INVESTIGATING THE ROLE OF FC GAMMA RECEPTORS (FcRS) IN DENGUE VIRUS NEUTRALIZATION TANU CHAWLA MSc (Biochemistry) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY YONG LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2015 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. __________________________ Tanu Chawla 20/01/2015 ii Acknowledgements I would like to take this opportunity to express my heartfelt gratitude to all those people who made this thesis possible. First and foremost, I am highly thankful to my supervisor, Associate Professor Eng Eong Ooi for giving me an opportunity to research in his lab, under his able guidance. His enthusiastic support and suggestions has remarkably aided the progress of my project. His patient mentorship throughout my research has provided excellent scientific training in terms of critical thinking and writing skills, for which I am very much obliged to him. I would like to extend my thanks to my thesis advisory committee members Associate Professor Paul MacAry, Professor Soman Abraham and Associate Professor Shee Mei Lok for their valuable suggestions during my research. I would like to express my heartfelt appreciation to my lab members Kuan Rong Chan, Hwee Cheng Tan, Summer Zhang, Eugenia Ong and Jason Tang for technical assistance, motivating discussions and friendly lab environment. Special thanks to my other colleagues from DUKE-NUS for providing important reagents required for the project, to FACS core facility for cell sorting and especially to my friend Elena Okina for encouraging dialogues. Finally, I would like to thank my family for their invaluable support. I am highly grateful to love of my life, my husband Sudhir Pasumarty, who has continuously supported and encouraged me and made my PhD a happy, smooth and successful journey. I am thankful to my parents from bottom of my heart for trusting in me and allowing me to follow my dream. They have always motivated me even though being far away in other country. I am indebted to them for their unconditional love. Lastly, I am thankful to my brother, Karan who made me laugh at times when I was stressed. Not forgetting my friend, Nimrata, Swati and Soka association friends with whom I share my joys and sorrows, I am glad to thank all. iii Table of Contents Acknowledgements … .………………………… . iii Table of contents …… .………………………… . iv Summary …………… ………………………… . viii List of tables …… .………………………… x List of figures …… .………………………… . xi List of abbreviations … .………………………… xiii List of publications …… ………………………… xvii S.No. Topic Page Chapter 1: An Introduction to Dengue … .……………………… 1.1 1.2 1.3 Dengue …….………………….……………… . 1.1.1 Dengue epidemiology …………………………… .……… 1.1.2 Dengue disease manifestations …………………… …… 10 1.1.3 Dengue virus genome and structure ……………… …… 13 Dengue prevention and control ……………….………… 18 1.2.1 Vector control ……………… …………………… .……… 18 1.2.2 Antivirals ……………… …………………… .…………… 23 1.2.3 Vaccine ……………… …………… …………… .……… 25 1.2.4 Challenges in vaccine development …………… .……… 28 Immune correlates of DENV infection ……………… … 32 1.3.1 T cell responses ……… …………………… .…………… 32 Pathogenesis by T cells ………………… ……………. 33 Protection by T cell responses … ………… .…………… 34 1.3.2 Antibodies are protective or pathogenic …….……………. 36 Protection by antibodies ………………….… .……………. 39 iv Antibody mediated DENV neutralization …… …………… 42 Pathogenesis by antibodies ……………… .…………… 44 1.4 FcRs Utilization in DENV enhancement and neutralization ….…. 49 Study aims ……………… .…………………………………………….… 57 Chapter 2: Methods … .……………………… 59 2.1 Cells …….……………………….……………… . 60 2.2 Antibodies ……………………….……………… 60 2.3 Virus culture and purification ……………… .…….…………… 62 2.4 Plaque assay ………………………………….…….…………… 62 2.5 Titration of h3H5 antibody for complete neutralization in THP-1, K562 shControl and shFcRIIA cells …………………………… . 