Molecular insights into neutralization and enhancement of dengue virus infection in monocytes

MOLECULAR INSIGHTS INTO NEUTRALIZATION AND ENHANCEMENT OF DENGUE VIRUS INFECTION IN MONOCYTES CHAN KUAN RONG B.Science (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR PHILOSOPHY NUS Graduate School for Integrative Sciences and Engineering National University of Singapore 2012 i 07/03/2013 i Acknowledgements I would like to express my heartfelt gratitude to Associate Professor Ooi Eng Eong for his patient guidance, advice and encouragement throughout my research His continued support and mentorship significantly aided the progress of the project I would also like to thank Professor Mariano Garcia-Blanco for his kind encouragement and suggestions My sincere appreciation is extended to my thesis advisory committee, Professor Chan Soh Ha, Associate Professor Subhash Vasudevan and Dr Justin Wong for the stimulating discussions and critical suggestions during my research Special thanks to Tan Hwee Cheng, Summer Zhang, Eugenia Ong, Tanu Chawla, Ryan Wu, Dr Brendon Hanson, Angeline Lim, Nivashini Kaliaperumal, Zhang Qian and Angelia Chow for providing technical assistance and support in my research And not forgetting colleagues from Duke-NUS who have made this a very conducive environment for research Lastly, I would like to thank my family and friends for their encouragement all these years I am glad to be able to share my successes and failures with them ii Table of contents Acknowledgements … …………………………………………………… ………… i Table of contents …………………………….………………………….….……….… ii Summary … …… ……………………… ………….………………… …….… viii List of tables …………………………………………………………………………… x List of figures ………………………………………………………………………… xi List of abbreviations ……………………………………………………… ……….… xvi List of publications …………………………………………………………………… xx Chapter 1: Introduction 1.1 Dengue …….………………… ………………………… 1.1.1 Dengue epidemiology ………………………………………………… 1.1.2 Clinical presentation and progression ………………………………… 1.1.3 Relationship between disease pathogenesis and immunity …………… 11 1.2 Dengue virus structure and genome ….……………….………….………… 12 1.3 Prevention and control of dengue ……………….……………………… …… 15 1.3.1 Limitations in vector control programs……………………………… 15 1.3.2 Vaccines in clinical development ……………………………………….18 Chimeric vaccines ………………………………………………… … 18 19 Live attenuated vaccines ………………………………….… ……… iii Sub-unit vaccines and nucleic acid vaccines ……………………… … 20 1.3.3 1.4 Challenges in dengue vaccine development ………………….……… 24 Immune responses and their consequences to dengue infection……………… 28 1.4.1 Antibody responses in protection and pathogenesis …….… ……… 28 Protective antibody responses…………………………………… … 30 Antibody responses in dengue pathogenesis ……………………… … 33 1.4.2 T-cell responses in protection and pathogenesis ……………………… 34 Protective T-cell responses …………………………… … .……….34 T-cell responses in dengue pathogenesis ………… …………… … 34 1.4.3 Cytokine responses involved in pathogenesis ………… ……….…… 36 1.5 Molecular insights of Dengue infection …… …………………………….… 39 1.5.1 Dengue life cycle ……………….………….………………….……… 39 1.5.2 Subversion of innate immunity to establish infection in monocytes…….42 1.6 Molecular insights of neutralization and enhancement of DENV infection … 45 1.6.1 The role of FcγR DENV neutralization …………………………….… 45 1.6.2 The role of FcγR in ADE of dengue infection ……………………… 50 1.6.3 Does ADE of DENV infection suppress innate immune responses? .50 1.7 Gaps in knowledge in DENV neutralization and ADE… 54 iv Chapter 2: Methods 2.1 Cells ………………… ………….…………… ……………………… .… 57 2.2 THP-1 subclones …………….…….…….…… ……….……………… … 58 2.3 Antibodies …….