Searching for an HIV Vaccine: a Heterologous Prime-Boost System Using Replicating
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Searching for an HIV Vaccine: A Heterologous Prime-boost System using Replicating Vaccinia Virus and Plant-produced Virus-like Particles by Lydia Rebecca Meador A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Approved July 2016 by the Graduate Supervisory Committee: Tsafrir Leket-Mor, Co-Chair Bertram Jacobs, Co-Chair Joseph Blattman Hugh Mason ARIZONA STATE UNIVERSITY August 2016 ABSTRACT The HIV-1 pandemic continues to cause millions of new infections and AIDS-related deaths each year, and a majority of these occur in regions of the world with limited access to antiretroviral therapy. Therefore, an HIV-1 vaccine is still desperately needed. The most successful HIV-1 clinical trial to date used a non-replicating canarypox viral vector and protein boosting, yet its modest efficacy left room for improvement. Efforts to derive novel vectors which can be both safe and immunogenic, have spawned a new era of live, viral vectors. One such vaccinia virus vector, NYVAC-KC, was specifically designed to replicate in humans and had several immune modulators deleted to improve immunogenicity and reduce pathogenicity. Two NYVAC-KC vectors were generated: one expressing the Gag capsid, and one with deconstructed-gp41 (dgp41), which contains an important neutralizing antibody target, the membrane proximal external region (MPER). These vectors were combined with HIV-1 Gag/dgp41 virus-like particles (VLPs) produced in the tobacco-relative Nicotiana benthamiana. Different plant expression vectors were compared in an effort to improve yield. A Geminivirus- based vector was shown to increase the amount of MPER present in VLPs, thus potentially enhancing immunogenicity. Furthermore, these VLPs were shown to interact with the innate immune system through Toll-like receptor (TLR) signaling, which activated antigen presenting cells to induce a Th2-biased response in a TLR-dependent manner. Furthermore, expression of Gag and dgp41 in NYVAC-KC vectors resulted in activation of antiviral signaling pathways reliant on TBK1/IRF3, which necessitated the use of higher doses in mice to match the immunogenicity of wild-type viral vectors. VLPs and NYVAC-KC vectors were tested in mice, ultimately showing that the best antibody and Gag-specific T cell responses were generated when both components were administered simultaneously. Thus, plant-produced VLPs and poxvirus vectors represent a highly immunogenic HIV-1 vaccine candidate that warrants further study. i This document is dedicated to my family. To my parents, who always pushed me to reach for the stars and follow my dreams. To my sisters, who drove me to be my best self. To my grandparents, who always believed in me, especially when I needed a reminder. ii ACKNOWLEDGMENTS I would like to acknowledge my advisors, Tsafrir Mor and Bert Jacobs, without whom the completion of this dissertation would not have been possible. Additionally, my committee members, Joe Blattman and Hugh Mason, who offered sound guidance that resulted in much higher quality experiments. I would also like to acknowledge the members of each lab that were prominent in my training, and taught me the necessary skills to succeed in lab. Trung (Joe) Huynh, whose stark honesty was a constant reminder we can always improve, and there’s always another way to complete a task. Karen Kibler, who always ensured experiments were carried out in the most ethical manner. I would like to thank Latha Kanna, who was always there for me to help me through those rough days. Sarah Kessans, who entrusted me with continuing on her work upon her graduation. Megan McAfee and Kavita Manhas, who helped me learn the essentials of immunological assays. Arpan Deb, whose antics in the lab kept me sane and always on my toes! I would especially like to acknowledge Kathy Larrimore, an amazing friend and colleague throughout my time at Arizona State University. A friend who not only helped me through struggles with experiments, but was always there to listen when I needed someone. I would also like to acknowledge Hansa Done, though not a member of my lab, became one of my closest friends and made sure I always set aside time to relax and have fun. Additionally, I want to acknowledge my boyfriend, Adam Gushgari, who supported me and pushed me relentlessly through finishing up my dissertation. I could not have done this without him. I have mentored many undergraduate students throughout my PhD, and I would like to thank all of them here for their efforts in data collection, dealing with all of my intense demands, and making me a better teacher/mentor: David Beaumont, Patrick Cervantes, Rebekah Dickey, Marissa Kaufman, Will Martelly, Carly Snyder, Siavosh Naji-Talakar, and Michelle Di Palma. There are members of the ASU faculty and staff whom I would like to thank as well. Dave Lowry, for teaching me electron microscopy. Jacki Kilbourne, without whom my animal studies would not have been such a success! Jacki taught me everything I know about handling animal experiments, and without her wonderful stories the experiments would have been much more iii dull. I would like to acknowledge the staff in the Biological Design program for their guidance and trying to maintain stability for us: Maria Hanlin, Laura Hawes, Joann Williams, and our director Tony Garcia. I would also like to thank Petra Fromme, though she did not serve on my PhD committee, her excellent questions in my comprehensive exam really taught me to be a better scientist. Lastly, I would like to thank my funding sources. The Biological Design Graduate Program, the NSF Graduate Research Fellowship Program, the Philanthropic Education Organization Scholar Award, and the ASU Dissertation Fellowship supported me with a stipend and tuition throughout my doctoral studies. I also received travel awards from the Graduate College, the Graduate and Professional Student Association, the NSF GRFP, the Biological Design Graduate Program, and the International Society for Antiviral Research. Thank you to everyone for their guidance and friendship throughout this process. I could not have completed my dissertation without any of these people. iv TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................................ xi LIST OF FIGURES ......................................................................................................................... xii CHAPTER 1 INTRODUCTION ................................................................................................................ 1 The HIV-1 Pandemic and Treatment Options: Why a Vaccine is Necessary .............. 1 Vaccination Strategies and Correlates of Protection ................................................... 2 Vaccinia Virus as a Vaccine Vector ............................................................................. 6 Plant-produced VLPs ................................................................................................. 11 VLPs and Innate Immunity ......................................................................................... 14 Dissertation Overview ................................................................................................ 16 2 ENHANCING EXPRESSION AND YIELD OF HIV-1 GAG/DGP41 VIRUS-LIKE PARTICLES PRODUCED IN NICOTIANA BENTHAMIANA WITH TRANSIENT, DECONSTRUCTED TMV AND GEMINIVIRUS-BASED VECTORS ............................... 23 Abstract .......................................................................................................................... 23 Introduction .................................................................................................................... 24 Materials and Methods ................................................................................................... 26 Cloning Geminivirus-based Vectors........................................................................... 26 Kinetics Time Course ................................................................................................. 27 Optimization of VLP Purification ................................................................................ 28 Large-scale VLP Production and Purification ............................................................ 29 VLP Quantification ..................................................................................................... 29 Mouse Immunizations ................................................................................................ 29 v CHAPTER Page Results ........................................................................................................................... 30 Construction of Transient Geminivirus Vectors ......................................................... 30 Geminivirus Vectors Display Accelerated Expression ............................................... 31 Removal of Rubisco in VLP Preparations .................................................................. 32 VLP Purification and Yield ......................................................................................... 32 VLPs Elicit Gag and Dgp41-specific Antibodies in Mice ............................................ 33 Discussion ...................................................................................................................... 34 3 PLANT-PRODUCED HIV-1 VLPS STIMULATE A TH2 RESPONSE THROUGH TOLL- LIKE RECEPTOR DEPENDENT ACTIVATION OF INNATE IMMUNE CELLS .............. 46 Abstract