The Mapping and Characterization of a Novel Binding Site on HIV-1 Gp120 for The

The mapping and characterization of a novel binding site on HIV-1 gp120 for the broadly neutralizing monoclonal antibody IgG1 b12 by Jillian Waruk A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements of the degree of DOCTOR OF PHILOSOPHY Department of Medical Microbiology University of Manitoba Winnipeg Copyright © 2011 by Jillian Waruk Abstract HIV infects target cells via fusion events following surface envelope glycoprotein binding to the CD4 receptor and a chemokine co-receptor. Despite the high sequence variability of envelope across and within HIV-1 subtypes, this process requires conserved sequences and structures on gp120, which also represent good targets for HIV-1 neutralizing antibodies. Few examples of HIV-1 broadly neutralizing antibodies exist, but these antibodies may hold the key to a protective HIV-1 vaccine. One such antibody, IgG1 b12 (b12), binds the CD4 binding site on the HIV-1 envelope glycoprotein gp120. To date, no vaccine preparations have been able to elicit a b12-like response. A complete understanding of the mechanism of b12 binding to gp120 is essential to successful design of an b12-like immune response. Until now, strategies to map the b12 binding site on gp120 have utilized indirect techniques and/or core gp120 and have shown that b12 binds to a site on gp120 that overlaps the CD4 binding site. To more directly map the b12 epitope on intact gp120, epitope excision mass spectrometry mapping was carried out in the MALDI QqTOF platform. The putative epitope sequence was confirmed by tandem mass spectrometry sequencing. Epitope mapping revealed a novel binding site for IgG1 b12 at the gp120 amino terminus called Nterm. b12 bound a synthesized peptide of the epitope and the nature of the epitope was explored by ELISA. Although the Nterm epitope is involved in b12-gp120 interactions, ELISAs also show that the epitope does not make up the entire binding site on gp120. Rabbits immunized with a peptide version of the Nterm epitope II do express antibodies that bind monomeric gp120, but these antibody responses do not neutralize HIV-1 in vitro. These data indicate that the b12 binding site on gp120 is much more complex than previously thought. The b12 binds the Nterm sequence of gp120, perhaps in conjunction with the CD4 binding site. It has been shown that another HIV-1-neutralizing antibody, 4E10, also binds this novel Nterm epitope, and this may indicate a similar mechanism of action utilized by these two different antibodies. Though not able to elicit neutralizing antibodies on its own, this epitope may be an important element of the neutralizing b12 epitope and an important component of HIV-1 neutralizing antibody responses. III Dedication This thesis is dedicated to all the teachers in my life who have inspired me. I admire your passion for education and respect for those whom you teach. Cette thèse est dédiée à tous les enseignants dans ma vie qui m'ont inspirée. J'admire votre passion pour l'éducation et votre respect pour vos étudiants. Monsieur Gauvin, your devotion to promoting sciences to youth, especially females, was truly the encouragement I needed to get me here. Merci. Monsieur Gamache, your humour engaged me, and your end devastated me. Your life provided me with focus. Je serai toujours un de tes petits bouts de choux. Katie Waruk, sister, there are not enough words. I have gained so much from our friendship. Your support and dedication to me is inextricably tied to all my successes in life. Thank you. IV Acknowledgements I’d like to thank way too many people than I can fit into a <2 page long acknowledgements section. This is by no means an exhaustive list of the people I would like to acknowledge and thank for their support over the years, so please excuse me if your name does not appear herein, I do thank you all from the bottom of my heart. I wish to first thank my advisor, Dr. Frank Plummer. His sometimes insane capacity for knowledge coupled with his drive to combat infectious disease here in Canada and in the Developing World make him a pleasure and an inspiration to work with. I consider myself lucky to have had Frank as my advisor. On a day-to-day basis, I was managed by Dr. Blake Ball. Thank you, Blake, for your supervision and friendship. You taught me to be a worthy and thoughtful researcher. My advisory committee provided both support and invaluable scientific advice throughout this process. Dr. Keith Fowke, your leadership in the collective HIV group has been invaluable. I will always think of you as my third advisor and almost as good a hockey player as me. Thank you Dr. Jody Berry for your guidance, especially with the inception of this project. Dr. Kevin Coombs, your adherence to the scientific process