ABSTRACT Title of Document: REVERSE GENETICS OF INFLUENZA B AND THE DEVELOPMENT OF A NOVEL LAIV VACCINE Courtney L. Finch, Doctor of Philosophy, 2014 Directed By: Professor Daniel R. Perez, Department of Veterinary Medicine Due to the disease burden of influenza virus types A and B, vaccines, which are manufactured as formalin-inactivated killed virus (KV) and live-attenuated virus (LAIV), are produced to provide coverage against currently circulating influenza A (IAV) and B (IBV) viruses. Although the licensed LAIV vaccine provides enhanced coverage over the KV vaccine, it is only licensed for immunocompetent individuals ages 2-49 years without pre-existing conditions, so individuals who are most at risk cannot receive it. Previously, our lab showed that incorporation of an 8 amino acid HA tag in frame at the C-terminus of the PB1 open-reading frame (ORF) in addition to the mutations found in the PB2 and PB1 segments of the licensed LAIV vaccine yielded a stable, efficacious alternative LAIV vaccine for IAV; however, to develop a complete vaccine, a corresponding IBV candidate is required. Towards this goal, a contemporary IBV strain, B/Brisbane/60/2008, was cloned and recovered by reverse genetics (RG-B/Bris). Subsequently, it was demonstrated that the parental and RG- B/Bris show similar growth kinetics in vitro. An initial vaccine attempt, which combined PB2 cap-binding mutants with the HA tag in PB1, was made but led to the realization of the PB2 cap-binding mutations, PB2 W359F and F406Y, as virulence factors. In a subsequent vaccine attempt, mutations analogous to those found only in segment 2 of the A/Ann Arbor/6/60 cold-adapted LAIV backbone were introduced into the homologous segment of RG-B/Bris. The following mutations were introduced into the PB1 gene segment of RG-B/Bris, either in the presence or absence of a C-terminal HA tag: K391E, E580G, and S660A. Two viruses were rescued, referred to as RG-B/Bris ts and RG-B/Bris att, both containing the set of three amino acid mutations but differing in the absence or presence of the HA tag, respectively. Both viruses showed ideal attenuation, safety, and immunogenicity in DBA/2 mice and conferred protection against lethal IBV challenge. More importantly, RG-B/Bris att, but not RG-B/Bris ts, showed ideal stability with no reverting mutations over 8 passages in eggs. Taken together, a stable, immunogenic, and live attenuated virus alternative to the current live influenza B virus vaccine was produced. REVERSE GENETICS OF INFLUENZA B AND THE DEVELOPMENT OF A NOVEL LAIV VACCINE By Courtney L. Finch Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2014 Advisory Committee: Professor Daniel R. Perez, Chair Assistant Professor Georg Belov Professor Jeffery DeStefano Associate Professor Yanjin Zhang Professor Xiaoping Zhu © Copyright by Courtney L. Finch 2014 Dedication I dedicate this dissertation to my family, namely my parents, Joseph and Carolyn, my husband, Nathan, and my in-laws. Mom and Dad, you taught me long ago that I could achieve anything. During my graduate career whenever I was in doubt, you believed. You gave me the strength and encouragement to continue believing in myself. I hope that seeing the acquisition of a PhD finally come to fruition will give you great pride. I regret that this achievement has been at the expense of time with the family. I hope that as my career progresses I can rectify this. To Nathan, my ever supportive, everloving husband, your unrelenting optimism and your unwavering belief in me have meant everything over these years. You alone know how truly difficult this process has been at times. You know my joys and my sorrows. You have been there for me the entire way. Thank you for tolerating the long nights and early mornings all of these years, thank you for never complaining and always understanding. This achievement also belongs to you. To the Finch family, especially Bud, Pat and Justin, you welcomed me into your family with open arms. You are more than just my in-laws; you are an extension of my immediate family. The love, support and acceptance you have shown me have been immeasurable. Thank you for always treating me like one of your own. ii Acknowledgements There are many who deserve thanks for the support and guidance provided to me during the course of my graduate career. Firstly, I would like to thank Dr. Daniel Perez. Thank you for the opportunity to work, learn and mature as a scientist in your laboratory. Thank you for the freedom you gave me to pursue my own ideas and to direct my own research. I know that I am a better scientist for having been in your laboratory. Few labs can rival the expertise in reverse genetics, transmission and animal models that the Perez lab offers, and I am proud to say that my most formative years as a scientist were spent in your lab. Secondly, I would like to thank my committee members, Dr. Belov, Dr. Culver, Dr. DeStefano, Dr. Zhang and Dr. Zhu. Thank you for your invaluable input in guiding my research progress. Thank you for pushing me to be better. Thank you for your time. Each committee meeting gave me more to consider and opened my eyes to new perspectives, strengthening my research. Thirdly, I would like to thank all members of the Perez lab, past and present, for the knowledge and skills that were imparted to me. In particular, I would like to thank Dr. Troy Sutton. The time you spent in mentoring and training me is unparalleled. I will never forget the role you played in my professional development. I will forever use 70% ethanol in excess and I have you to thank. In all sincerity, thank you. Additionally, I would like to thank Ana Silvia Gonzalez Reiche. It is difficult to describe how much your friendship and support have meant throughout the years. Having someone in the same year, someone with similar work ethic and interests has been amazing. iii Table of Contents Dedication ..................................................................................................................... ii Acknowledgements ...................................................................................................... iii Table of Contents ......................................................................................................... iv Abbreviations List ...................................................................................................... viii List of Tables ................................................................................................................ x List of Figures .............................................................................................................. xi Chapter 1: Introduction to Influenza B, Reverse Genetics and LAIV Vaccines .......... 1 1.1. General Introduction to Orthomyxoviridae ....................................................... 1 1.2. General introduction to influenza B ................................................................... 2 1.3. Genomic organization and encoded proteins of influenza B ............................. 5 1.3.1. Genomic organization ................................................................................. 5 1.3.2. Influenza B encoded proteins ...................................................................... 8 1.4. Influenza B virus life cycle and immune response to infection ....................... 11 1.4.1. Virus attachment and entry ....................................................................... 11 1.4.2. Nuclear import .......................................................................................... 14 1.4.3. Transcription, replication and translation ................................................. 15 1.4.4. Nuclear export ........................................................................................... 16 1.4.5. Virus assembly and budding ..................................................................... 17 1.4.6. Host immune response to infection ........................................................... 18 1.5. Influenza B host-range, evolution and restriction ............................................ 23 1.5.1. Host range ................................................................................................. 23 1.5.2. Evolution ................................................................................................... 23 1.5.3. Host restriction of influenza B .................................................................. 25 1.6. Introduction to Influenza B Reverse Genetics ................................................. 27 1.6.1. HA receptor binding, egg-adapted mutations and the significance of the HA cytoplasmic tail ............................................................................................ 30 1.6.2. Antiviral resistance of NA and the role of NB ......................................... 32 1.6.3. Transcription of BM1 and BM2 and implications for vaccine development and experimentation ............................................................................................ 34 1.6.4. BNS1 antagonism of the innate immune response ................................... 37 1.7. Introduction to LAIV Vaccines ....................................................................... 39 1.7.1. Review of the B/Ann Arbor ca vaccine backbone .................................... 43 1.7.2. Modifying the NS Gene Segment ............................................................