Investigations on the immunopathology of enterovirus 71 1

INVESTIGATIONS ON THE IMMUNOPATHOLOGY OF ENTEROVIRUS 71 KHONG WEI XIN (B. Sc. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Acknowledgements This thesis could not have been written without Dr. Sylvie Alonso, who not only served as my supervisor but also encouraged and challenged me throughout my academic program. Thank you for being such a fantastic teacher and for guiding me patiently throughout the dissertation process, never accepting less than my best effort. The impact of your help is significant, and will benefit me for the rest of my life. I truly can't thank you enough and will be forever grateful. Special gratitude to Associate Professor Vincent Chow and Associate Professor Kevin Tan. Thank you for all the much-appreciated advice and guidance. Thank you a million times over to my past and present lab mates from the SA lab. I find myself so fortunate to have such wonderful friends working alongside me. Thanks a ton for making the lab a blissful working environment and for returning my endless complains with support and understanding. You are a great source of strength to me over the past years. To Michelle, Wenwei, Regina, Vanessa, Zarina and Fiona, thank you for the wonderful time. We made it! Every happy moment we had together has been seared in my memory, which I'll never forget. I'm forever indebted to Jowin, Grace, Andrew, Boon King and Eng Lee, who offered so much valuable insights to my work, and for always being there, in big ways and smalls. A most loving and special thank you to my family and Adrian. Words alone cannot express what I owe them for their encouragement and whose patient care enabled me to complete this daunting yet well-worth journey. Special thanks to Adrian who read and corrected every single draft of this thesis, for putting up with me all, and for cracking me up, time after time, always knowing when it's most needed. Because of you, I feel lucky everyday. ii Publications Articles Khong WX, Chow VTK and Alonso S (2010). Exploring the versatility of the autotransporter BrkA for the presentation of enterovirus 71 vaccine candidates at the surface of attenuated Bordetella pertussis. Procedia in Vaccinology. 2:66-72. Khong WX, Yan B, Yeo H, Tan EL, Lee JJ, Ng JK, Chow VT, and Alonso S (2012). A non-mouse-adapted enterovirus 71 (EV71) strain exhibits neurotropism, causing neurological manifestations in a novel mouse model of EV71 infection. J. Virol. 86: 2121-31. Khong WX, Foo DGW, Trasti SL, Tan EL, and Alonso S (2011). Sustained high levels of IL-6 contribute to the pathogenesis of enterovirus 71 in a neonate mouse model. J. Virol. 85: 3067-76. Lin XF, Jia Q, Khong WX, Yan B, Premanand B, Alonso S, Chow VT, and Kwang J (2012). Characterization of an isotype-dependent monoclonal antibody against linear neutralizing epitope effective for prophylaxis of enterovirus 71 infection. PLoS One. 7:e29751. Review Khong WX, Yeo H and Alonso S (2012). Enterovirus 71: Pathogenesis, Control and Models of Disease. Future Virology. Accepted. iii Table of Contents ACKNOWLEDGEMENTS II PUBLICATIONS . III LIST OF FIGURES . IX LIST OF TABLES . XII SUMMARY XIII LIST OF ABBREVIATIONS XVI CHAPTER LITERATURE REVIEW 1.1 VIROLOGY 1.1.1 Classification 1.1.2 Genomic and organization of EV71 1.1.3 Virus entry and spread in humans 1.1.4 Life cycle and replication 1.2 EPIDEMIOLOGY 11 1.2.1 Clinical epidemiology .11 1.2.2 Molecular epidemiology .13 1.3 CLINICAL FEATURES 18 1.3.1 Mucocutaneous and respiratory 18 1.3.2 Neurological and systemic manifestations .19 1.3.3 Pathological observations .22 1.4 PATHOGENESIS 26 1.4.1 Viral determinants of virulence 26 1.4.2 Host genetic factors 28 1.4.3 Immunopathogenesis 29 1.4.3.1 Cytokine and chemokine-induced bystander damage .31 1.4.3.2 Lymphocyte depletion 33 1.4.3.3 Virus spread using immune target cell 33 1.4.3.4 Antibody-dependent enhancement 36 1.5 CONTROL OF VIRAL INFECTIONS 37 1.5.1 Virus surveillance and social distancing 37 1.5.2 EV71 Vaccine development 40 1.5.3 Treatment against EV71 45 1.6 ANIMAL MODELS . 