63 2.6 Fluorescent Labeling of Viruses ……………… .………… … 2.7 Infection for localization studies in THP-1 or K562 cells … … 64 2.8 Immunofluorescence Assay (IFA) ………………… .………… . 65 2.9 Fluorescence-activated cell sorting (FACS) ………………… . 66 2.10 siRNA transfection in THP1 or K562 ………………… .…… . 66 2.11 Flow Cytometry to determine surface expression of FcRs … 68 2.12 Western Blot ………………… .………………………………… . 68 2.13 Human Phospho-kinase array ………………… .………… . 69 2.14 Lentiviral particles transduction in THP-1 cells ………………… 69 2.15 Rapamycin drug treatment ………………… .………… 70 2.16 Statistical analysis ………………… .………… . 70 v 63 Chapter 3: Results … .……………………… . 71 3.1 Stoichiometric antibody requirements for DENV neutralization, when antibody-opsonized DENV is ligated with different activating Fc gamma receptors (FcRI and FcRIIA) 73 3.1.1 FcRIIA mediated phagocytosis requires higher antibody concentration for DENV neutralization …… . 74 3.1.2 Inhibition of phagocytosis in K562 cells mediated by FcRIIB ……………………………………………… . 78 3.1.3 FcRI requires less antibodies for complete neutralization and lowered virus yield in sub-neutralizing concentrations 80 3.1.4 DENV is preferentially phagocytosed by FcRI when opsonized with neutralizing antibody but not sub-neutralizing levels ……………………………………… 82 3.2 Analysis of signaling events regulated by FcRI and FcRIIA involved in antibody-mediated neutralization in monocytes … 87 3.2.1 Akt-protein kinase pathway is activated following infection with the h3H5 neutralizing antibody opsonized DENV in the presence of FcRI …………… . 89 3.2.2 Deriving a stable FcRI and FcRIIA knockdown cell line 95 3.2.3 Akt and 4EBP-1 phosphorylation in shControl and shFcRIIA Cells ……………………………… ……… . 100 3.2.4 ISG translation in shFcRIIA cells …………………… . 103 3.2.5 Low expression of eiF4E does not change antibody requirement for DENV neutralization ……… ……… . 104 3.2.6 Inhibiting of mechanistic target of rapamycin (mTORC1) activity did not change the antibody requirement in DENV neutralization ……… 106 vi Chapter 4: Discussion … .……………………… . 109 4.1 Lower antibody concentration required for DENV neutralization if immune complex is phagocytosed through FcRI compared to FcRIIA …….……………………….……………… 112 4.2 Preferential interaction of FcRI and DENV opsonized with antibody at neutralizing but not sub-neutralizing levels … 116 4.3 Analysis of FcRI mediated signaling to unravel the path for DENV clearance . 118 4.4 Future directions 4.5 Conclusions . 123 121 Chapter 5: Bibliography . 124 Appendices . 155 vii Summary Dengue is the most prevalent mosquito borne viral disease globally and is hence a cause of disease burden on all tropical nations. Currently there is no licensed vaccine or therapeutic for dengue and vector control has not been sustainable in stemming the spread of disease. In humans, DENV has been shown to infect monocytes, macrophages and dendritic cells, all of which express activating Fc gamma receptors (FcR). Consequently, neutralization of DENV by antibodies should be understood in the context of these cell types. Using human acute monocytic leukemia cells expressing FcRI and FcRII (THP-1) and human myelogenous erythroleukemic cells (K562) expressing FcRII, this thesis examined how different activating FcRs influence the amount of antibody required to completely neutralize DENV. Experimental data indicates that the amount of antibody needed for complete DENV neutralization is significantly lower when immune complexes are phagocytosed by FcRI than by FcRIIA. Moreover, the data also demonstrates that FcRI is preferentially engaged by DENV when opsonized with neutralizing levels of antibody by clustering this receptor. Finally, we also show that lower antibody concentration required when antibody-opsonized DENV is phagocytosed by FcRI instead of FcRIIA cannot be explained by an antiviral response triggered by FcRI signaling that controls viral uncoating due to insufficient neutralization. 