……………….…….……… ……………… 58 2.4 Human sera…………….……………… ……………….…………….……… 60 2.5 Viruses …….……………….………… …………………………………… 60 2.6 Affinity measurements by indirect ELISA …………….….…….……… 61 2.7 Plaque assay …….……………………….…………………………… … 61 2.8 Plaque reduction neutralization test……….…….………… ………………… 62 2.9 Virus infection in THP-1 cells …………….……… ………… …….… 62 2.10 Real-time PCR …………….….…………… …………… ……….………… 62 2.11 Fluorescent labeling of viruses …………………… ………….……… … 63 2.12 Visualization and quantification of DiD-virus uptake ………….…………… 63 2.13 Sucrose gradient analysis of DENV immune complex sizes …………….…… 64 2.14 Western blot ………….………………………….…………………… ……… 65 2.15 Immunoprecipitation ……….… ………………………………………… 65 2.16 siRNA transfection of THP-1 and K562 …………….….…….… 66 2.17 Overexpression in THP-1 …………… …… ………….……………… 66 2.18 Receptor blocking studies ……….…….……… ………………….……….… 66 v 2.19 Assessing surface monocytic marker expression …………… ………… 66 2.20 Microarray analysis…….….…………… ……….……….…….… …….… 67 2.21 Interferon and drug treatment …………………… ………….… … 68 2.22 Co-immunoprecipitation ………….…….………………………… ……… 68 2.23 ELISA to assess LILRB1 binding to DENV…………………………………… 69 2.24 Statistical analysis …………….……………………………………… ……… 69 Chapter 3: Results 3.1 Early interactions in antibody-mediated neutralization of dengue virus 3.1.1 Homologous DENV serotypes are neutralized despite FcγR-mediated uptake but heterologous DENV serotypes are neutralized only when 57 73 FcγR-mediated uptake is inhibted………………….…………… 57 3.1.2 Increasing antibody concentrations inhibits FcγR-mediated uptake of immune complexes …………………………………………………… 3.1.3 58 82 Size of DENV immune complex is dependent on the concentration of 58 antibody.……………………………………………………………… 86 58 3.1.4 Aggregation of DENV enables antibodies to cross-link the inhibitory 22 FcγRIIB.…………………………….………………………………… 3.1.5 90 25 Dengue neutralization in the presence of phagocytosis distinguishes 26 serotype-specific from cross-neutralizing antibodies with better accuracy than PRNT…………………………………………………… 28 95 vi 31 32 3.2 The use of FcγR in DENV neutralization 3.2.1 FcγRI ligation is required for uptake of neutralized DENV immune complexes……………………………………………………………… 103 3.2.2 FcγR-mediated phagocytosis following FcγRIIB knockdown………… 106 3.2.3 Preferential engagement of FcγRI in monocytes results in neutralization of DENV immune complexes………………………… 108 3.3 Regulators of FcγR-signalling in antibody-dependent enhancement of dengue infection 3.3.1 Isolation of two THP-1 subclones with increased uptake of dengue immune complexes…………………………………………………… 115 3.3.2 Two subclones of THP-1 with differential susceptibility to antibodydependent enhancement of dengue infection……………………… … 120 3.3.3 IFN signaling pathway contributes minimally to ISG induction…….… 123 3.3.4 ISG induction following ADE of DENV infection is Syk-dependent… 126 3.3.5 ISG induction following ADE of DENV infection can be attenuated by increased SHP-1 phosphorylation……………………………………… 3.3.6 128 Antibody opsonised DENV co-ligates LILRB1 with activating FcR for enhanced DENV replication……………………………………… 131 vii 54 Chapter 4: Discussion 4.1 Preface……………………………………………………………………… 138 4.2 Viral aggregates co-ligate inhibitory receptor FcγRIIB to inhibit uptake of dengue immune complexes………………………………………………… 4.3 139 Assessment of FcγR-mediated phagocytosis to distinguish protective humoral immunity from cross-reactive immune response………………………….…… 143 4.4 Preferential engagement of FcγRI during dengue neutralization results in uptake and neutralization of dengue immune complexes……………………… 145 4.5 LILRB1 in ADE of dengue infection…………………………………… …… 147 4.6 Concluding remarks…………………………………………….