53 iv 1.6.1 Non-human primate animal model 53 1.6.2 Mouse models 54 1.7 SPECIFIC AIMS . 57 CHAPTER MATERIALS AND METHODS . 59 2.1 MOLECULAR BIOLOGY . 59 2.1.1 Detection of specific IgM and IgG antibodies 59 2.1.2 Cytokine quantification by ELISA 60 2.2 VIRUS WORK 60 2.2.1 Virus strains 60 2.2.2 Virus propagation .62 2.2.3 Purification and concentration of virus .62 2.2.4 Virus quantification 63 2.2.4.1 Virus quantification by 50% tissue culture infective dose (TCID50) assay .63 2.2.4.2 Virus quantification by real-time PCR .64 2.2.4.3 Virus quantitation by plaque assay .65 2.3 CELL BIOLOGY . 66 2.3.1 The rhabdomyosarcoma cell line 66 2.3.1.1 Maintenance and storage 66 2.3.1.2 Plaque reduction neutralization test (PRNT) 67 2.3.2 Primary cells 68 2.3.2.1 Isolation and differentiation of mouse bone-marrow derived dendritic cells (BMDCs) .68 2.3.2.2 Isolation of murine splenocytes .68 2.3.2.3 Isolation of cells from lymph nodes .69 2.3.2.4 Isolation of T-lymphocytes .70 2.3.3 BMDC infection .70 2.3.4 Quantification of cell viability .71 2.3.4.1 XTT assay 71 2.3.4.2 PI staining .72 2.3.5 Allogeneic mixed lymphocyte reaction .72 2.3.6 Measurement of cell proliferation via 3H-thymidine incorporation 73 2.3.7 Flow cytometric analysis .73 2.3.7.1 Surface marker expression .73 2.3.7.2 Carboxyfluorescein succinimidyl ester (CFSE) staining 74 2.4 ANIMAL WORK 75 2.4.1 Ethics statement .75 2.4.2 Neonatal mice 76 v 2.4.2.1 EV71 infection of neonatal mice .76 2.4.2.2 Anti-IL-6 monoclonal antibody treatment 76 2.4.2.3 Isolation of intestinal RNA for viral quantification .76 2.4.3 AG129 mice .77 2.4.3.1 EV71 infection of AG129 mice 77 2.4.3.2 Passive transfer of antibody 77 2.4.3.3 Ribavirin treatment .78 2.4.3.4 Quantification of blood and tissue viral loads .78 2.4.4 Histology 79 2.4.5 Adoptive transfer of BMDC .80 2.5 STATISTICS 80 CHAPTER 3: ROLE OF INTERLEUKIN-6 IN THE IMMUNOPATHOGENESIS OF EV71 INFECTION . 82 3.1 INTRODUCTION . 82 3.2 RESULTS 84 3.2.1 Systemic and local levels of IL-6 are elevated in EV71-infected mice .84 3.2.2 Suppression of serum IL-6 levels in EV71-infected mice by antibodies 85 3.2.3 Anti-IL-6 treatment protects mice from lethal EV71 infection 88 3.2.4 Anti-IL-6 antibody treatment prevents tissue damage in EV71-infected mouse neonates .91 3.2.5 Anti-IL-6 antibody treatment did not affect the viral load 95 3.2.6 Anti-IL-6 antibody treatment increased serum IL-10 production 97 3.2.7 Anti-IL-6 treatment at the time of infection is detrimental to the mice 99 3.3 DISCUSSION 105 CHAPTER 4: EV71 INFECTION OF BONE-MARROW DERIVED DENDRITIC CELLS (BMDCS) . 111 4.1 INTRODUCTION 111 4.2 RESULTS . 113 4.2.1 BMDCs are permissive to EV71 infection 113 4.2.2 EV71 infection increases BMDC viability 115 4.2.3 Cytokine profiles in BMDCs infected with EV71 118 4.2.4 Differential phenotypic modulation of BMDCs infected with live EV71 and heat-inactivated EV71 120 4.2.5 EV71-infected BMDCs show defects in the activation of TH1 cells in vitro 122 4.2.6 EV71-infected BMDCs show defects in the activation of TH1 cells in vivo 125 4.2.7 EV71 infection increases BMDCs mobility . 128 4.3 DISCUSSION 131 vi CHAPTER DEVELOPMENT OF A NOVEL MOUSE MODEL OF EV71 INFECTION 136 5.1 INTRODUCTION 136 5.2 RESULTS . 140 5.2.1 Two-week-old or younger AG129 mice develop fatal EV71 infection 140 5.2.2 AG129 mice are susceptible to EV71 infection via ip. and oral route in a dose-dependent manner 142 5.2.3 EV71 strain 41 displays neurotropism in AG129 mice 144 5.2.4 Histopathological examination of EV71-infected mice . 148 5.2.5 Pro-inflammatory cytokines are up-regulated in EV71-infected mice . 151 5.2.6 Adaptive immune response in EV71-infected AG129 mice 153 5.2.7 Model validation . 