Adaptive Evolution of the SIV Envelope Protein During Early SIV Infection The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Ita, Sergio. 2016. Adaptive Evolution of the SIV Envelope Protein During Early SIV Infection. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493399 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Adaptive Evolution of the SIV Envelope Protein during Early SIV Infection A dissertation presented by Sergio Ita to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Virology Harvard University Cambridge, Massachusetts January 2016 © 2016 Sergio Ita All rights reserved. Dissertation Advisor: Dr. Welkin E. Johnson Sergio Ita Adaptive Evolution of the SIV Envelope Protein during Early SIV infection Abstract Primate lentiviruses (PLVs), including human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), and the simian immunodeficiency viruses (SIVs), cause persistent lifelong infections despite the presence of virus-specific adaptive immune responses. The target of antibodies is the viral envelope glycoprotein (Env), which is expressed on the surface of virions and infected cells as a trimer of gp120:gp41 heterodimers. SIV env sequence variation arising from evasion of antibodies is well established and closely mimics the pattern observed in HIV-infected human patients, yet despite the experimental advantages of the macaque model, the viral dynamics during acute and early infection leading to escape within env have not been well defined. To examine the evolutionary dynamics of SIV env during early infection (< 6 months post-infection), we obtained longitudinal plasma samples from a small cohort of SIVmac251-infected rhesus macaques. We deep-sequenced the env gene from longitudinal samples spanning acute and early infection (2-29 weeks post-infection) from four SIV-infected rhesus macaques and the inoculum (a swarm stock of uncloned SIVmac251) that was used to infect the cohort (by repeated low-dose challenge). Using high-resolution next generation sequencing (NGS), we captured a population bottleneck at the point of transmission from the stock into each animal, tracked the subsequent emergence of Env diversity from the initially homogeneous population, and correlated changes with the onset of Env-specific antibodies. We identified a pattern of common substitutions, insertions, and deletions in env of animals with antibody responses, which repeatedly emerge in SIV-infected primate hosts, and found that adaptive changes clustered within short regions of the V1 and V4 loops of gp120. Surprisingly, we found multiple large in-frame deletions in V4 emerge to become dominant in the viral population in two animals with detectable antibody responses to Env. Furthermore, we developed a novel deep sequencing based viral fitness assay (Fit-Seq) and measured the relative fitness of several key in vivo iii antibody escape adaptations. Using Fit-Seq, we found that Env adaptations in V1 and V4 that confer antibody escape had no associated fitness costs in the absence of antibody, but rather, replicated to the same level as SIVmac239 WT. Surprisingly, we also found that Fit-Seq was able to detect antibody- mediated neutralization of SIVmac239 even in cases where activity was undetectable by standard neutralization assays. Our observation of reproducible patterns of Env variation clustered in V1 and V4, together with measurements of relative fitness, suggest that antibody responses can select for mutations that confer viral escape yet have little or no associated replicative fitness cost in the absence of antibody, even while providing a clear fitness advantage in the environment of constant antibody selection encountered in vivo. Thus, sites that can change with little or no impact on relative fitness may have evolved as an immune evasion mechanism to facilitate rapid and early escape from Env-specific antibody. iv Acknowledgments I would like to begin my acknowledgments with the faculty members of the Virology program, including the admissions committee the year that I applied to the program and the faculty members who I interviewed with that year, including Dr. Lee Gehrke, Dr. R. Paul Johnson, and Dr. Welkin Johnson. As a first-generation college graduate, a minority from a disadvantaged background, and a military veteran, each of the members who saw in me the potential to not only be a student within the program, but a colleague within the ranks of other Harvard scientists changed the course of my career and life. I would also like to thank the members of my dissertation advisory committee, Dr. Lee Gehrke, Dr. Karl Munger, and Dr. Miti Kaur for steering me through my graduate studies and providing invaluable guidance on my research. Next I would like to thank all the members of the Virology program family including all of the other graduate students who I got a chance to meet and interact with during my time in the program and the faculty members who took the time out of their schedules to teach the courses we took in our first and second years. I would especially like to thank my entering class of 2009, who over the course of our time before joining labs became a family to me. I would like to acknowledge all of the members of the Johnson lab who I’ve gotten to know over the last five years. I especially want to thank Dr. Ben Fofana. He has been a tremendously valuable mentor to me in the lab, who patiently taught me all he knew about antibodies, protein purification, and neutralization assays when I first started in the lab!! But more than that he has become a dear friend to me. We have had conversations on just about every possible topic ranging from politics to religion to raising our families in Boston. On that note, I would also like to thank Alison Hill, who, if I can use a military analogy, went through bootcamp with me in Longwood, before we both got stationed at Camp Welkin Johnson!! I would first like to thank Alison for playing a role in putting my research project on the right track and introducing me to the wonders of next-generation sequencing, the unidirectional flow of sample handling, and those amazing excel sheets!! We have witnessed each other mature as scientists v and in life, even if we didn’t want to admit it. Thank you for the friendship you have shown my family and me over the years. I know somehow we will see you in Kentucky soon!! I want to thank Kevin for showing me what passion for science looked like and teaching my kids how to curse! I think you will always be the only track-running death-metalhead I know. Also I would like to thank Annabel who sat next to me for just under 4 years and showed my how efficient I could have been in the lab, but was not. Finally, I would like to thank Welkin first for accepting me into his group and taking on the challenging role of being a graduate student mentor and for his continuous support through my entire time in his lab. I want to thank you for allowing me to pursue my line of research in the lab and mentoring me through the earliest stages of the project forcing me to think critically about my work. Also, thank you for giving me the flexibility I needed to maintain my responsibilities as a graduate student and my responsibilities as a father. I need to thank my family starting with my Mom. Thank you for instilling in me a sense to go out into the world and pursue something bigger then myself, for teaching me the importance of an education, and teaching me to believe in myself. You will always be my first inspiration for all that I have accomplished in my life. I want to thank my brother Louie. We would probably never have predicted growing up in New Mexico that we would end up together for so many years here in New England, but here we are! Thank you for producing the artwork for the 2012 program T-shirt that is now worn by over 50 Harvard virologists! I would like to thank each one of my children-Ava, Amaya, and Santi. You give my life meaning and everyday since you were all born have taught me how to be more patient and more loving. Last, I must thank my wife April. When we first met I was just about ready to leave the military and all I had to share with you was a dream that I was going to be a doctor one day. It only took me 11 years, but I did it! You have been with me at every step of this journey. We had no idea what we were getting ourselves into when we came to Boston, but in our time year we have become stronger together in almost every way. I thank you for doing everything!!! For providing a home to the kids, for pushing me when I thought that I couldn’t keep up, for keeping me together! vi Table of Contents Abstract ........................................................................................................................................................iii! Acknowledgments......................................................................................................................................... v! List of Figures ............................................................................................................................................... x! List of Tables .............................................................................................................................................xiii! CHAPTER 1 Introduction-Replication and Persistence of Primate Lentiviruses........................................ 1! 1.1 Primate lentiviruses are complex retroviruses................................................................................. 2! 1.2 The Persistence of PLVs through Immune Evasion and Viral Sequence Diversity.....................