……………… 151 Bibliography ……… ………………………………………………… …………… 153 Appendices …………………………………………………………………………… 190 viii Type- and subcomplex-specific neutralizing antibodies against domain III of dengue virus type envelope protein recognize adjacent epitopes J Virol, 81, 12816-26 SUN, S F., PAN, Q Z., HUI, X., ZHANG, B L., WU, H M., LI, H., XU, W., ZHANG, Q., LI, J Y., DENG, X M., CHEN, J W., LIAN, Z X & LI, N 2008 Stronger in vitro phagocytosis by monocytes-macrophages is indicative of greater pathogen clearance and antibody levels in vivo Poult Sci, 87, 172533 SYAM, S., MERO, P., PHAM, T., MCINTOSH, C A., BRUHNS, P & BOOTH, J W 2010 Differential recruitment of activating and inhibitory Fc gamma RII during 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ROSSMANN, M G & CHEN, J 2008 Structure of the immature dengue virus at low pH primes proteolytic maturation Science, 319, 1834-7 ZELLWEGER, R M., PRESTWOOD, T R & SHRESTA, S 2010 Enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibodyinduced severe dengue disease Cell Host Microbe, 7, 128-39 ZHANG, S L., TAN, H C., HANSON, B J & OOI, E E 2010 A simple method for Alexa Fluor dye labelling of dengue virus Journal of Virological Methods, 167, 172-7 ZOMPI, S., MONTOYA, M., POHL, M O., BALMASEDA, A & HARRIS, E 2012 Dominant Cross-Reactive B Cell Response during Secondary Acute Dengue Virus Infection in Humans PLoS Negl Trop Dis, 6, e1568 190 Appendices 191 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 192 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 193 ELISA 2.1 ELISA coating buffer 0.212g sodium carbonate (Sigma) 0.252g sodium bicarbonate (Sigma) Dissolve in 50ml deionized water, adjust pH to 9.66 and filter-sterilize 2.2 ELISA wash buffer (PBST) Add 500μl of Tween-20 to 1L of 1xPBS 2.3 ELISA blocking buffer (1%) Dissolve 0.5g skim milk in 50ml of 1x PBS 2.4 ELISA antibody diluents (0.5%) Dissolve 0.25g skim milk in 50ml of 1x PBS 194 Virus labelling and immuno-assays 3.1 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 3.2 Wash buffer 10ml FBS Top up to 1L with 1xPBS 3.3 Permeabilization solution Dissolve 1g saponin (Sigma) in 1L of 1x PBS 3.4 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 3.5 3% pFA Thaw a vial of 12% pFA and dilute in with 1x PBS 195 Plaque assay 4.1 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 4.2 0.5% crystal violet in 25% formaldehyde 5g crystal violet (Sigma) 676ml 37% Formaldehyde Top up to 1L with 1XPBS 196 Western blot 5.1 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 5.2 Lysis buffer for co-immunoprecipitation 4.383g sodium chloride 0.5ml Nonidet-P40 buffer 3.03g Tris Dissolve in 500ml with milli-Q water Adjust to pH 8.0 Protease inhibitor (Sigma) added fresh at 1:100 197 5.3 Immunoprecipitation buffer 1.51g Tris 4.383g sodium chloride Dissolve in 500ml with milli-Q water Adjust to pH 7.2 5.4 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 5.5 Transfer buffer 3g Tris 14.4g glycine 200ml methanol Top up to liter with de-ionized water 198 ... monocytes? ??….42 1.6 Molecular insights of neutralization and enhancement of DENV infection … 45 1.6.1 The role of FcγR DENV neutralization …………………………….… 45 1.6.2 The role of FcγR in ADE of dengue infection. .. and enhancement of dengue infection would be important in the design of an effective dengue vaccine that protects against all four DENV serotypes while minimizing the risk of ADE of DENV infection. .. engagement of FcγRI during dengue neutralization results in uptake and neutralization of dengue immune complexes……………………… 145 4.5 LILRB1 in ADE of dengue infection? ??………………………………… …… 147 4.6 Concluding