155 5.3 DISCUSSION 159 CHAPTER INVESTIGATIONS ON EV71 VIRULENCE DETERMINANTS IN THE AG129 MOUSE MODEL . 163 6.1 INTRODUCTION 163 6.2 RESULTS . 166 6.2.1 Comparison of clinical outcomes following infection in AG129 mice 166 6.2.2 Fatality was associated with tissue damages in CNS of AG129 mice . 169 6.2.3 Fatal strains displayed neurotropism in AG129 mice 171 6.2.4 Pro-inflammatory cytokines were up-regulated in mice infected with fatal-causing strains 174 6.2.5 Adaptive immune response in EV71-infected AG129 mice 176 6.2.6 Fatal-causing strains induced greater cytotoxicity in vitro . 180 6.2.7 Comparative genomic analysis of EV71 strains 184 6.3 DISCUSSION 186 CHAPTER CONCLUSION AND FUTURE WORK 192 7.1 ROLE OF INTERLEUKIN-6 IN THE IMMUNOPATHOGENESIS OF EV71 INFECTION . 192 7.2 ROLE OF DC IN EV71 INFECTION 196 7.3 DEVELOPMENT OF A NOVEL MOUSE MODEL FOR EV71 INFECTION . 199 7.4 INVESTIGATIONS ON EV71 VIRULENT DETERMINANTS IN THE AG129 MOUSE MODEL 202 CHAPTER REFERENCES 206 APPENDIX I: REAGENTS FOR GROWTH MEDIA I vii APPENDIX II: MISCELLANEOUS BUFFERS II APPENDIX III: TCID50 ASSAY IV APPENDIX IV: PUBLICATIONS V viii List of Figures CHAPTER Figure 1.1 Enterovirus 71 (EV71) structure and genome structure of the virion. Figure 1.2 Intracellular life cycle of EV71. 10 Figure 1.3 Distribution of EV71 isolates identified globally from 1970 to 2000. 16 Figure 1.4 Distribution of inflammation in brain sections of EV71 patients. 23 Figure 1.5 The postulated pathology of EV71-associated acute pulmonary oedema.25 CHAPTER Figure 3.1 Systemic IL-6 levels in EV71-infected mouse neonates.80 Figure 3.2 IL-6 productions in the brain, muscle, intestines, spleen, and lungs from EV71-infected mouse neonates. 85 Figure 3.3 Survival rate and clinical score of EV71-infected mouse neonates either untreated or treated with anti-IL-6 antibodies post-infection. 90 Figure 3.4 Histological examination of the muscles, intestines, and spleen from EV71infected mice either untreated or treated with anti-IL-6 antibodies postinfection. 93 Figure 3.5 Spleen cell composition in EV71-infected mice either untreated or treated with anti-IL-6 antibodies post-infection. 94 Figure 3.6 Viral load in the intestines of EV71-infected mice either untreated or treated with anti-IL-6 antibodies post-infection. 96 Figure 3.7 Serum IL-6 and IL-10 levels in EV71-infected mice either untreated or treated with anti-IL-6 antibodies post-infection. 98 Figure 3.8 Survival rate and clinical score of EV71-infected neonatal mice either untreated or co-treated with anti-IL-6 antibodies. 101 Figure 3.9 Histological examination of the limb muscle, intestines, and spleen from EV71-infected mice either untreated or co-treated with anti-IL-6 antibodies. 102 ix APPENDIX IV XXXI APPENDIX IV XXXII APPENDIX IV XXXIII APPENDIX IV XXXIV APPENDIX IV XXXV APPENDIX IV XXXVI APPENDIX IV XXXVII APPENDIX IV XXXVIII APPENDIX IV XXXIX APPENDIX IV XL APPENDIX IV XLI APPENDIX IV XLII APPENDIX IV XLIII APPENDIX IV XLIV APPENDIX IV XLV APPENDIX IV XLVI [...]... REVIEW Table 1. 1 Human enterovirus species and serotype Enterovirus species A Enterovirus species B Enterovirus species D 1- 3 Polioviruses Coxsackie A viruses Enterovirus species C 2-8, 10 , 12 , 14 , 16 9 Coxsackie B viruses 1- 6 Echoviruses 1, 11 , 13 , 15 , 17 -22, 24 1- 9, 11 - 21, 24-27, 29-33 Enteroviruses 71, 76, 89-92 69, 73-75, 77-88, 93, 97, 98, 10 0, 10 1, 10 6, 10 7 95, 96, 99, 10 2, 10 4, 10 5, 10 9, 11 6 68, 70,... via the oral route 14 7 Figure 5.5 Histological examination of EV 71- infected mice 14 9 Figure 5.6 Detection of EV 71 particles in the brain by immunohistochemistry 15 0 14 3 x Figure 5.7 Systemic cytokine profile in EV 71- infected AG129 15 2 Figure 5.8 Adaptive immune response in EV 71- infected AG129 15 4 Figure 5.9 Passive protection of EV 71- infected AG129 mice 15 7 Figure 5 .10 Effect of ribavirin treatment on. .. formation assay 18 5 Figure 6.7 In vitro analysis of EV 71 strain virulence 18 3 xi List of Tables CHAPTER 1 Table 1. 