Cellular immune responses of dengue patients in singapore

CELLULAR IMMUNE RESPONSES OF DENGUE PATIENTS IN SINGAPORE DENNIS BERBULLA BELA-ONG BSc (Biology), University of the Philippines in the Visayas MSc (Molecular Biology and Biotechnology), University of the Philippines at Los Baños A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE IN INFECTIOUS DISEASES, VACCINOLOGY, AND DRUG DISCOVERY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE AND BIOZENTRUM DER UNIVERSITÄT BASEL, SWITZERLAND 2009 Acknowledgments I am greatly indebted to my supervisor Dr Katja Fink for her trust, patience, guidance, and support throughout the conduct of this project and beyond Thank you for opening up for me a challenging yet exciting world of immunology research and for inspiring me to pursue this field in the future The time that I spent in the lab was truly a great scientific adventure! I gratefully acknowledge the support of Drs Thomas Keller, Marcel Tanner, and Markus Wenk, which enabled me to pursue and successfully complete this program I would like to thank Drs Feng Gu and Martin Lloyd Hibberd for the very helpful discussions, motivation, and support, and for trusting me to work on this project I express my gratitude to the EDEN team for the wonderful collaboration; to the polyclinic nurses and to the staff of the Singapore Tissue Network, for collecting and providing us the samples; to Angelia Chow at the DSO National Laboratories, for providing us with patient data I thank all the patients for their generosity which made this study possible I express my grateful appreciation to all the people at the Dengue Unit of the Novartis Institute for Tropical Diseases for welcoming me at NITD, for the generous help in the lab, and for making my stay truly worthwhile I thank Liu Wei for the guidance in working at the virus room; Swee Hoe Ong for the amino acid sequence alignments; and Drs Celine Nkenfou and Thai Leong Yap for all the help and the very nice conversations I thank Mr Anthony Tanoto Tan at the Hepatic Viral Diseases Laboratory, Singapore Institute for Clinical Sciences, for helping me with the ELISPOT reader It was truly very nice to have Paul Murima as roommate and Martin Rao and Martin Bratschi as flatmates I thank the rest of my colleagues in this program: Peiling Yap, Boon Zhi Quek, Zhenying Song, Vincent Pang Junxiong, Cristina Mueller, and Meliana Riwanto for making my time both in Switzerland and Singapore truly an enjoyable, wonderful, and unforgettable experience I thank all our professors at the University of Basel, Swiss Tropical Institute, National University of Singapore, and Novartis Institute for Tropical Diseases for imparting your knowledge and expertise I thank Dr Gerd Pluschke for his support of my research pursuits and Dr Niklaus Weiss for his kindness 2|P ag e I greatly appreciate the reliable and very kind assistance of Christine Mensch at the Swiss Tropical Institute, for making our stay in Basel comfortable and the best that it can be I am grateful to my fellow Filipinos for the very warm welcome and accommodation you gave me while I was in Switzerland deprived of my family and friends Thank you for making me a part of your own families while I was away from home: Tita Sienna Tschopp, Lin Laube, Laura Marty, Marife Alday, and Janise Millan I thank all my friends who always find time to communicate in order to give encouragement and inspiration I thank Albert, Arman, Hazel, Martha, Nino, and Nicole for the fun and companionship I acknowledge the support of my former colleagues at the Institute of Biological Sciences, University of the Philippines Los Banos I thank everybody who in one way or another had contributed to the completion of this work I am most grateful to my family for the love, support, encouragement, and inspiration: my parents Godofredo and Lermie; my brothers Joseph, Wilfred, Ferdie, Edson, and John Kevin; my sister-in-law Hernila and niece Jonhel; my cousins Ma Victoria and Mary Grace; my nephew Virgil; my aunt Tita Emilia; and grandmother Lola Loting This work is dedicated to the memory of my grandfather Matias, and to my Tiyay Thelma, my first teacher, for the love and support, without which my dreams would have never been realized I also