UNIVERSITY of CALIFORNIA SAN DIEGO Responses of Germline
Total Page：16
File Type：pdf, Size：1020Kb
UNIVERSITY OF CALIFORNIA SAN DIEGO Responses of Germline Targeting Immunogens to Influenza A Thesis submitted in partial satisfaction of the requirements for the degree Master of Science in Biology by Justin Romualdo Abadejos Committee in charge: Professor Stephen Hedrick, Chair Professor Li-Fan Lu, Co-Chair Professor Elina Zuniga 2018 Copyright Justin Romualdo Abadejos, 2018 All rights reserved. The Thesis of Justin Romualdo Abadejos is approved and it is acceptable in quality and form for publication on microfilm and electronically: Co-Chair Chair University of California San Diego 2018 iii DEDICATION I would like to thank everyone at the old Kirak lab: Oktay Kirak, Manching Ku, Nathaniel Phillips, Niklas Münchmeier, Shih-En Chang, Julio Hernandez, Jesus Zaragoza, Amy Nham, and Brett Freeman for taking me in and introducing me to the world of science. I would like to also thank Dr. Stephen Hedrick, Dr. Li-Fan Lu, and Dr. Zuniga for allowing me to continue my research at TSRI and for acting as my committee. I would also like to thank everyone at the Nemazee lab: David Nemazee, Amanda Gavin, Patrick Skog, Tanya Blane, Deli Huang, Sabrina Volpi, Miyo Ota, Terri Thinnes, and Wenjuan Li for taking me in to a wonderful family and helping me along the way. A special thank you to David Nemazee, Takayuki Ota, Shanshan Lang for being wonderful mentors and for guiding me throughout my time in science. I want to take the time to thank Nicole Aguirre, for continuously pushing me to be my best and supporting me in everything I do. A huge thank you and odes of appreciation to my parents, Efren and Tessie Abadejos for putting up with me and being my foundation, words can’t explain how grateful I am for you two. I’d like to dedicate this thesis to my grandfather and my late grandmother, Felix and Isadora Romualdo. iv EPIGRAPH “Never doubt that a small group of thoughtful, committed citizens can change the world; indeed, it's the only thing that ever has.” -Margaret Mead “If you want to go fast, go alone. If you want to go far, go together.” -African Proverb v TABLE OF CONTENTS Signature Page ..……………………………………………………………………. iii Dedication ...……………………………………………………………………….. iv Epigraph …………………………………………………………………………… v Table of Contents ...………………………………………………………………... vi List of Abbreviations ………………………………………………………………. vii List of Figures ……………………………………………………………………… ix List of Tables ………………………………………………………………………. xi Acknowledgements ………………………………………………………………... xii Vita…………………………………………………………………………………. xiii Abstract of the Thesis ...…………………………..……………………………….. xiv Chapter 1: Introduction…………………………………………………………….. 1 1.1: Figures………………………………………………………………… 9 Chapter 2: Testing the ability of anti-idiotype K1-18 and engineered HA 587H1-X to induce a broadly neutralizing HA stem response in a 9114 gH mouse model…….. 16 2.1: Generation of a 9114 gH mouse model……………………………….. 16 2.2: Testing of anti-idiotypic antibody K1-18 as a germline targeting immunogen…………………………………………………………………. 17 2.3: Testing of engineered HA 587H1-X as a germline targeting immunogen…………………………………………………………………. 20 2.4: Discussion……………………………………………………………… 24 2.5: Methods………………………………………………………………... 30 2.6: Figures and Tables…………………………………………………...… 42 References…………………………………………………………………………... 57 vi LIST OF ABBREVIATIONS BLI biolayer inferometry bnAb broadly neutralizing antibody BSA bovine serum albumin CDR complementarity determining region CDRH complementarity determining region heavy chain ELISA Enzyme linked immunosorbent assay FAB fragment antigen binding FACS fluorescence-activated cell sorting Fc fragment crystallizable FR framework region gH germline heavy chain GL germline HA hemagglutinin IgG immunoglobulin G Kd dissociation constant M molar MHC major histocompatibility complex NA neuraminidase Ni-NTA nickel-nitrilotriacetic acid PBMC peripheral blood mononuclear cells PBS phosphate buffered saline vii PDB Protein Data Bank PEG polyethylene glycol SSRL Stanford Synchrotron Radiation Lightsource UCA unmutated common ancestor viii LIST OF FIGURES Figure 1.1. The influenza life cycle and potential immune responses……………… 9 Figure 1.2. Structures and subtypes of hemagglutinin……………………………… 10 Figure 1.3. Isolation, identification, and theoretical development of broadly neutralizing antibodies………………………………………………………………. 11 Figure 1.4. Crystal structures revealing HA stem binding contacts of VH1-69 bnAb CR6261……………………………………………………………………….. 12 Figure 1.5. VH1-69 bnAbs utilizing key CDRH2 residues to bind to HA stem…..... 13 Figure 1.6. Binding ability of various influenza bnAbs to the different HA subtypes 14 Figure 1.7. DNA Vaxibody dimer structure………………………………………... 15 Figure 2.1. Design of 9114 gH knock-in mice……………………………………... 