University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2018 Dissecting Human Antibody Responses Against Influenza A Viruses And Antigenic Changes That Facilitate Immune Escape Seth J. Zost University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Allergy and Immunology Commons, Immunology and Infectious Disease Commons, Medical Immunology Commons, and the Virology Commons Recommended Citation Zost, Seth J., "Dissecting Human Antibody Responses Against Influenza A Viruses And Antigenic Changes That Facilitate Immune Escape" (2018). Publicly Accessible Penn Dissertations. 3211. https://repository.upenn.edu/edissertations/3211 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3211 For more information, please contact [email protected]. Dissecting Human Antibody Responses Against Influenza A Viruses And Antigenic Changes That Facilitate Immune Escape Abstract Influenza A viruses pose a serious threat to public health, and seasonal circulation of influenza viruses causes substantial morbidity and mortality. Influenza viruses continuously acquire substitutions in the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). These substitutions prevent the binding of pre-existing antibodies, allowing the virus to escape population immunity in a process known as antigenic drift. Due to antigenic drift, individuals can be repeatedly infected by antigenically distinct influenza strains over the course of their life. Antigenic drift undermines the effectiveness of our seasonal influenza accinesv and our vaccine strains must be updated on an annual basis due to antigenic changes. In order to understand antigenic drift it is essential to know the sites of antibody binding as well as the substitutions that facilitate viral escape from immunity. In this dissertation, we explore both the epitopes targeted in human antibody responses and how influenza viruses ve ade these responses. We first demonstrate that prior exposure shapes the sites targeted in human antibody responses, and show that many middle-age adults mounted an antibody response against H1N1 viruses that is focused against sites on HA conserved between contemporary strains and strains that circulated in early childhood. In addition, we demonstrate that a viral substitution in this epitope allows influenza viruses ot evade neutralizing antibody responses. We next demonstrate that an H3N2 HA substitution introducing a glycosylation site prevents the binding of neutralizing antibodies present in a large number of individuals. Importantly, our egg-based vaccines lack this glycosylation due to culture-adaptive substitutions, but a vaccine containing this glycosylation motif more potently induced antibody responses against circulating strains. Finally, we identify and characterize antibodies that target conserved residues in the receptor- binding site (RBS) of HA. We demonstrate that in some individuals RBS antibodies in sera contribute to neutralization of antigenically distinct strains, even in the case of an antigenically mismatched vaccine. Overall, the work presented here helps address the complex interaction of influenza viruses and human immunity. Importantly, our work identifies shortcomings with our current process of vaccine strain selection and production and investigates epitopes of interest for universal influenza accinev efforts. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Scott E. Hensley Keywords antibody, evolution, immunity, influenza, virus Subject Categories Allergy and Immunology | Immunology and Infectious Disease | Medical Immunology | Microbiology | Virology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/3211 DISSECTING HUMAN ANTIBODY RESPONSES AGAINST INFLUENZA A VIRUSES AND ANTIGENIC CHANGES THAT FACILITATE IMMUNE ESCAPE Seth J. Zost A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2018 Supervisor of Dissertation __________________ Scott E. Hensley, PhD Associate Professor of Microbiology Graduate Group Chairperson _____________________ Daniel S. Kessler, PhD Associate Professor of Cell and Developmental Biology Dissertation Committee Paul F. Bates, PhD, Professor of Microbiology Frederic D. Bushman, PhD, Professor of Microbiology Laurence C. Eisenlohr, PhD, VMD, Professor of Pathology and Laboratory Medicine George M. Shaw, MD, PhD, Professor of Medicine DISSECTING HUMAN ANTIBODY RESPONSES AGAINST INFLUENZA A VIRUSES AND ANTIGENIC CHANGES THAT FACILITATE IMMUNE ESCAPE COPYRIGHT 2018 Seth Julius Zost This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-sa/3.0/us ACKNOWLEDGMENTS I would first like to thank my thesis advisor Scott Hensley for his mentorship and support during my time in graduate school. I learned a great deal during my time in the lab and had the opportunity to work on exciting questions and develop and refine techniques to address them. It was exciting when the path seemed very clear and we agreed about the steps to take. However, I think some of my strongest work arose from our disagreements, because I was pushed to come up with better experiments and explanations. The environment in the lab was enhanced by many outstanding labmates who provided valuable advice, reassurance, and assistance. I would like to acknowledge Kaela Parkhouse and Megan Gumina in particular for their invaluable help with conducting experiments, and I would like to thank all the members of the lab for helping foster a collegial and fun environment. I would also like to thank former graduate students Susi Linderman and Ben Chambers for their help and support as I joined the lab. I would like to thank the members of my thesis committee – Paul Bates, Rick Bushman, George Shaw, and Ike Eisenlohr. I enjoyed discussing my progress with them at my committee meetings and valued their insights. I would like to thank the members of the Department of Microbiology for fostering a environment that encouraged collaboration and mentorship. I was fortunate to be able to collaborate with a number of excellent scientists on the studies described here, including Jesse Bloom, Patrick Wilson and Sarah Cobey. They all provided valuable insights and expertise that I lacked and generously shared their time and resources. My parents have helped foster my independence and love of learning and have always encouraged me to pursue my interests. Mom and Dad, words cannot express how grateful I am for your unconditional love and support and how it has shaped the person I am today. I am also grateful to my sister Mary for her encouragement and support. Finally, I would like to thank my fiancé Ashley for her love and support over the last eight years, especially during the past four years in which we were studying in different cities. Your enthusiasm and devotion in everything you do is infectious and it never fails to inspire me. I am excited to take our next steps together in the same city! iii ABSTRACT DISSECTING HUMAN ANTIBODY RESPONSES AGAINST INFLUENZA A VIRUSES AND ANTIGENIC CHANGES THAT FACILITATE IMMUNE ESCAPE Seth Julius Zost Scott E. Hensley Influenza A viruses pose a serious threat to public health, and seasonal circulation of influenza viruses causes substantial morbidity and mortality. Influenza viruses continuously acquire substitutions in the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). These substitutions prevent the binding of pre-existing antibodies, allowing the virus to escape population immunity in a process known as antigenic drift. Due to antigenic drift, individuals can be repeatedly infected by antigenically distinct influenza strains over the course of their life. Antigenic drift undermines the effectiveness of our seasonal influenza vaccines and our vaccine strains must be updated on an annual basis due to antigenic changes. In order to understand antigenic drift it is essential to know the sites of antibody binding as well as the substitutions that facilitate viral escape from immunity. In this dissertation, we explore both the epitopes targeted in human antibody responses and how influenza viruses evade these responses. We first demonstrate that prior exposure shapes the sites targeted in human antibody responses, and show that many middle-age adults mounted an antibody response against H1N1 viruses that is focused against sites on HA conserved between contemporary strains and strains that circulated in early childhood. In addition, we demonstrate that a viral substitution in this epitope allows influenza viruses to evade neutralizing antibody responses. We next demonstrate that an H3N2 HA substitution introducing a glycosylation site prevents the binding of neutralizing antibodies present in a large number of individuals. Importantly, our egg-based vaccines lack this glycosylation due to culture-adaptive substitutions, but a vaccine containing this glycosylation motif more potently induced antibody responses against circulating strains. Finally, we identify and characterize antibodies that target conserved residues in the receptor-binding site (RBS) of iv HA. We demonstrate that in some individuals RBS antibodies