dedicate this work to all my teachers, whose mentorship, inspiration, and selfless dedication to their vocation made me the person that I am May this work bring honor and glory to GOD, in Whom we live, move, and have our being Thank you for blessing me with talent Thank you for the gift of all the wonderful people Thank you for making all things possible, for “unless You build the house, the builders labor in vain” MARAMING SALAMAT PO SA INYONG LAHAT! (And as we say in my homeplace: Madamo 3|P ag e gid nga salamat! Table of Contents Acknowledgments…………………………………………………………………… Table of Contents…………………………………………………………………… Summary……………………………………………………………………………… List of Tables………………………………………………………………………… 12 List of Figures………………………………………………………………………… 13 18 Introduction……………………………………………………………………… 1.1 Objectives and Significance of the Study………………………………… 23 Review of Literature…………………………………………………………… 25 2.1 The dengue virus…………………………………………………………… 26 2.1.1.Virion structure and genome organization………………………… 26 2.1.2 Replication cycle……………………………………………………… 27 2.1.3 Transmission cycle of DENV ………………………………………… 30 2.2 Dengue epidemiology 32 2.3 Clinical manifestations of dengue disease 35 2.3.1 Undifferentiated fever 35 2.3.2 Classic dengue fever………………………………………………… 36 2.3.3 Dengue hemorrhagic fever and dengue shock syndrome……… 36 2.3.4 Other disease manifestations………………………………………… 38 2.4 Dengue diagnosis…………………………………………………………… 39 2.4.1 Diagnostic tools………………………………………………………… 39 2.4.2 The WHO classification scheme…………………………………… 40 4|P ag e 2.5 Early events in the host after DENV infection………………………… 42 2.6 Risk factors for severe disease…………………………………………… 44 2.6.1 Viral factors…………………………………………………………… 45 2.6.1.1 Viral serotype…………………………………………………… 45 2.6.1.2 Viral genotype…………………………………………………… 45 2.6.1.3 Genetic determinants of virulence …………………………… 46 2.6.2 Host factors……………………………………………………………… 47 2.6.2.1 Age………………………………………………………………… 47 2.6.2.2 Genetic factors ………………………………………………… 48 2.7 Protective immune responses during dengue infection……………… 2.7.1 Innate immune responses…………………………………………… 50 50 2.7.1.1 Dendritic cells……………………………………………………… 50 2.7.1.2 Natural killer cells………………………………………………… 51 2.7.1.3 T cells expressing CD56………………………………………… 53 2.7.1.4 Interferons………………………………………………………… 57 2.7.2 Adaptive immune responses…………………………………… 58 2.7.2.1 Antibody-based protective immunity …………………………… 58 2.7.2.2 T cell-mediated protective immunity …………………………… 60 2.8 Immunopathogenesis……………………………………………………… 62 2.8.1 Antibody-mediated pathogenesis: the antibody-dependent enhancement hypothesis………………………………………………… 5|P ag e 62 2.8.2 T cell-mediated pathology…………………………………………… 2.8.2.1 T cell activation………………………………………………… 64 64 2.8.2.2 T cell original antigenic sin: the role of cross-reactive DENV-specific T cells……………………………………………… 66 2.9 Dengue in Singapore………………………………………………………… 69 2.10 The Early DENgue Infection (EDEN) and Outcomes Study …………… 74 Materials and Methods…………………………………………………………… 77 3.1 Patients……………………………………………………………………… 78 3.2 Antibody labeling of whole blood and FACS analysis…………………… 80 3.3 Statistical analysis…………………………………………………………… 82 3.4 Isolation of peripheral blood mononuclear cells (PBMCs)……………… 82 3.5 Plasma isolation……………………………………………………………… 83 3.6 IFN-γ T cell enzyme-linked immunospot (ELISPOT)…………………… 83 3.6.1 Peptide synthesis……………………………………………………… 83 3.6.2 ELISPOT………………………………………………………………… 85 3.7 Stimulation of PBMCs with DENV peptide pools, surface staining for T cell markers, and intracellular staining for IFN-γ………………………… 86 3.7.1 PBMC stimulation……………………………………………………… 86 3.7.2 Staining for cell surface antigens and intracellular IFN-γ………… 87 3.8 DENV-specific T cell line establishment…………………………………… 88 Results……………………………………………………………………………… 90 4.1 Activation and phenotypic changes in natural killer cells, natural killer T cells, and T cells in patient PBMCs during acute dengue infection and convalescence………………………………………………………………… 6|P ag e 91 4.1.1 Natural killer cells………………………………………………………… 91 4.1.2 CD3+ lymphocytes expressing CD56 99 4.1.3 T lymphocytes…………………………………………………………… 108 4.1.3.1 Phenotyping of DENV-induced effector T cells………………… 114 4.1.3.2 Changes in T cell subset phenotypes and differentiation stages