THE ANALYSIS OF THE HUMAN ANTIBODY RESPONSE TO FILOVIRUS INFECTION By Andrew I

214 Pages·2016·46.09 MB·English

by  Andrew Flyak

THE ANALYSIS OF THE HUMAN ANTIBODY
RESPONSE TO FILOVIRUS INFECTION

By
Andrew I. Flyak

Dissertation
Submitted to the Faculty of the
Graduate School of Vanderbilt University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
in
Microbiology and Immunology
August, 2016
Nashville, Tennessee

Approved:
James W. Thomas, M.D.
Terence S. Dermody, M.D.
D. Borden Lacy, Ph.D.
Melanie D. Ohi, Ph.D.
James E. Crowe, Jr., M.D.
To survivors of Marburg and Ebola virus infections
who generously donated their blood for this study.

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ACKNOWLEDGEMENTS

This work was financially supported by the Defense Threat Reduction Agency (grant
HDTRA1-13-1-0034) and U.S. NIH grant U19AI109711. The Vanderbilt Medical Center Flow
Cytometry Shared Resource is supported by NIH grants P30 CA68485 and DK058404. I
thank all these funding sources; this work would not have been possible without their
support.
First and foremost, I would like to thank my mentor, James Crowe for entrusting me
with the project that resulted in this thesis. From the very first day in the Crowe Lab, Jim
gave me a freedom to explore and supportive environment to learn. There were no limits, no
boundaries of what we can do as a team in pursuit of discovery. Jim has taught me to dream
big and commit 100% to everything you love.
I would like to express my sincere gratitude to my dissertation committee, Tom
Thomas (Chair), Terry Dermody, Borden Lacy, and Melanie Ohi for their scientific guidance
over the years. I thank you all for your excellent scientific suggestions and personal advises
you shared with me. Your leadership and support enabled me to become a better scientist.
This work would not be possible without numerous people outside the Vanderbilt, who
helped me tremendously to advance this project. I use the pronoun “we” throughout this
thesis to reflect the collaborative nature of the work I present. First, I would like to thank
Alexander Bukreyev’s group at UTMB (including Philipp Ilinykh, Xiaoli Shen, Natalia
Kuzmina and a former trainee Tania Garron) for their help with neutralization studies, animal
experiments and escape mutants generation. Erica Saphire’s group at Scripps (including
Marnie Fusco, former trainees Zachary Bornholdt and Takao Hashiguchi) for providing
Ebola and Marburg glycoproteins and advancing this project with X-ray crystallography
studies. Andrew Ward’s team at Scripps (including Daniel Murin, Hannah Turner and
Joshua David) for mapping antibody-binding sites using electron microscopy techniques.
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Benjamin Doranz and Edgar Davidson from Integral Molecular for mapping Ebola-specific
antibodies using alanine-scanning mutagenesis.
I would like to thank all past and present members of the Crowe Lab. An early thanks
goes to the Hybridoma Master Scott Smith, a brilliant scientist and friend, who taught me the
holy grail of human hybridoma generation and helped make me a better scientist. I would
like to thank my friend and colleague Natalie Thornburg who would answer every question I
had about science and parenting. I would like to thank my scientific colleagues who helped
me with my work, including Nurgun Kose who helped me with hybridoma generation and
Hannah King who took good care of my hybridomas. I thank present and former lab
technicians Rebecca Lampley, Gloria Fritz, Vidisha Singh and Leland Brown who helped me
with antibody purification as well as Stephen Graham and Leah Loerinc who provided help
with protein expression. I thank Robin Bombardi, Jessica Finn and Andre Branchizio who
provided much needed assistance with high-throughput antibody sequencing. I also thank
Frances House and Gopal Sapparapu for their excellent technical support.
I would like to thank Pavlo Gilchuk and Sebastian Joyce, my first scientific mentors
who helped me to become a scientist and who gave me an opportunity to work here. I also
thank the Division of Pediatric Infectious Diseases and Department of Pathology,
Microbiology, and Immunology for their dedication to training students. The Vanderbilt Flow
Cytometry Core provided much needed assistance with hybridoma sorting. Christopher
Gulka, a former graduate student in David Wright’s lab, provided help with peptide
synthesis.
Finally, I would like to thank my family. All my family in Ukraine, my parents,
grandparents and my younger brother, I thank you all for your constant support and
encouragement. I thank my wife Solomiia, for supporting me at all times. For bringing light,
joy and purpose in my life. To my beautiful daughter Sofia, your smiles and love are worth
more than you will ever know.
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TABLE OF CONTENTS

