Protection from Cytomegalovirus Infection by Glycoprotein-specific Monoclonal Antibodies

141 Pages·2013·15.67 MB·English

Protection from Cytomegalovirus Infection by
Glycoprotein-specific Monoclonal Antibodies

Schutz vor Infektion mit dem Cytomegalievirus durch
glykoproteinspezifische monoklonale Antikörper

DER NATURWISSENSCHAFTLICHEN FAKULTÄT
DER FRIEDRICH-ALEXANDER-UNIVERSITÄT
ERLANGEN-NÜRNBERG
ZUR ERLANGUNG DES
DOKTORGRADES DR. RER. NAT.

VORGELEGT VON
ASTRID KARBACH
AUS BONN

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 16.10.2012

Vorsitzender der
Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatter: Prof. Dr. Thomas Winkler
Zweitberichterstatter: Prof. Dr. Thomas Stamminger

I
Acknowledgements
I owe huge gratitude to my advisor, Prof. Dr. Michael Mach, for offering me the opportunity to work on this
interesting, challenging, and rewarding project and for always providing me with constructive feedback, support,
and encouragement. I would also like to thank him for letting me present my results to the scientific community at
several national and international meetings.
I would also like to thank Prof. Dr. Thomas Winkler, for always supporting me. Thank you for all the helpful
discussions and reviewing this thesis.
I am very grateful to Prof. Dr. Heinrich Sticht and Pia Rücker for modeling the 3D-structure of mCMV gB and
suggesting the alanine scan mutants and several peptides. Your input was invaluable for this project. Thank you Pia
for not only being a colleague, but also a great friend.
I would like to acknowledge Prof. Dr. Jutta Eichler for kindly providing several peptides for this study and also
for her valuable support and for taking the time to be one of the examiners at my oral doctoral examination.
I thank Prof. Dr. Thomas Stamminger for reviewing my dissertation.
My thanks go to PD Dr. Brigitte Biesinger for her immense encouragement throughout my study. Thank you
and the whole GRK1071 for the wonderful research excursions to Boston and Schlaiffhausen.
I am very grateful to Barbara Kropff, who contributed immeasurably to this work, for example by cloning of
pcM55. Thank you for always answering my recurrent laboratory questions.
My thanks go also to Monika Dietz, Anna Wiegers, Anna Bootz, and Nadja Spindler for the fruitful discussion in
our group’s seminar, for their input, for the familiar atmosphere in the lab, and for all the great group excursions.
I would also like to thank the students, whom I was mentoring while I was working on my thesis, in particular
Ina Ersing and Eva-Maria Schilling. Thank you for your invaluable contribution to this study.
My thanks also go to Philipp Schuster for not only helping me with all flow cytometric assays, but also for
always having time to answer my questions and for being a friend.
I am intensely grateful to my family and friends for their constant support throughout my PhD thesis. Thank
you for always believing in me and supporting me. I owe immense gratitude to Jennifer Karbach and Doris Karbach
for revising and correcting pages over pages of the first drafts of this thesis.
I deeply thank Fabian Haag, who not only edited this thesis, but also read many parts of it. He always
encouraged me and made me dinner when my fridge was empty.
In the end I want to thank everybody, who was not specifically mentioned in this acknowledgement, but who
contributed to the success of this project.