always steered me in the right direction. Dr. John Wilkins, you and your lab staff provided much needed help when I ran in to technical difficulties. Thanks also to Robbin Shattock for providing feedback on my thesis. I would not have been able to complete the research study detailed herein without the help of out amazing technical staff, including John, Leslie, Sue, and Steve. They all have displayed large amounts of patience with me and their scientific input was and is valued. I performed the MS studies alongside Keding Cheng and would like to acknowledge the contribution of the other staff at the Manitoba Centre for Proteomics. Dr. David Montefiori and staff at the Laboratory of Immune Measurements performed the pseudovirus assays and were always available for discussions. I’d like to thank Ma, Binhua, and Ralph for invaluable discussions and Trent Bjorndahl and Gary for continued collaborations. Without the generous donations of reagents by Drs. Dennis Burton and Carlos Barbas III, this project never would have began. I’d like to thank Nicole and staff at Animal Care at the NML for taking good care of the animals that were sacrificed in the name of HIV vaccine research. Thanks to Jen for stats and thesis discussions. Thank you to the Department of Medical Microbiology, in particular, Joanne Embree, Angela, Carol, Jude, Lynn, Eva, and Sharon. Without you I would not have graduated or been paid or even had a chair to sit on, and I’m not exaggerating. I have seen many other students come and go from both the Plummer and Fowke labs, most who have made both major and minor contributions to my work and overall happiness. I was extremely lucky to end up with a bunch of students who made the lab a fun and congenial place to work. In moderately chronological order, I’d like to thank my V friends Lyle, Paul, Sandy, Tammy, Shehzad, Hezhao, Françoise, Lindsay, Mariel, Allison, Crystal, Catherine, Meika, Caitlin (my antibuddy), JJ, Nadine, and Melissa, Aida, Derek, Lindsay, Winnie, and Were. I’m really fortunate I got to work with you all and look forward to our future collaborations. I’d like to acknowledge and thank the colleages and staff at the University of Nairobi. Though my work did not involve the clinics in Kenya, their dedication to the eradication of HIV is praiseworthy and without their tireless efforts, my project would not have been funded. I would be remiss if I did not also mention the women of the Pumwani cohort. Their continued cooperation and contribution to this project will never be forgotten, not by any of us. Thanks to all my friends outside the lab, your encouragement has meant a lot to me. Thanks to Leanne for allowing me to vent long-distance and for the trip last year. Thank you Lindsay for the lunches, thanks Laura for always being there. Jill#2, thanks for insisting that I be Jill#1 and, with Andy, for your willingness to go for beers. Thank you to Dale and the Wiebes. I will always remember how much you supported me. Thanks to my sports team families who always provided me with an outlet. Thanks to my health care team. Thank you, my chosen family, Jaime, Kerri, Judy, Marsha, Natalie, Caitlin, and JJ. Last, but certainly not least, I’d like to thank each and every one of my family. They grounded me by never letting a chance to make fun of me go by and lifted me up with their interest in the value of my work and attempts to understand the jillitope. Thanks Aunts, Uncles, Cousins. Thank you Ivy. You just missed meeting the 1st Dr. Waruk. Thank you Shayla. Thanks Dad. Thank you Katie. Thanks Mom, keeper of my cats. My last thank you is reserved for my Alana. Everyday you remind me of the value of life and the science of compassion. VI Table of Contents Abstract II Dedication IV Acknowledgements V Table of Contents VII List of Tables XII List of Figures XIV List of Copyrited Materials for which Permission was Obtained XVI Section 1.0 Introduction 1 1.1 Human Immunodeficiency Virus 1 1.1.1 History of HIV/AIDS and HIV epidemiology 1 1.1.2 Transmission and prevention 3 1.1.3 HIV classification, infection, and protection 5 1.1.4 The HIV-1 envelope glycoprotein and mechanism of HIV-1 entry 9 1.1.4.1 The envelope trimer 9 1.1.4.2 Envelope 10 1.1.4.3 Entry 12 1.1.5 HIV infection kinetics, clinical manifectations, and treatment 14 1.2 Immune system – general 17 1.2.1 Innate immunity 17 1.2.2 Adaptive immunity 18 1.2.2.1 T cells 18 1.2.2.2 B cells and antibodies 20 1.3 Immune system – HIV-specific immunity 25 1.3.1 T cell responses 25 1.3.2 B cells and antibodies 26 1.3.2.1 B cells in infection 26 1.3.2.2 HIV-specific antibody responses 27 1.3.2.3 HIV-specific monoclonal antibodies 32 1.3.2.3.1 IgG1 b12 and CD4 binding site monoclonal 33 antibodies 1.3.2.3.1.i IgG1 b12 activity 33 1.3.2.3.1.ii

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