Page

DEDICATION .................................................................................................................. ii

ACKNOWLEDGMENTS ................................................................................................. iii

LIST OF TABLES .......................................................................................................... vii

LIST OF FIGURES ....................................................................................................... viii

LIST OF ABBREVIATIONS ............................................................................................ x

I. Introduction ................................................................................................................. 1

Thesis overview .......................................................................................................... 1
Epidemiology of Marburg virus infection ..................................................................... 2
Epidemiology of Ebola virus infection ......................................................................... 5
2013-2015 Ebola virus outbreak in West Africa .......................................................... 7
Filovirus genes and proteins ...................................................................................... 9
The structure of filovirus glycoprotein ...................................................................... 10
Entry of filoviruses .................................................................................................... 13
Human humoral response to filovirus infection ........................................................ 16
Ebola virus-specific antibodies .................................................................................. 17
Marburg virus-specific antibodies .............................................................................. 22
Successful treatments with cocktails of antibodies .................................................. 23

II. Mechanism of human antibody-mediated neutralization of Marburg virus ................ 24

Introduction ............................................................................................................... 24
Isolation of human monoclonal antibodies against Marburg virus ............................ 25
Neutralization activity of human monoclonal antibodies ........................................... 28
Recognition of varying forms of GP .......................................................................... 30
Competition-binding studies ...................................................................................... 30
Electron microscopy studies of antigen-antibody complexes .................................... 33
Antibody neutralization escape mutant viruses ......................................................... 34
Cross-reactive binding of Marburg virus antibodies with Ebola glycoprotein ............ 35
In vivo testing of Marburg virus antibodies ................................................................ 36
Discussion ................................................................................................................. 39
Materials and Methods .............................................................................................. 40

III. Cross-reactive neutralizing antibodies from survivors of Ebolavirus infection ......... 52

Introduction ............................................................................................................... 52
Isolation of human mAbs ........................................................................................... 53
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Binding and neutralizing activity of human mAbs ...................................................... 56
Major antigenic sites recognized by human mAbs .................................................... 61
Diverse patterns of molecular recognition defined by negative stain electron
microscopy ................................................................................................................ 63
Epitope mapping of Group 3A mAbs using saturation mutagenesis and negative
strain electron microscopy ........................................................................................ 66
Therapeutic efficacy of human mAbs in small animal models of EBOV infection ..... 68
Discussion ................................................................................................................. 69
Materials and Methods .............................................................................................. 71

IV. Cross-reactive human antibodies to the HR2/MPER region of Ebola virus
glycoprotein .................................................................................................................... 81

Introduction ............................................................................................................... 81
Cross-reactive neutralizing antibodies bind a unique region on GP surface ............. 82
Electron microscopy studies of cross-reactive mAbs from Group 3B ....................... 86
Analysis of GP residues important for mAb cross-reactivity and neutralization ........ 87
Discussion ................................................................................................................. 90
Materials and Methods ............................................................................................. 91

V. Summary and Future Directions ............................................................................... 97

Thesis summary ........................................................................................................ 97
Future directions ..................................................................................................... 100

List of publications ...................................................................................................... 106

Bibliography ............................................................................................................... 107

Appendix

A. Mechanism of human antibody-mediated neutralization of Marburg virus

B. Structural basis for Marburg virus neutralization by a cross-reactive human antibody

C. Chimeric filoviruses for identification and characterization of monoclonal antibodies

D. Cross-reactive and potent neutralizing antibody responses in human survivors of
natural Ebolavirus infection

E. Host-primed Ebola virus GP exposes a hydrophobic NPC1 receptor-binding pocket,
revealing a target for broadly neutralizing antibodies

