 ATTENUATED B. PERTUSSIS FOR HETEROLOGOUS ANTIGEN DELIVERY . 67 2.10.1 Live Bacteria as Vaccine Delivery System 67 2.10.2 Attenuated B. pertussis as a Live Recombinant Nasal Delivery Vector 68 2.10.3 FHA as an Antigen Carrier 70 2.10.4 Other Antigen Carriers . 72 CHAPTER MATERIALS AND METHODS 74 (I) ESCHERICHIA COLI WORK 74 3.1 BACTERIAL STRAINS, PLASMIDS AND GROWTH CONDITIONS . 74 3.1.1 E. coli Strains and Plasmids . 74 3.1.2 Growth Conditions . 75 3.2 MOLECULAR BIOLOGY . 76 3.2.1 Oligonucleotides and Primers 76 3.2.1.1 List of oligonucleotides and primers . 76 3.2.1.2 Hybridization of oligonucleotides . 76 3.2.2 Plasmid Extraction . 79 3.2.3 Polymerase Chain Reaction (PCR) 79 3.2.3.1 DNA amplification 79 3.2.3.2 Colony PCR screening 79 vi 3.2.4 Restriction Enzyme Digestion . 80 3.2.5 Agarose Gel Electrophoresis 80 3.2.5.1 Gel migration 80 3.2.5.2 Gel extraction 81 3.2.6 DNA Cloning . 81 3.2.7 Transformation of Chemically Competent E. coli . 82 3.2.8 DNA Sequencing . 82 (II) BORDETELLA PERTUSSIS WORK . 83 3.3 BACTERIAL STRAINS AND GROWTH CONDITIONS . 83 3.3.1 B. pertussis Strains . 83 3.3.2 Growth Conditions . 84 3.4 MOLECULAR BIOLOGY . 84 3.4.1 List of Primers 84 3.4.2 Construction of Recombinant B. pertussis Strains 85 3.4.2.1 Construction of Recombinant B. pertussis Strains Expressing M2e, HA1-1, HA2-1 and HA2-2 85 3.4.2.1.1 Design and synthesis of optimized m2e (opm2e), ha1-1(opha1-1), ha21(opha2-1) and ha2-2(opha2-2) . 85 3.4.2.1.2 Cloning opm2e, opha1-1, opha2-1 and opha2-2 into fhaB . 87 vii 3.4.2.1 Construction of Recombinant B. pertussis Strains Expressing ∆NA and NP . 90 3.4.2 Transformation of B. pertussis . 92 3.4.2.1 Preparation of electrocompetent cells . 92 3.4.2.2 Electroporation of plasmid DNA into B. pertussis . 92 3.4.4 Screening for True Recombinants . 93 3.5 PROTEIN EXPRESSION STUDIES . 94 3.5.1 Preparation of B. pertussis Samples . 94 3.5.1.1 Supernatant . 94 3.5.1.2 Cell extract 94 3.5.2 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis 95 (SDS-PAGE) . 95 3.5.3 Coomassie Blue Staining . 95 3.5.4 Western Blot 96 (III) ANIMAL WORK 98 3.6 MOUSE STRAINS . 98 3.7 PRODUCTION OF POLYCLONAL ANTI-M2E IMMUNE SERA 98 3.8 INTRANASAL B. 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CC APPENDICES APPENDIX I: REAGENTS FOR GEL ELECTROPHORESIS 1.1 1.1.1 DNA Electrophoresis 50 × Tris-Acetate-EDTA (TAE) Buffer Per L Tris base 242 g Glacial acetic acid 57.1 ml 0.5 M EDTA (pH 8) 100 ml Final pH adjusted to 7.8 1.1.2 Agarose Gel 1% 1.5% Agarose 0.5 g 0.75 g × TAE 50 ml 50 ml × TAE was prepared by adding 20 ml of 50 × TAE buffer to 980 ml of ddH2O. 1.1.3 × DNA Loading Dye Bromophenol blue 0.25% Xylene cyanol 0.25% Ficoll (type 400) in ddH2O 1.2 25% Protein Electrophoresis A APPENDICES 1.2.1 SDS-PAGE 1.2.1.1 × SDS/Glycine Electrophoresis Buffer Per L Tris-base 15.1 g Glycine 72 g SDS 5g 1.2.1.2 10% or 12% Separating Gel Per 30 ml 30% Acrylamide-bisacrylamide (29:1) 8.75 ml or 10.5 ml M Tris-HCl (pH 8.8) 7.5 ml 10% SDS 300 µl 20% APS 400 µl TEMED 40 µl ddH2O 13.01 ml or 11.26 ml 1.2.1.3 4% Stacking Gel Per 10 ml 30% Acrylamide-bisacrylamide (29:1) ml M Tris-HCl (pH 8.8) 1.25 ml 10% SDS 100 µl 20% APS 400 µl TEMED 40 µl ddH2O 11.26 ml 1.2.1.4 × Protein Loading Buffer B APPENDICES Per 50 ml M Tris-HCl (pH 6.8) 6.25 ml 100% glycerol ml SDS powder 5g Bromophenol blue drop β-mercaptoethanol ml 1.2.2 1.2.2.1 Coomasie Staining of SDS Gel Coomasie Blue Staining Solution Coomasie brilliant blue 0.2% Ethanol 45% Acetic acid 10% ddH2O 45% 1.2.2.2 Destaining Solution Ethanol 25% Acetic acid 10% ddH2O 65% C APPENDICES APPENDIX II: REAGENTS FOR GROWTH MEDIA 2.1 2.1.1 E. coli Culture Media Luria-Bertani (LB) Agar Per L Tryptone 10 g Yeast extract 5g NaCl 10 g Agar 15 g Autoclaved at 121ºC for 15 mins. 2.1.2 