DETERMINANTS OF NEUTRALIZATION IN ENVELOPE PROTEINS OF HEPATITIS C VIRUS by Lisa Naomi Wasilewski A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland May 2016 ABSTRACT Hepatitis C Virus (HCV) is a chronic viral infection of humans which poses a significant global health threat. Millions of affected individuals are at risk for cirrhosis and hepatocellular carcinoma and as yet, no vaccine exists. Despite recent significant advances in HCV treatment, development of an HCV vaccine is a vital component in the effort to eradicate the virus. It has been demonstrated that broadly neutralizing antibodies (bNAbs) can prevent HCV infection despite a broad range of genetic variability and, as such, may serve as a useful tool for vaccine development. However, current understanding of bNAbs is still limited, particularly in regard to specific determinants of neutralization. In this thesis, we seek to gain a better understanding of viral polymorphisms which confer resistance to neutralization by both polyclonal plasma and broadly neutralizing monoclonal antibodies. Here, we identify a polymorphism which renders Bole1a, a computationally derived, ancestral genotype 1a HCV strain resistant to neutralization by both polyclonal plasma and multiple broadly neutralizing monoclonal antibodies. A considerable amount of work has gone towards developing research models which effectively mimic aspects of the viral life cycle in infected individuals. It is critical to define the relative benefits and limitations of different model systems in order to understand the effects of each. Two commonly used models are the HCV pseudoparticle system (HCVpp) and HCV cell culture (HCVcc). This thesis undertakes to describe the development of an efficient means by which to expand the repertoire of HCVcc constructs and to systematically compare the ii relative fitness and neutralization resistance of HCV envelope proteins in both the HCVpp and HCVcc model systems. In doing so, we show differences in HCV envelope fitness measured using the two systems but demonstrate strong agreement between antibody neutralization measured using HCVpp or HCVcc. iii Stuart C. Ray, M.D. (Advisor, Reader) Justin R. Bailey, M.D., Ph.D. (Committee Member, Reader) Alan Scott, Ph.D. (Committee Member) Joel Blankson, M.D., Ph.D. (Committee Member) iv ACKNOWLEDGEMENTS First I would like to thank my advisors, Stuart C. Ray and Justin R. Bailey for giving me the opportunity to learn and grow as a scientist under their guidance. The privilege of working with people for whom I have such profound respect both professionally and personally has made my experience here deeply gratifying. For their boundless patience, equanimity, and generosity, I am sincerely grateful. I would also like to thank my classmates and labmates, both students and technicians, for their constant support and friendship. They have enriched my time at Hopkins both in and outside the lab and this sense of camaraderie has brought joy to my education. Thank you to my coauthors and those who have laid the foundation for my project work. The members of the Viral Hepatitis Center have contributed invaluably over the years with thoughtful suggestions and contributions of their own time and expertise. Other members of the Hopkins community have been instrumental in bringing this work to bear, particularly John Gibas at the Ross Fluorescence Imagine Center and Christopher Thorburn at the Human Immunology Core. For feedback, helpful suggestions, and flexibility in scheduling, I thank the members of my thesis committee, Alan Scott, PhD and Joel Blankson, MD, PhD. Finally, the loving encouragement of my family throughout my life, in endeavors both academic and personal, has been the cornerstone of my every achievement. v TABLE OF CONTENTS ABSTRACT .......................................................................................................... ii ACKNOWLEDGMENTS....................................................................................... v LIST OF TABLES .............................................................................................. vii LIST OF FIGURES ............................................................................................ viii CHAPTER I: INTRODUCTION ............................................................................. 1 CHAPTER II: A HEPATITIS C VIRUS ENVELOPE POLYMORPHISM CONFERS RESISTANCE TO NEUTRALIZATION BY POLYCLONAL SERA AND BROADLY NEUTRALIZING MONOCLONAL ANTIBODIES ................... 11 ABSTRACT ..................................................................................................... 12 IMPORTANCE ................................................................................................ 13 INTRODUCTION ............................................................................................. 14 MATERIALS AND METHODS......................................................................... 16 RESULTS ........................................................................................................ 23 DISCUSSION .................................................................................................. 30 FIGURES ........................................................................................................ 33 CHAPTER III: AGREEMENT BETWEEN HEPATITIS C VIRUS NEUTRALIZATION MEASURED USING REPLICATION COMPETENT VIRUS AND PSEUDOPARTICLES ............................................................................... 49 ABSTRACT ..................................................................................................... 50 IMPORTANCE ................................................................................................ 52 INTRODUCTION ............................................................................................. 53 MATERIALS AND METHODS......................................................................... 56 RESULTS ........................................................................................................ 62 DISCUSSION .................................................................................................. 67 FIGURES ........................................................................................................ 73 CHAPTER IV: DISCUSSION ............................................................................. 88 APPENDIX REFERENCES ................................................................................................ 92 CURRICULUM VITAE ................................................................................... 114 vi LIST OF TABLES Table 3.1 E1E2-matched HCVpp and HCVcc IC50 values Table 3.2 HCVpp Fraction Unaffected and HCVcc IC50 values vii LIST OF FIGURES Figure 2.1 Codons 242, 424, and 570 show evidence of deep phylogenetic variation, and evolution at the 424 position is constrained Figure 2.2 Mutation of arginine (R) 424 to serine (S) confers resistance to neutralization by plasma Figure 2.3 Mutation of arginine (R) 424 to serine (S) confers resistance to neutralization by plasma Figure 2.4 Mutation of arginine (R) 424 to serine (S) confers resistance to binding of plasma antibodies Figure 2.5 Mutation of arginine 424 to serine confers resistance to neutralization by a diverse array of broadly neutralizing monoclonal antibodies Figure 2.6 Mutation of arginine 424 to serine confers resistance to binding of broadly neutralizing monoclonal antibodies Figure 2.7 Bole1a_S424 carries a fitness cost Figure 3.1 Cloning strategy to generate HCVcc and HCVpp with matched E1E2 proteins Figure 3.2 E1E2 genes used to generate E1E2-matched HCVcc and HCVpp are genetically diverse Figure 3.3 No correlation between relative specific infectivities of E1E2-matched HCVcc and HCVpp viii Figure 3.4 E1E2-matched HCVcc and HCVpp show the same hierarchy of neutralization resistance to a panel of broadly neutralizing antibodies targeting distinct epitopes Figure 3.5 Positive correlation between the rank orders of IC50 values measured using E1E2-matched HCVpp and HCVcc Figure 3.6 Positive correlation between the rank orders of Fraction Unaffected values measured using HCVpp and IC50 values measured using E1E2-matched HCVcc Supplemental Figure 3.1 All HCVcc and HCVpp neutralization curves used to calculate IC50 values Supplemental Table 3.1 HCVcc and HCVpp specific infectivity measurements ix CHAPTER I: INTRODUCTION 1 The natural history of hepatitis C virus Hepatitis C Virus (HCV) is found in virtually every region of the world, posing an important global health problem and affecting an estimated 170 million people (1). In the United States, there are approximately four million people with persistent hepatitis C infection and more than 10 thousand HCV-related deaths each year (2). Acute HCV infection progresses to chronic infection in 60-85% of cases. A large percentage of those who become infected will remain asymptomatic; however, of those chronically infected for two decades, 5-20% will develop cirrhosis of which 7% will annually progress to end-stage liver disease and hepatocellular carcinoma. HCV remains the leading indication for liver transplant in the United States, Japan, and Australia (3). In a minority of cases, infected persons will spontaneously clear HCV infection. Little is understood about why certain individuals become chronically infected while others spontaneously resolve the viral infection. Some predictors of the course of infection include