1 Human enterovirus species and serotype 2 Table 1. 2 Enterovirus 71 genotypic subgroups reported to be circulating in the AsiaPacific region between 19 70 and 2 010 17 Table 1. 3 Neurological syndromes associated with EV 71 infection 21 Table 1. 4 Anti-EV 71 activity of selected compounds 49 Table 1. 5... infection enhanced BMDC migration by increased expression of CCR7 13 0 11 7 12 1 CHAPTER 5 Figure 5 .1 Age-dependent mortality of AG129 mice intraperitoneally infected with EV 71 1 41 Figure 5.2 Survival rate of AG129 mice infected with a dose range of EV 71 Figure 5.3 Virus titers in organs from AG129 infected with EV 71 via the ip and oral route 14 6 Figure 5.4 Viral RNA in organs from AG129 infected with EV 71. .. Stimulation index xix S10 Strain 10 S 41 Strain 41 TNF Tumor necrosis factor TCID50 50% of Tissue Culture Infective Dose TLR Toll-like receptor WBC White blood cell WHO World health organization xx CHAPTER 1 LITERATURE REVIEW Chapter 1 Literature Review 1. 1 Virology 1. 1 .1 Classification Taxonomically, the major etiological agent of the Hand, Foot and Mouth disease (HFMD), Enterovirus 71 (EV 71) belongs to... stimulation Figure 4.3 Differential cytokine profiles by BMDCs stimulated with live and heatinactivated EV 71 119 Figure 4.4 EV 71 infection impairs responsiveness of BMDCs to TLR ligands Figure 4.5 In vitro proliferative response of lymphocytes against EV 71- infected BMDCs 12 4 Figure 4.6 T cells from mice receiving EV 71- infected BMDCs showed diminished response to re-stimulation with EV 71 127 Figure 4.7 EV 71. .. VPg (3B protein) The 3 ′UTR region contains a pseudo-knot like structure and is important for the replication of EV 71 The polyprotein is subdivided into three regions, namely P1, P2 and P3 (Fig 1. 1) The P1 region encodes four viral structural (VP1 to VP4), while the other two regions encode seven non-structural proteins (2A to 2C and 3A to 3D) (Brown & Pallansch, 19 95) Once synthesized, the nascent polyprotein... arranged on an icosahedral lattice (Fig 1. 1) Among them, VP1, VP2 and VP3 are the main structural components of the virion, whereas VP4 is completely internalized and is not, therefore, exposed to the host antibody response (Hogle et al, 19 85) The capsid proteins play the roles of not only receptor binding on the surface from susceptible host cells but also contain the antigenic determinants of the virus...Figure 3 .10 Spleen cell composition in EV 71- infected mice either either untreated or co-treated with anti-IL-6 antibodies 10 3 Figure 3 .11 Viral load and systemic IL-6 levels in EV 71- infected mice either untreated or co-treated with anti-IL-6 antibodies 10 4 CHAPTER 4 Figure 4 .1 Virus production upon infection of bone marrow-derived dendritic cells with EV 71 114 Figure 4.2 BMDCs increase viability upon stimulation... cytopathic effect of the virus but correlate with the ability of the virus to induce brain damage A full genome comparison of these EV 71 isolates could potentially lead to the identification of genetic determinants underlying the neurovirulence of EV 71 xiv Overall, our work has contributed to a better understanding of the mechanisms involved in EV 71 pathogenesis with the development of a novel mouse . Classification 1 1. 1.2 Genomic and organization of EV 71 3 1. 1.3 Virus entry and spread in humans 6 1. 1.4 Life cycle and replication 7 1. 2 EPIDEMIOLOGY 11 1. 2 .1 Clinical epidemiology 11 1. 2.2. at the time of infection is detrimental to the mice 99 3.3 DISCUSSION 10 5 CHAPTER 4: EV 71 INFECTION OF BONE-MARROW DERIVED DENDRITIC CELLS (BMDCS) 11 1 4 .1 INTRODUCTION 11 1 4.2 RESULTS 11 3. model of EV 71 infection. J. Virol. 86: 212 1- 31. Khong WX, Foo DGW, Trasti SL, Tan EL, and Alonso S (2 011 ). Sustained high levels of IL-6 contribute to the pathogenesis of enterovirus 71 in