42 Figure 2.2. Characterization of the B6 9114 gH Mouse Model……………………. 43 Figure 2.3. Structure and paratope of K1-18……………………….………….…… 44 Figure 2.4. K1-18 activates B6 9114 gH B cells…………………………………… 45 Figure 2.5. K1-18 Immunizations show 587H1-X IFY enhancing mutation-specific response via Octet…………………………………………………………………… 46 Figure 2.6. K1-18 Immunizations show 9114-specific, 587H1-X IFY enhancing mutation-specific response via competitive ELISA……………………………..…. 47 Figure 2.7. Vaxibody DNA immunizations with K1-18 scFv as the antigenic unit show responses to K1-18 and 587H1 via endpoint titre ELISA………………………….. 48 Figure 2.8. Structures outlining mutations in Cali09/H1 that improve binding to germline VH1-69 bnAb CR6261……………………………………………………………… 50 Figure 2.9. In vitro binding and activation of 587X-H1 to B6 9114 gH B cells…… 51 Figure 2.10. 587H1-X Immunizations show IFY motif enhancing mutation-specific response via octet and ELISA assays………………………………………………. 52 Figure 2.11. Immunizations of B6 9114 gH mice with 587H1-X generate mutations in 9114 heavy chain…………………………………………………………………. 54 ix Figure 2.12. Future directions in testing germline targeting immunogens for development of influenza broadly neutralizing antibodies………………………………………… 56 x LIST OF TABLES Table 2.1. Mutations in Cali09/H1 that improve binding to germline VH1-69 bnAb CR6261……………………………………………………………………………… 49 xi ACKNOWLEDGEMENTS I’d like to thank Shanshan Lang (TSRI) for developing our germline targeting immunogens, providing and creating protein for analysis, for structures of K1-18, 587H1- X, CR6261, CR9114, and 27F3; Takayuki Ota (TSRI) for generating the 9114 gH mouse model, for help characterizing the mouse model, for proving mutation analysis data and in general for all his help and mentorship on all aspects of this project; Stephen M. Hedrick, Li-Fan Lu, and Elina Zuniga for allowing me to continue my research at TSRI and for gratefully serving on my committee; Patrick Skög for his help with optimizing immunizations; Tanya Blane for her help with mouse techniques and immunizations; Gunnveig Grødeland for mentorship and providing the vaxibody construct; TSRI’s FACS ++ core for help with flow cytometry and providing resources for FACS analysis and Ca flux; TSRI’s department of animal resources for outstanding services. Lastly, I’d like to thank Dr. David Nemazee for his oversight of the entire project and his mentorship and guidance throughout these past two years. xii VITA 2016 Bachelor of Science, Human Biology, University of California San Diego 2016-2017 Teaching Assistant, Department of Biological Sciences University of California San Diego 2018 Master of Science, Biology, University of California San Diego PUBLICATIONS Ku, M., S. E. Chang, J. Hernandez, J. R. Abadejos, M. Sabouri-Ghomi, N. J. Muenchmeier, A. Schwarz, A. M. Valencia and O. Kirak (2016). "Nuclear transfer nTreg model reveals fate-determining TCR-beta and novel peripheral nTreg precursors." Proc Natl Acad Sci U S A 113(16): E2316-2325. FIELDS OF STUDY Major Field: Human Biology Studies in Immunology Professors Oktay Kirak and David Nemazee xiii ABSTRACT OF THE THESIS Response of Germline Targeting Immunogens to Influenza by Justin Romualdo Abadejos Master of Science in Biology University of California San Diego, 2018 Professor Stephen Hedrick, Chair Professor Li-Fan Lu, Co-Chair Influenza virus is a constant threat to international public health. The current, seasonal vaccine generates a response to epitopes of influenza virus that have a high rate xiv of mutation leading to antigenic drift and antigenic shift leaving current memory responses useless and potentially resulting in a pandemic. The need for a universal influenza vaccine is evident. High affinity, broadly neutralizing antibodies (bnAbs) against a conserved epitope of the influenza virion, such as the stem of hemagglutinin, show potential to protect against a wide breadth, if not all influenza viruses. Identifying germline precursors of these bnAbs and generating immunogens that drive the maturation of a heterosubtypic response could provide a universal answer to influenza. In this thesis, I show the development and characterization of a mouse model consisting of B cells containing the germline heavy chain of the influenza bnAb CR9114. Furthermore, I outline the responses of two germline targeting immunogens to influenza: an anti-idiotypic antibody known as K1-18 and an engineered variant of an H1- hemagglutinin protein known as 587H1-X. Both K1-18 and 587H1-X are shown via calcium flux and activation markers to simulate B cells of our 9114 gH mouse model. Immunization studies with both immunogens show their ability to generate an IgG response that cross binds to wild type hemagglutinin. 587H1-X in particular shows mutation capabilities that drive these gl9114 antibodies towards