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The Journal of general virology 89: 3047-3051 154 APPENDICES 1. Cell culture medium 1.1 Fetal bovine serum (FBS) Heat-inactivate at 56°C for 30mins Aliquot into 50ml Falcon tubes. Store at -20°C till further use. 1.2 BHK-21 growth medium, 500ml 5ml of L-glutamine (200nM, Gibco) 5ml of sodium pyruvate (100mM, Gibco) 5ml of Penicillin/Streptomycin (100X liquid, Gibco) 50ml FBS Top up to 500ml with RPMI-1640 (Gibco) and filter-sterilize 1.3 BHK-21 maintenance medium, 500ml 5ml of L-glutamine (200nM, Gibco) 5ml of sodium pyruvate (100mM, Gibco) 5ml of Penicillin/Streptomycin (100X liquid, Gibco) 10ml FBS Top up to 500ml with RPMI-1640 (Gibco) and filter-sterilize 1.4 Vero growth medium, 500ml 5ml of sodium pyruvate (100mM, Gibco) 5ml of Non-essential amino acids (10mM, Gibco) 50ml FBS Top up to 500ml with Medium-199 (Gibco) and filter-sterilize 155 1.5 Vero maintenance medium, 500ml 5ml of sodium pyruvate (100mM, Gibco) 5ml of Non-essential amino acids (10mM, Gibco) 10ml FBS Top up to 500ml with Medium-199 (Gibco) and filter-sterilize 1.6 THP-1 growth medium, 500ml 5ml of HEPES (1M, Gibco) 50ml FBS Top up to 500ml with RPMI-1640 (Gibco) and filter-sterilize 1.7 THP-1 maintenance medium, 500ml 5ml of HEPES (1M, Gibco) 10ml FBS Top up to 500ml with RPMI-1640 (Gibco) and filter-sterilize 156 2. Virus labelling and immuno-staining assays 2.1 HNE buffer, pH 7.4 0.651g HEPES (Sigma) 4.38g sodium chloride (Sigma) 0.019g EDTA (Sigma) Top up with 500ml deionized water, adjust pH to 7.4 and filter-sterilize 2.2 Wash buffer 10ml FBS Top up to 1L with 1xPBS 2.3 Permeabilization solution Dissolve 1g saponin (Sigma) in 1L of 1x PBS 2.3 12% paraformaldehyde (pFA) Dissolve 12g of pFA (Sigma) in 90ml of deionized water. Adjust pH to 7.2 and top up to 100ml with 1x PBS. Filter-sterilize and store in -80°C in aliquots. 2.4 3% pFA Thaw a vial of 12% pFA and dilute in with 1x PBS. 157 3. Plaque assay 3.1 0.8% methycellulose overlay (A) x RPMI Dissolve RPMI-1640 powder (Gibco) in 500ml deionized water 10ml sodium bicarbonate (7.5%, Gibco) 5ml sodium pyruvate (100mM, Gibco) 20ml FBS Filter-sterilize (B) 1.6% carboxy-methyl cellulose (CMC) Dissolve 8g of CMC (Calbiochem) in deionized water Autoclave-sterilize at 121°C, 20min Add part of 2X RPMI (A) to part of 1.6% CMC (B) in 50ml Falcon tubes. Shake vigorously to mix. Store at 4°C. 3.2 0.5% crystal violet in 25% formaldehyde 5g crystal violet (Sigma) 676ml 37% Formaldehyde Top up to 1L with 1XPBS 158 4. Western blot 4.1 Lysis buffer for western blot 4.383g sodium chloride 5ml Nonidet-P40 buffer 3.03g Tris Dissolve in 500ml with milli-Q water Adjust to pH 8.0 Protease inhibitor (Sigma) added fresh at 1:100 4.2 10x SDS running buffer 288g glycine 60.4g Tris base 20g SDS Dissolve in liters with de-ionized water To use, dilute in 10 to use at 1x SDS running buffer 4.3 Transfer buffer 3g Tris 14.4g glycine 200ml methanol Top up to liter with de-ionized water 159 [...]... from patients during the 1943 dengue epidemic in Nagasaki, Japan A year later, Albert B Sabin and Walter Schlesinger also isolated DENV, and since then there has been a sharp rise in the spread of disease that is correlated with the increase in urbanization, international trade and travel (Gubler, 2002; Gubler, 2011; Tatem et al, 2012) Climatic change is also influencing the spread of dengue at higher... genus Flavivirus in the family Flaviviridae, which includes other viruses that are clinically relevant such as West Nile virus (WNV), Yellow fever virus (YFV) and Japanese encephalitis virus (JEV) The four DENV serotypes show 65-70% homology in their genome sequence The virion of dengue is ~50nm in diameter and is composed of a single, positive-strand RNA genome of ~11Kb with a single