DEVELOPMENT OF LIVE BACTERIAL SYSTEMS FOR PRESENTATION OF DENGUE EDIII TO THE MUCOSAL IMMUNE SYSTEM LAM JIAN HANG NATIONAL UNIVERSITY OF SINGAPORE 2014 DEVELOPMENT OF LIVE BACTERIAL SYSTEMS FOR PRESENTATION OF DENGUE EDIII TO THE MUCOSAL IMMUNE SYSTEM LAM JIAN HANG B.SC (HONS), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously _ Lam Jian Hang 20 August 2014 PUBLICATIONS Hoo, R., Lam, J.H., Huot, L., Pant, A., Li, R., Hot, D., Alonso, S (2014) Evidence for a role of the polysaccharide capsule transport proteins in pertussis pathogenesis PLoS One 9(12): e115243 Ng, J.K., Zhang, S.L., Tan, H.C., Yan, B., Maria Martinez Gomez, J., Tan, W.Y., Lam, J.H., Tan, G.K., Ooi, E.E., Alonso, S (2014) First experimental in vivo model of enhanced dengue disease severity through maternally acquired heterotypic dengue antibodies PLoS Pathog 10(4): e1004031 PRESENTATION AT INTERNATIONAL CONFERENCES Lam, J.H., Alonso, S (2012) Expression and delivery of dengue EDIII by Bordetella pertussis BPZE1 via the BrkA autotransporter Oral presentation prize In: 4th Australasian Vaccines and Immunotherapeutics Development Meeting, Brisbane, Australia i ACKNOWLEDGEMENTS I would like express to my heartfelt thanks to my supervisor Assoc Prof Sylvie Alonso for first giving me the chance to join her lab in 2008, and subsequently offering invaluable advice, guidance and opportunities to develop my mind and skills as a researcher Over the years, I have known her to be the most patient and encouraging supervisor I could ask for and, once again, I offer my sincere appreciation for all she has taught me To my lab mates, past and present, I thank you all for making the working environment lively, entertaining, exciting and, frequently, edible I really treasure the friendships that we’ve built and the time we spent as a lab A big thank you to all the names listed in Table A below :) Past Jowin Ng Kai Wei Regina Hoo May Ling Annabelle Lim Rui Fen Xu Wei Zhen Grace Tan Kai Xin Khong Wei Xin Zarina Li Rui Aakanksha Present Lin Wenwei Julia Maria Martinez Gomez Michelle Ang Vanessa Koh Emily Ang Ong Li Ching Chionh Yok Hian Ng Sze Wai Issac Too Anna Ker Yeo Huimin Eshele Table A: List of awesome SA lab mates encountered over the course of my PhD candidature I would also like to express my appreciation to my TAC Dr Ooi Eng Eong and Dr Ratha Mahendran for offering useful comments and advice during our meeting Lastly, I would like to express my deepest gratitude to my parents and my sister who have been hugely supportive and understanding when I had to shift my residential address to CeLS during my most intensive months None of this work would have been possible without them ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii SUMMARY xii LIST OF TABLES xiv LIST OF FIGURES xv LIST OF ABBREVIATIONS xviii CHAPTER 1: INTRODUCTION 1.1 DENGUE: VIROLOGY, DISEASE AND EPIDEMIOLOGY 1.1.1 Virion structure and assembly 1.1.2 Disease and epidemiology 1.2 THE ADAPATIVE IMMUNE RESPONSE FOLLOWING A DENV INFECTION 1.2.1 The anti-DENV immune response in protection and disease enhancement 1.2.1.1 Antibodies 1.2.1.1.1 Antibody-dependent enhancement (ADE) of infection 1.2.1.2 1.3 T cells PROSPECTS FOR A DENGUE VACCINE 10 1.3.1 Challenges of vaccine development 10 1.3.1.1 Unbalanced immune responses 11 1.3.1.2 Immune correlates of protection 11 1.3.1.3 Animal model 12 1.3.2 Vaccine candidates in development 15 1.3.2.1 LATVs 15 1.3.2.2 Subunit vaccines as an alternative 15 1.4 DENV ENVELOPE GLYCOPROTEIN 19 1.4.1 Structural and serological characteristics 19 1.4.2 EDIII as a vaccine candidate: some considerations 20 1.4.2.1 20 Production and protective efficacy iii 1.4.2.2 Relevance to the human disease 20 1.4.2.3 Quality of the anti-EDIII antibody response 21 1.5 LIVE BACTERIAL VECTORS FOR ANTIGEN DELIVERY 22 1.5.1 Rationale for using a live bacterial vector 22 1.5.2 Attenuated pathogens 24 1.5.2.1 B pertussis as a live vector for nasal delivery of heterologous antigens 25 1.5.2.1.1 Resurgence of pertussis despite vaccination 25 1.5.2.1.2 Developing a live attenuated B pertussis vaccine 26 1.5.2.1.3 Virulence factors 26 1.5.2.1.3.1 FHA 27 1.5.2.1.3.2 PTX 28 1.5.2.1.3.3 BrkA 30 1.5.2.1.4 B pertussis as a live delivery system 31 1.5.2.1.4.1 Integration of foreign genes into B pertussis genome 31 1.5.2.1.4.2 