after dengue infection………………………………………………………… 119 4.2 IFN-γ T cell enzyme-linkedimmunospot (ELISPOT) responses to peptides spanning the DENV-2 NS3 antigen……………………………… 123 4.2.1 IFN-γ responses from patients and dengue-immune healthy individuals……………………………………………………………… 123 4.2.2 Correlation of disease severity with T cell response ……………… 130 4.2.3 Comparison of ELISPOT with direct ex vivo plating and restimulated cells………………………………………………………… 135 4.3 T cell line establishment…………………………………………………… 138 4.4 Flow cytometry analysis of intracellular IFN-γ T cell responses to DENV-2 NS3 antigen peptides after ex vivo re-stimulation……………… 152 159 Discussion 5.1 Phenotypic analyses of DENV-induced NK cells, CD56-expressing T cells, and T ymphocytes 161 5.1.1 Activation of NK cells, CD56-expressing T cells, and T lymphocytes ……………………………………………………… 161 5.1.1.1 NK cells and CD56-expressing T cells ……………………… 161 5.1.1.2 T lymphocytes …………………………………………………… 166 5.1.2 Dengue-induced changes in T cell subpopulation phenotypes and in T cell differentiation stage………………………………………………… 169 5.1.3 Concluding remarks and outlook…………………………………… 172 7|P ag e 5.2 DENV-specific T cell responses to DENV-2 NS3 peptides……………… 175 5.2.1 Concluding remarks and outlook……………………………………… 179 5.3 T cell line establishment…………………………………………………… 180 5.3.1 Concluding remarks and outlook……………………………………… 182 5.4 Flow cytometry analysis of intracellular IFN-γ T cell responses to DENV-2 NS3 antigen peptides after ex vivo re-stimulation……………… 183 5.4.1 Concluding remarks and outlook……………………………………… 184 Bibliography………………………………………………………………………… 185 8|P ag e Summary Dengue virus (DENV) infection is a major cause of morbidity and mortality in tropical and subtropical areas worldwide Pathogenesis of dengue disease is not completely understood due to a lack of suitable experimental models for DENV infection, which hampers attempts to develop antiviral drugs and effective vaccines It has been reported that the magnitude of immune responses correlate with severe dengue disease We therefore wanted to characterize the DENVspecific T cell, NK cell, and CD56+CD3+ T cell responses in patients with acute dengue infection to find potential targets for intervention and prognostic markers As part of the on-going Early DENgue Infection and Outcomes (EDEN) Study in Singapore, fresh whole blood samples were analyzed from dengue patients longitudinally from fever onset to convalescence We found that dengue patients exhibited significantly lower absolute counts of T cells, NK cells, and CD56+ T cells early after infection and significantly higher levels of T cell, NK cell, and CD56+ T cell activation compared to febrile non-dengue controls in the acute phase, which reflects an efficient activation of the immune response early after infection The percentage of total activated cells did not significantly differ between dengue patients with primary and secondary infection, indicating that naïve T cells are as efficiently activated as memory T cells We also show changes in the T cell compartments of dengue patients potentially leading to the 9|P ag e establishment of high numbers of memory cells These changes did not significantly differ between primary and secondary dengue infection Activation of naïve T cells and reactivation of memory T cells may be protective or diseaseenhancing However, since all our cases were mild, we cannot address the question whether re-activation of T cell memory is potentially immunopathogenic, as suggested by others Next, we established methods to detect dengue-specific T cells Therefore, peripheral blood mononuclear cells from dengue patients and healthy dengueimmune individuals were investigated by IFN-γ T cell ELISPOT and flow cytometry for their DENV-specific responses to peptides spanning conserved regions of the immunodominant NS3 epitope of DENV serotype majority consensus sequence The specificity of the T cell responses was donordependent IFN-γ responses of cultured PBMCs (in the absence of irradiated autologous PBMCs) from dengue-immune individuals after re-stimulation with peptide pools were analyzed by intracellular IFN-γ staining and flow cytometry CD4+ and CD8+ T cell 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dengue