open reading frame... domains: Domain I (DI), Domain II (DII), and Domain III (DIII) (Modis et al, 2003; Zhang et al, 2004) DIII is believed to play a critical role in binding host cell receptors DII consist of the fusion loop that interacts with the endosomal membrane for fusion of the virus during entry into the cell (Rey et al, 1995) The hinge region that joins DI to DII is quite flexible and a low pH environment in the endosome... studies in Nicaragua indicated that a clade change in the circulating virus was associated with increased fitness in human and mosquito cells, along with greater rates of severe dengue (OhAinle et al, 2011) These studies suggest that with intensive transmission, the likelihood of selection for an epidemiologically fitter virus will increase However, genomic changes can also reduce the epidemic potential of. .. Chan KR, Zhang SL, Tan HC, Lim AP, Hanson BJ and Ooi EE (2013) Dengue Virus Neutralization in Cells Expressing Fc Gamma Receptors Plos one, Volume 8 /Issue 5 / e65231 2 Review article Chawla T, Wilder-Smith A and Ooi EE Dengue, an expanding neglected tropical disease Neglected tropical diseases in East Asia (in press) xvii CHAPTER 1 AN INTRODUCTION TO DENGUE 1.1 Dengue Dengue is an acute viral infection... resulted in unexpected growth of urban centers and housing with improper water supply, sanitation and sewage systems that aided the breeding of Aedes mosquitoes The presence of susceptible human hosts along with the mosquito vector led to the emergence of cyclical dengue epidemics (Ooi & Gubler, 2009) The Philippines had two DHF outbreaks in a gap of two years, in 1954 and 1956 and Thailand had an epidemic... for many years Insecticides such as pyrethroid deltamethrin or metofluthrin have been used and shown to kill mosquitoes (Ritchie & Devine, 2013) However there are problems related to the usage of insecticides such as dosage, method of delivery and the emergence of 18 insecticide resistance in mosquitoes (Maciel-de-Freitas et al, 2014) Most importantly, the use of insecticides involves high cost and the. .. not, then the eggs laid do not contain Wolbachia and hence they do not hatch However if the female insect contain Wolbachia but the male does not then the eggs would contain Wolbachia and they will hatch producing new offspring (Bordenstein & Werren, 1998; Sinkins et al, 1995) A study done in La Reunion Island had shown that Aedes albopictus naturally infected with Wolbachia restricts virus density in. .. time after the Second World War had an outbreak of dengue fever in August 2014 with nearly 150 confirmed cases of DF (CDC, 2014; WHO, 2014) These trends suggest that the burden of dengue will likely continue to increase in Asia in the coming years (Figure 1.1) 4 Figure 1.1: South East Asian countries with dengue epidemic, 2014 5 The increase in both vector and virus distribution geographically in areas... To preclude the spread of dengue, it is essential to understand the pattern of occurrence, including the factors that may have changed with time in endemic regions Dengue cases, both endemic and imported, have been increasing worldwide Though the Second World War facilitated the geographical expansion and population densities of Aedes aegypti, the unplanned urbanization later provided the optimum conditions . INVESTIGATING THE ROLE OF FC GAMMA RECEPTORS (Fc RS) IN DENGUE VIRUS NEUTRALIZATION TANU CHAWLA MSc (Biochemistry) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. expressing Fc RI and Fc RII (THP-1) and human myelogenous erythroleukemic cells (K562) expressing Fc RII, this thesis examined how different activating Fc Rs influence the amount of antibody. following infection with the h3H5 neutralizing antibody opsonized DENV in the presence of Fc RI …………… 89 3.2.2 Deriving a stable Fc RI and Fc RIIA knockdown cell line 95 3.2.3 Akt and 4EBP-1