III
Table of Contents
1 Zusammenfassung ...................................................................................... 1
2 Summary .................................................................................................... 2
3 Introduction ............................................................................................... 3
3.1 The Human Cytomegalovirus .......................................................................................... 3
3.1.1 Structure and Morphology ...................................................................................................... 3
3.1.2 Epidemiology, Pathogenesis, and Clinical Manifestations ...................................................... 4
3.2 The Humoral Immune Response to Antigens ................................................................... 5
3.2.1 Generation and Structure of Antibodies ................................................................................. 6
3.2.2 Function of Antibodies ............................................................................................................. 7
3.2.3 Monoclonal Antibodies ............................................................................................................ 8
3.2.4 Fcγ Receptors ........................................................................................................................... 9
3.2.5 Vaccination .............................................................................................................................. 9
3.3 The Immune Response to Cytomegalovirus Infection..................................................... 10
3.3.1 The Cellular Immune Response to Cytomegalovirus Infection .............................................. 10
3.3.2 The Humoral Immune Response to Cytomegalovirus Infection ............................................ 11
3.4 Glycoprotein B of the Human Cytomegalovirus ............................................................. 11
3.4.1 Characterization of Glycoprotein B........................................................................................ 11
3.4.2 The Immune Response to Glycoprotein B ............................................................................. 12
3.5 The Murine Cytomegalovirus and its Glycoprotein B...................................................... 13
3.6 Therapy for and Prophylaxis against hCMV ................................................................... 14
3.6.1 Antiviral Chemotherapy ......................................................................................................... 14
3.6.2 Passive Immunization ............................................................................................................ 14
3.6.3 Active Immunization .............................................................................................................. 15
4 Project Rationale ...................................................................................... 18
5 Results ...................................................................................................... 19
5.1 Cloning of Glycoprotein B of the Murine Cytomegalovirus ............................................. 19
5.2 Generation of Hybridoma Cells ..................................................................................... 19
5.3 Screening of Hybridoma Cells........................................................................................ 20
5.3.1 Screening for Recognition of mCMV Particles ....................................................................... 20
5.3.2 Screening for Glycoprotein B Recognition and for Neutralization of Free Virus ................... 20
5.4 Determination of Antibody Isotype ............................................................................... 22
5.5 Recognition of Virions .................................................................................................. 23
IV Table of Contents
5.5.1 Recognition of Virions by Monoclonal Antibodies by ELISA .................................................. 23
5.5.2 Recognition of Virions by Monoclonal Antibodies by Western Blotting ............................... 24
5.6 Localization of Epitopes within mCMV Glycoprotein B ................................................... 25
5.6.1 Structural Predictions and Bioinformatic Model for mCMV Glycoprotein B ......................... 25
5.6.2 Cloning of gB Substructures in Eukaryotic and Prokaryotic Expression Vectors ................... 26
5.6.3 Expression and Detection of Eukaryotically Expressed Protein Domains.............................. 27
5.6.4 Purification and Detection of GST-Tagged Protein Fragments .............................................. 29
5.6.5 Seropositivity Rate against Antigenic Domains on mCMV gB ............................................... 33
5.7 Neutralization Capacity of gB-Specific Antibodies .......................................................... 34
5.7.1 Neutralization Capacity of gB-Specific Antibodies on Different Cell Types ........................... 34
5.8 A gN-Specific Antibody with Extraordinary Neutralization Capacity ............................... 38
5.8.1 Monoclonal 32.22 Recognizes a mCMV Protein .................................................................... 38
5.8.2 32.22 Specifically Binds mCMV gN ......................................................................................... 39
5.8.3 Neutralization Capacity of the gN-Specific Antibody ............................................................. 41
5.8.4 Seropositivity Rate against gN ............................................................................................... 42
5.9 Large Scale Purification and Concentration of Antibodies .............................................. 43
5.9.1 Binding of Antibodies to Protein A and Elution ..................................................................... 43
5.10 Therapy of Infected Rag-/- Mice with mCMV-Specific Antibodies .................................... 45
5.10.1 Reduction of Vial Load Following Therapy with mCMV-Specific Antibodies ......................... 45
5.10.2 In Vivo Kinetics of Monoclonal Antibodies ............................................................................ 47
5.10.3 Survival Following Therapy with mCMV-Specific Antibodies ................................................ 48
5.10.4 Therapy of Rag-/- Mice with Fcγ-Receptor Knock Out ............................................................ 49
5.11 Prophylactic Treatment of Rag-/- Mice with mCMV-Specific Antibodies .......................... 50
5.11.1 Reduced Viral Replication after Prophylactic Treatment with anti-gB Antibodies ................ 50
5.11.2 Reduced Viral Replication after Prophylactic Treatment with 32.22 .................................... 51
5.12 Analysis of Sterilizing Immunity Following Passive Immunization .................................. 53
5.12.1 Determination of an Appropriate Virus Inoculum ................................................................. 53
5.12.2 Survival Following Passive Immunization .............................................................................. 53
5.12.3 Detection of Viral DNA in Organs ........................................................................................... 54
5.13 Immunization with UV-inactivated Virions or DomII of mCMV gB .................................. 56
5.13.1 Experimental Setup ................................................................................................................ 56
5.13.2 Analysis of Immunization ....................................................................................................... 57
5.13.3 Prophylactic Transfer of mCMV-Specific and DomII-Specific B-cells ..................................... 59
5.13.4 Prophylactic Transfer of DomII-Specific Sera ......................................................................... 62
5.13.5 Addition of ISCOM and CpG to the Vaccine and Boost of Infected Mice .............................. 64
5.13.6 Immunofluorescence Analysis with Sera of Immunized Mice ............................................... 65
5.13.7 Competition of DomII Specific Sera and a DomII Specific Monoclonal (1F11) ...................... 67
Table of Contents V
5.13.8 DomII Peptide Scanning and Recognition of Peptides by DomII Specific Sera ...................... 68