Virulence factors as carriers for antigen presentation 33 1.5.3 Lactic acid bacteria 1.5.3.1 36 L lactis as a live vector for delivery of heterologous antigens 36 1.5.3.1.1 Characteristics of L lactis 36 1.5.3.1.2 The L lactis molecular toolbox 38 1.5.3.1.2.1 Promoters 38 1.5.3.1.2.1.1 Constitutive promoters 38 1.5.3.1.2.1.2 Inducible promoters 39 1.5.3.1.2.2 41 Protein targeting 1.5.3.1.2.2.1 Cytoplasmic expression 41 1.5.3.1.2.2.2 Secretion 41 1.5.3.1.2.2.3 Cell wall associated 42 1.5.3.1.3 46 1.6 L lactis as live mucosal vaccine HETEROLOGOUS PRODUCTION OF EDIII IN B PERTUSSIS AND L LACTIS – SOME CONSIDERATIONS 48 1.6.1 B pertussis – a gram negative organism iv 48 1.6.2 L lactis – a gram positive organism 49 1.7 51 OBJECTIVES OF THIS PROJECT CHAPTER 2: MATERIALS AND METHODS 2.1 52 ASSESSING THE SUITABILITY OF EDIII AS A SUBUNIT VACCINE CANDIDATE 53 2.1.1 Escherichia coli work 53 2.1.1.1 E coli strains, plasmids and culture conditions 53 2.1.1.1.1 Bacterial strains and plasmids 53 2.1.1.1.2 Culture conditions 54 2.1.1.2 Molecular biology 54 2.1.1.2.1 List of primers 54 2.1.1.2.2 DNA amplification 54 2.1.1.2.3 Restriction enzyme digest, agarose gel electrophoresis, gel extraction and generation of recombinant DNA 2.1.1.2.4 55 E coli transformation, plasmid extraction and DNA sequencing 2.1.1.3 55 Production and purification of recombinant EDIII (rEDIII) 55 2.1.1.3.1 Expression of ediii by IPTG induction 55 2.1.1.3.2 Inclusion body isolation 55 2.1.1.3.3 Solubilisation of inclusion body and refolding of rEDIII 56 2.1.1.3.4 Purification of rEDIII using Ni-NTA chromatography 56 2.1.2 Animal work 57 2.1.2.1 Mouse strains 57 2.1.2.2 Subcutaneous injection 57 2.1.2.3 Assessment of serum antibody responses 57 2.1.2.3.1 Enzyme-linked immunosorbent assay (ELISA) 57 2.1.2.3.1.1 Preparation of coating antigens 57 2.1.2.3.1.2 Indirect ELISA 58 2.1.3 Virus work 2.1.3.1 58 Virus strain, cell lines, growth conditions and virus quantitation 58 v 2.1.3.1.1 Virus strain and cell lines 58 2.1.3.1.2 Growth conditions 59 2.1.3.1.3 Virus quantitation 59 2.1.3.1.4 Plaque reduction neutralisation test (PRNT) 60 2.1.4 Statistical analysis 2.2 60 BORDETELLA PERTUSSIS AS A LIVE VECTOR FOR THE PRODUCTION AND MUCOSAL DELIVERY OF DENV2 EDIII 61 2.2.1 Escherichia coli work 61 2.2.1.1 E coli strains, plasmids and culture conditions 61 2.2.1.1.1 Bacterial strains and plasmids 61 2.2.1.1.2 Culture conditions 63 2.2.1.2 Molecular biology 63 2.2.1.2.1 List of primers 63 2.2.1.2.2 Polymerase chain reaction (PCR) 64 2.2.1.2.2.1 DNA amplification for cloning work 64 2.2.1.2.2.2 Colony PCR screening 65 2.2.1.2.3 Restriction enzyme digest 65 2.2.1.2.4 Agarose gel electrophoresis 65 2.2.1.2.5 Gel extraction 66 2.2.1.2.6 Generation of recombinant DNA 66 2.2.1.2.7 E coli heat shock transformation 66 2.2.1.2.8 Plasmid extraction 67 2.2.1.2.9 DNA Sequencing 67 2.2.2 Bordetella pertussis work 67 2.2.2.1 B pertussis strains and culture conditions 67 2.2.2.1.1 Bacterial strains 67 2.2.2.1.2 Culture conditions 68 2.2.2.2 Bacterial transformation 69 2.2.2.2.1 Preparation of electrocompetent cells 69 2.2.2.2.2 Electroporation 69 2.2.2.2.3 Double homologous recombination 69 2.2.2.3 Analysis of protein production 70 2.2.2.3.1 Western blot 70 vi 2.2.2.3.1.1 Preparation of bacterial lysate and culture supernatant 2.2.2.3.1.2 70 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 70 2.2.2.3.1.3 Coomassie Blue staining 71 2.2.2.3.1.4 Electroblotting 71 2.2.2.3.1.5 Immunoblotting 71 2.2.2.3.2 Flow cytometry 72 2.2.2.4 Animal work 73 2.2.2.4.1 Mouse strains 73 2.2.2.4.2 Intranasal inoculation 73 2.2.2.4.3 Lung colonisation study 73 2.2.2.4.4 Immunisation study 74 2.2.2.4.4.1 Immunisation and blood collection schedule 74 2.2.2.4.4.2 Assessment of serum IgG response by enzyme-linked immunosorbent assay (ELISA) 74 2.2.2.4.4.2.1 Preparation of coating antigens 74 2.2.2.4.4.2.2 Indirect ELISA 74 2.2.3 Statistical analysis 75 2.2.4 Virus work 75 2.2.4.1 Virus strain, cell lines, growth conditions, virus quantitation and PRNT 2.3 75 LACTOCOCCUS LACTIS AS A LIVE VECTOR FOR THE PRODUCTION AND MUCOSAL DELIVERY OF DENV2 EDIII 76 2.3.1 Escherichia coli work 76 2.3.1.1 E coli strains, plasmids and culture conditions 76 2.3.1.1.1 Bacterial strains and plasmids 76 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