A novel method for measuring IgG-dependent triggering of host {FcγRs [FcgammaRs] CD16, CD32 and CD 64 reveals a selective inhibition through herpesviral {FcγRs [FcgammaRs] [Elektronische Ressource] / von Eugenia Corrales-Aguilar

Eugenia Corrales-Aguilar

Sujets

• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2008
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

A Novel Method for Measuring IgG-Dependent Triggering of
Host FcγRs CD16, CD32 and CD64 Reveals a Selective
Inhibition through Herpesviral FcγRs

Dissertation
Zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat)
im Fach Biologie
eingereicht an der


Mathematisch-Naturwissenschaftlichen Fakultät I
Der Humboldt-Universität zu Berlin


von


Eugenia Corrales-Aguilar
geboren am 22.03.1978 in San José, Costa Rica




Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Christian Limberg




Gutachter: 1. Prof. Dr. Hartmut Hengel
2. Prof. Dr. Richard Lucius
3. Prof. Dr. Günther Schönrich


Tag der mündlichen Prüfung: 16. Oktober, 2008





















Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes
2













“Around here, however, we don’t look
backwards for very long. We keep moving
forward, opening up new doors and doing
new things… and curiosity keeps leading
us down new paths.”

Walt Disney




3
4
Table of Contents

Summary..................................................................................................................................8

Zusammenfassung.................................................................................................................10

Abbreviations..........................................................................................................................12

1 Introduction..........................................................................................................................15
1.1 Viruses .........................................................................................................................15
1.1.1 Herpesvirus Family................................................................................................16
1.1.2 Human Cytomegalovirus (HCMV) .........................................................................18
1.1.3 Herpes simplex virus (HSV) ..................................................................................21
1.2 IgG and IgG-mediated effector functions for viral control.............................................22
1.3 Host Fcγ Receptors (FcγRs) .........................................................................................25
1.3.1 CD64 (FcγRI).........................................................................................................27
1.3.2 CD32 (FcRγII)........................................................................................................28
1.3.3 CD16 (FcγRIII).......................................................................................................28
1.3.4 Mouse FcγRs30
1.4 Immune evasion by herpesviruses...............................................................................31
1.4.1 NK immune evasion by CMV.................................................................................32
1.5 Viral FcγRs ...................................................................................................................35
1.5.1 HSV vFcγRs ..........................................................................................................35
1.5.2 CMV vFcγRs38
1.6 Methods for Measuring Antiviral IgG ............................................................................40
1.7 Aim of the Thesis42

2 Results ................................................................................................................................43
2.1 Establishment of a novel assay system for detection and quantification of virus specific
IgG antibodies triggering host FcγRs ...........................................................................43
2.1.1 Stable expression of FcγR-ζ constructs and activation of BWFcγR-ζ transfectants43
2.1.2 IgG dependent activation of the BWFcγR-ζ transfectants .....................................47
2.1.3 Fc of IgG is required for activation for the BWFcγR-ζ Assay.................................50
2.1.4 Immune IgG activation of chimeric hCD32-ζ and hCD64-ζ receptors...................51
2.1.5 Opsonization of MCMV infected target cells by polyclonal virus-specific IgG
activates FcγR-ζ transfectants ..............................................................................54
2.1.6 Opsonization of HCMV infected target cells by pic IgG γR-ζ transfectants56
2.1.7 Universal applicability of the assays to detect virus-specific IgG ..........................58
2.1.8 IgG binding to virus-infected target cells is required before activation of FcγR-ζ
effector cells ..........................................................................................................60
2.1.9 Comparing CD16 activation in the BWCD16-ζ assay vrs CD107a degranulation
assay with primary human NK cells ......................................................................65
2.2 Diagnostic Application of the BWFcγR-ζ assay............................................................67
2.2.1 BWFcγR-ζ assay efficiencies for MV-specifc IgG..................................................67
2.2.2 BWFcγR-ζ assay efficiencies and reaction patterns assessed for CMV-IgG in
human sera ...........................................................................................................71
2.3 Selective inhibition of herpesviral FcγR of IgG mediated effector functions .................75
2.3.1 Herpesviruses encode vFcγR binding Fcγ on the surface of infected cells ...........75
2.3.2 Virion neutralization and complement mediated virolysis is slightly affected by HSV
gE but unaffected by HCMV gp68 and gp34.........................................................77
2.3.3 gp68 and gp34 do not influence complement mediated cell lysis .........................77
2.3.4 Inhibition of the host FcγRs through herpesviral FcγRs.........................................80
5
2.3.5 The HSV vFcγR gE inhibits CD16 and CD32 activation but fails to inhibit CD64..82
2.3.6 HCMV vFcγRs interfere with CD16 activation .......................................................84
2.3.7 HCMV vFcγRs interfere with CD16 downstream signalling...................................86
2.3.8 The HCMV vFcγRs inhibit antibody dependent NK cell degranulation..................88
2.3.9 HCMV vFcγRs interfere with CD32 and CD64 activation ......................................90
2.3.10 MCMV fcr-1/m138 diminishes IgG-dependent activation of the murine CD16 ....94
2.3.11 m138/fcr-1 mediates IgG bipolar bridging ...........................................................98
2.3.12 m138/fcr-1 blockade of CD16 activation by W6/32 IgG2a is extremely efficient102

3 Discussion.........................................................................................................................104
3.1 The novel FcγR activation assay opens a window for understanding the role of antiviral
antibodies105
3.1.1 Advantages of the FcγR activation assay............................................................105
3.1.2 Virus-specific IgG responses as a correlate of immunity.....................................106
3.1.3 The BWFcγR-z activating assay’s open questions and future needs for
optimization.........................................................................................................108
3.1.4 Applicability of the FcγR activation assay113
3.2 The Novel Method for Measuring IgG-Dependent Triggering of Host FcγRs CD16,
CD32 and CD64 Reveals a Selective Inhibition through Herpesviral FcγRs .............113
3.2.1 Not all herpesviral FcγRs interfere with neutralization and complement activation114
3.2.2 Herpes simplex virus vFcγR gE interferes with the host FcγRs CD16 and CD32 but
not with CD64 activation .....................................................................................115
3.2.3 Human Cytomegalovirus FcγRs interfere with the host FcRs activation...........117
3.2.4 Mouse CytoγR m138/fcr-1 interferes with the host CD16 activation121
3.2.5 vFcγRs selective interference of Host FcγRs.......................................................123
3.3 Herpesviruses and antibodies ....................................................................................124
3.4 Future Perspectives ...................................................................................................125

4 Materials............................................................................................................................128
4.1 Devices...128
4.2 Chemicals and biochemicals......................................................................................129
4.3 Kits .............................................................................................................................131
4.4 Solutions and Buffers ........................................................................................