LB Broth Per L Tryptone Yeast extract NaCl 10 g 5g 10 g Autoclaved at 121ºC for 15 mins. D APPENDICES 2.2 2.2.1 B. pertussis Culture Media Stainer-Scholte (SS) Medium Fraction A: Per L Na-L-Glutamate 11.84 g L-Proline 0.24 g NaCl 2.5 g KH2PO4 0.5 g KCl 0.2 g MgCl2.6H2O 0.1 g CaCl2.2H2O 0.02 g Tris 1.5 g Casamino acids 10 g Dimethyl-ß-Cyclodextrine 1g Final pH adjusted to 7.4. Autoclaved at 121ºC for 15 mins. Fraction B: Per 10 ml L-Cysteine 0.04 g FeSO4.7H2O 0.01 g Nicotinic acid 0.04 g Ascorbic acid 0.4 g Glutathione 0.15 g Filter-sterilize with 0.2 µm filter unit. Add 10 ml of filter-sterilized Fraction B to L of autoclaved Fraction A. E APPENDICES 2.2.2 Bordet-Gengou (BG) Agar Per L Potato infusion from 125 g 4.5 g NaCl 5.5 g Agar 20 g Glycerol 10 g Autoclaved at 121ºC for 15 mins. Prior to pouring into petri dish, 10% sterile, defibrinated sheep blood was added at 45ºC – 50ºC. F APPENDICES APPENDIX III: REAGENTS FOR ANIMAL WORK 3.1 Anaesthetic Cocktail for Nasal Administration Valium 6% Atropine 10% Ketamine 20% × PBS 64% Cocktail must be prepared under sterile conditions. 120 µl cocktail is injected intraperitoneally for a mouse of approximately 17 g of body weight. G PUBLICATIONS Articles 1. Li, R., Lim, A., Phoon, M. C., Narasaraju, T., Ng, J. K., Poh, W. P., Sim, M. K., Chow, V. T., Locht, C. & Alonso, S. (2010). Attenuated Bordetella pertussis protects against highly pathogenic influenza A viruses by dampening the cytokine storm. J Virol 84, 7105-7113. 2. Neo, Y., Li, R., Howe, J., Hoo, R., Pant, A., Ho, S. & Alonso, S. (2010). Evidence for an intact polysaccharide capsule in Bordetella pertussis. Microbes Infect 12, 238-245. Patent 1. Alonso S., Li R., Chow V. International patent “Influenza vaccine, composition and method of use.” Filed on June 15th, 2009. Conference papers 1. Li, R., Lim, A. R. F., Ow, T. L.S., Phoon, M. C., Narasaraju, T., Chow, V. T. K., Alonso, S. Development of Universal Influenza A Vaccines Using Attenuated Bordetella pertussis as Antigen Delivery. In: 8th Asia Pacific Congress of Medical Virology, Hongkong, China. February 26-28, 2009. 2. Li, R., Lim, A. R. F., Ow, T. L.S., Phoon, M. C., Narasaraju, T., Chow, V. T. K., Alonso, S. Development of Universal Influenza A Vaccines Using Attenuated Bordetella pertussis as Antigen Delivery. In: 2nd International Singapore Symposium, Singapore. January 19-20, 2009. Li, R., Narasaraju, T., Phoon, M. C., Chow, V. T. K., Alonso, S. Development of Universal Influenza A Vaccines Using Attenuated Bordetella pertussis as Antigen Delivery. In: 1st International Singapore Symposium, Singapore. January 14-16, 2008. Li, R., Narasaraju, T., Phoon, M. C., Chow, V. T. K., Alonso, S. Development of Universal Influenza A Vaccines Using Attenuated Bordetella pertussis as Antigen Delivery. In: 1st Global Vaccine Congress, Amsterdam, The Netherlands. December 9-11, 2007. [...]... of Bordetella pertussis, namely BPZE1, has been engineered (Locht, 2008; Mielcarek et al., 200 6a; Mielcarek et al., 2006b) and was shown to be a promising and attractive candidate for the delivery of heterologous vaccine antigens via the nasal route 3 CHAPTER 1 INTRODUCTION (Ho et al., 2008) In order to have a comprehensive understanding of the potential of attenuated B pertussis as a live nasal delivery... Volt vags virulence-activated genes Vras Virulence-repressed antigens vrgs virulence-repressed genes WHO World Health Organization xxii SUMMARY Bordetella pertussis, a strict human pathogen, is the causative agent of whooping cough As a pathogen naturally infecting the respiratory tract, B pertussis is particularly well adapted for the nasal delivery of heterologous vaccines candidates and has already... least 50 million people, justifying its description as “the