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LIST OF TABLES

Table Page

1. Protective mAbs against Ebolavirus glycoprotein ........................................ 20
2. Efficacy of combined mAb treatment in non-human primates ...................... 23
3. Percentages of lines secreting species-specific,
or cross-reactive antibodies ......................................................................... 56

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LIST OF FIGURES

Figure Page

1. Marburg virus outbreaks in Africa .................................................................... 4
2. Ebola virus outbreaks in Africa ........................................................................ 6
3. Structure of the filovirus virion particle ............................................................ 9
4. The structure of Ebola virus glycoprotein ...................................................... 11
5. Molecular envelopes of full-length filovirus glycoproteins ............................ 12
6. Model of filovirus entry pathway .................................................................... 15
7. Sites of vulnerability on EBOV GP for protective mAbs ............................... 18
8. Isolation of human mAbs against Marburg virus .......................................... 26
9. Neutralizing and binding activities of MARV mAbs ....................................... 28
10. Heatmap showing the neutralization potency of MARV GP-specific mAbs .. 29
11. MARV-neutralizing mAbs target a distinct antigenic region on the
GP surface .................................................................................................... 31
12. Binding patterns of MARV GP-specific antibodies ........................................ 32
13. Neutralizing mAbs from a human survivor of MARV bind to the receptor-
binding site of GP .......................................................................................... 33
14. Generation of escape mutants for MARV neutralizing antibodies ................. 35
15. Breadth of binding or neutralization of human MARV-specific mAbs for
diverse filoviruses .......................................................................................... 37
16. Survival and clinical overview of mice treated with MARV mAbs ................. 38
17. Cross-reactive B cell responses in filovirus immune donors ........................ 55
18. Cross-neutralizing antibodies from survivors of natural BDBV infection ...... 57
19. Binding and neutralizing activities of BDBV mAbs ....................................... 58
20. Antibodies from groups 1B, 2B and 3B recognize BDBV GP and BDBV
GPΔmuc but not BDBV sGP in ELISA binding assay ................................... 60
21. BDBV-neutralizing antibodies target at least two distinct antigenic regions
of the GP surface .......................................................................................... 62
22. BDBV-neutralizing antibodies bind to the glycan cap or base region of GP . 65
23. Epitope mapping of Group 3A mAbs using saturation mutagenesis and
negative stain electron microscopy ............................................................... 67
24. Survival and clinical signs of EBOV inoculated guinea pigs treated with
BDBV mAbs .................................................................................................. 70
25. Neutralizing and protective activity of Group 3B cross-reactive mAbs. ......... 83
26. Cross-reactive neutralizing antibodies from Group 3B bind a unique region
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on GP surface ............................................................................................... 84
27. Cross-reactive neutralizing antibodies from Group 3B bind near the
membrane proximal region of GP ................................................................. 86
28. Structural and functional analysis of GP residues important for mAb cross-
reactivity and neutralization ........................................................................... 88
29. Neutralization activity of Group 3B mAbs against recombinant BDBV
isolate and escape mutants ........................................................................... 90
30. Generation of bsAbs by Fab-arm exchange method ................................... 101
31. Proposed structural rearrangements in GP2 during entry .......................... 104

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LIST OF ABBREVIATIONS

Ab Antibody
BDBV Bundibugyo virus
EBOV Ebola virus
EC 50% maximum-effective concentration
50
Fab Fragment antibody-binding, obtained after papain digest
GP Glycoprotein
HR Heptad repeat
IC 50% maximum-inhibitory concentration
50
MAb Monoclonal antibody
MAbs Monoclonal antibodies
MARV Marburg virus
MLD Mucin-like domain
MPER Membrane proximal external region
NPC1 Niemann-Pick C1 protein
sGP Secreted/soluble glycoprotein
SUDV Sudan virus
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