5.13.9 Glycosylation Pattern of DomII .............................................................................................. 69
5.14 Immunization with mCMV gB AD-1 ............................................................................... 70
5.14.1 Analysis of Immunization with AD-1 GST............................................................................... 70
5.14.2 Neutralization Capacity of AD-1-Specific Sera ....................................................................... 71
6 Discussion ................................................................................................. 73
6.1 Generation of Monoclonal Antibodies Protecting against mCMV ................................... 73
6.1.1 Most Neutralizing Antibodies Induced by mCMV Infection Recognize gB ............................ 74
6.1.2 Protection from mCMV Infection by Glycoprotein-Specific Antibodies ................................ 74
6.1.3 Correlation of in vitro Neutralization Capacity with in vivo Protection ................................. 75
6.1.4 Transfer of Sterilizing Immunity by CMV-Specific Antibodies ............................................... 77
6.1.5 Influence of Fc Receptors on Antiviral Activity ...................................................................... 78
6.2 Vaccination against CMV-infection ................................................................................ 79
6.2.1 Seropositivity Rate Against Viral Glycoproteins and Protein Subdomains in Mice ............... 80
6.2.2 Immunization with Different gB-derived Antigens or UV-Inactivated Virions ...................... 80
6.2.3 Analysis of the Impact of Alternative Adjuvants on Antiviral Responses .............................. 82
6.3 Conclusions and Future Prospects ................................................................................. 83
7 Methods ................................................................................................... 84
7.1 Cell Culture Techniques ................................................................................................ 84
7.1.1 Maintenance of Cell Cultures ................................................................................................ 84
7.1.2 Cryo-Conserving of Eukaryotic Cells ...................................................................................... 84
7.1.3 Hybridoma Technique............................................................................................................ 84
7.1.4 Transfection of Cultured Cells................................................................................................ 85
7.2 Immunological Methods ............................................................................................... 85
7.2.1 Enzyme-Linked Immunosorbent Assay (ELISA) ...................................................................... 85
7.2.2 Immunofluorescence Analysis ............................................................................................... 87
7.2.3 Western Blotting .................................................................................................................... 87
7.2.4 Flow Cytometric Analysis and Single Cell Sorting .................................................................. 87
7.3 Virological Methods ..................................................................................................... 88
7.3.1 In vitro Amplification and Purification of mCMV Particles .................................................... 88
7.3.2 Determination of Viral Titers of mCMV Preparations ........................................................... 88
7.3.3 Luciferase-Based in vitro Neutralization Assay ...................................................................... 88
7.4 Molecularbiological Methods ....................................................................................... 89
7.4.1 Polymerase Chain Reaction ................................................................................................... 89
7.4.2 Cloning ................................................................................................................................... 90
7.4.3 Removing N-linked Oligosaccharides by PNGase F Cleavage ................................................ 91
VI Table of Contents
7.5 Proteinbiochemical Methods ........................................................................................ 91
7.5.1 Determining Protein Concentrations ..................................................................................... 91
7.5.2 SDS Polyacrylamide Gel Electrophoresis (PAGE) ................................................................... 92
7.5.3 Purification of Monoclonal Antibodies Utilizing Protein A Columns ..................................... 92
7.5.4 Purification of Prokaryotically Expressed Proteins ................................................................ 92
7.5.5 Biotinylation of Antibodies .................................................................................................... 93
7.6 In vivo and ex vivo Experiments .................................................................................... 93
7.6.1 Infection, Antibody Application, and Euthanasia................................................................... 93
7.6.2 Collection of Blood ................................................................................................................. 93
7.6.3 Bioluminescence Imaging ...................................................................................................... 94
7.6.4 Purification of B-Cells from Murine Spleens for Adoptive Transfer ...................................... 94
7.6.5 Determination of the Viral Load in Organ Samples ............................................................... 95
7.6.6 Adoptive B-Cell- or Serum Transfer ....................................................................................... 95
7.6.7 Immunization ......................................................................................................................... 96
8 Material .................................................................................................... 97
8.1 Cell Lines and Culture Media ........................................................................................ 97
8.2 Antibodies ................................................................................................................... 98
8.3 Oligonucleotides .......................................................................................................... 98
8.4 Peptides ....................................................................................................................... 99
8.5 Buffers and Solutions ................................................................................................... 99
8.6 Kits .... ….………………………………………………………………………………………………………………………100
8.7 Standards ................................................................................................................... 101
8.8 Virus .... ...…………………………………………………………………………………………………………………….101
8.9 Adjuvans ..................................................................................................................... 101
8.10 Protein A Column ........................................................................................................ 101
8.11 Consumables and Glassware ....................................................................................... 101
8.12 Chemicals and Reagents .............................................................................................. 101
8.13 Software ..................................................................................................................... 102
8.14 Instruments ................................................................................................................ 102
9 Bibliography ........................................................................................... 104
10 Appendix ................................................................................................ 121
10.1 List of Abbreviations ................................................................................................... 121
10.2 List of Tables ............................................................................................................... 123