last great plague of mankind” The subsequent pandemics in 1957 Asian flu (H2N2) and 1968 Hong Kong flu (H3N2) were milder, but nonetheless also caused a total of approximately 2 million deaths The recent spread of HPAI H5N1 virus across Asia and parts of Europe and the Middle East, as well as the occasional infections of humans with an overall... the nasal route offers several advantages over 2 CHAPTER 1 INTRODUCTION the oral route Intranasal vaccination generally elicits stronger immune responses than oral administration of the same vaccine In addition to local immunity, systemic immune responses are also achieved more easily by intranasal delivery However, although generally more effective than oral vaccination, intranasal vaccination usually... 5.1 Serum and BALFs anti-M2e IgG isotype 160 Table 5.2 Characteristics of recombinant B pertussis strains expressing H5 epitopes 162 xvii ABBREVIATIONS aa Amino acid AC Adenylate cyclase ACE 3-amino-9-ethyl-carbazole ACT Adenylate cyclase toxin ADCC Antibody-Dependent Cellular Cytoxicity ADP Adenosine diphosphate AMs Alveolar macrophages Amp Ampicillin APS Ammonium persulphate APC Antigen-presenting... detail in the following chapter Moreover, immunity induced by influenza virus infection and influenza vaccines have been covered in the first part of that chapter in order to provide a basis for discussing influenza vaccine design and development On the other hand, as influenza virus is a respiratory pathogen and influenza infections are initiated at mucosal surfaces, it is expected that mucosal vaccines... protective immunity against pertussis infection These features make BPZE1 strain not only an attractive live pertussis vaccine candidate but also a potential vehicle for vaccine delivery via the nasal route In this study, H5N1 specific antigen candidate NA, and several other antigens that are highly conserved among influenza A viruses, namely the ectodomain of M2 protein (M2e), 3 conserved neutralizing epitopes... nonspecific antiinflammatory properties of BPZE1 and suggested a potential prophylactic application to protect against highly pathogenic influenza A viruses xxiv We also investigated the potential of recombinant B pertussis strains expressing antigen candidates from influenza virus as live recombinant vaccines against influenza virus, thus combining the nonspecific anti-inflammatory properties of BPZE1 and. .. as a promising mucosal vaccine delivery system Recently, a highly attenuated B pertussis strain, BPZE1, has been engineered In addition, BPZE1 has entered phase I clinical trial in humans as live pertussis vaccine Although highly attenuated as evidenced by a markedly reduced lung inflammation in the infected animals, BPZE1 bacteria still maintain the ability to colonize the mice lungs efficiently and. .. exposure of the antigen vaccine candidate to the host‟s immune system, thereby inducing a strong specific immune response As a pathogen naturally infecting the respiratory tract, B pertussis, the causative agent of whooping cough, is particularly well adapted for the nasal delivery of heterologous vaccines which target respiratory pathogens such as influenza viruses Several heterologous antigens from various . DEVELOPMENT OF BORDETELLA PERTUSSIS AS A LIVE VEHICLE FOR HETEROLOGOUS ANTIGEN DELIVERY, AND ITS APPLICATION AS A UNIVERSAL INFLUENZA A VACCINE LI RUI NATIONAL UNIVERSITY OF. UNIVERSITY OF SINGAPORE 2010 DEVELOPMENT OF BORDETELLA PERTUSSIS AS A LIVE VEHICLE FOR HETEROLOGOUS ANTIGEN DELIVERY, AND ITS APPLICATION AS A UNIVERSAL INFLUENZA A VACCINE . Epitopes of HA protein as Universal Vaccine Candidates 48 (II) BORDETELLA PERTUSSIS AS A LIVE VEHICLE FOR HETEROLOGOUS VACCINE ANTIGENS DELIVERY THROUGH THE NASAL ROUTE 50 2.7 MUCOSAL VACCINATION