T Cell Immunity in Hepatitis C Virus Infection: Dissertation by Alex Hartlage, Ohio State University

T CELL IMMUNITY IN A SMALL ANIMAL SURROGATE OF HEPATITIS C VIRUS INFECTION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Alex Stephen Hartlage, BS Biomedical Sciences Graduate Program The Ohio State University 2020 Dissertation Committee: Amit Kapoor, PhD, Advisor Christopher M. Walker, PhD, Co-Advisor Arash Grakoui, PhD Mireia Guerau-de-Arellano, PhD Stefan Niewiesk, DVM, PhD Copyright by Alex Stephen Hartlage 2020 Abstract The hepatitis C virus (HCV) chronically infects 71 million people worldwide and is major cause of severe progressive liver diseases such as cirrhosis and hepatocellular carcinoma. Persistence of HCV in most infected persons is thought to be a consequence of failed virus-specific T cell immunity. Indeed, the only HCV vaccine candidate to progress to phase II clinical efficacy testing in humans is a virally vectored prime-boost regimen designed to induce virus-specific CD4 and CD8 T cell responses.

However, there is still uncertainty about the role of T cells in acute HCV control, why they tend to fail, and whether their induction via vaccine could prevent virus chronicity. Addressing these fundamental questions has been exceedingly difficult in the absence of relevant HCV animal models. Chimpanzees, the only species besides humans fully permissive to HCV infection, are no longer available for research, and current mouse models are immune- compromised and therefore unsuitable for detailed immunological studies. In recent years, a number of closely-related viral homologs of HCV have been discovered in diverse animal hosts, opening new possibilities for in vivo modeling.

The most promising of these is a rodent hepacivirus (RHV) discovered in wild Norway rats (Rattus norvegicus). RHV shares important virological features with HCV and, most notably, causes chronic hepatotropic infections in immune- competent laboratory rats. The central objective of these studies was to elucidate the importance of T cells during rat RHV infection and to identify mechanisms leading to their failure to eliminate persistent virus, in both naïve and immunized contexts. Using a recombinant adenovirus vaccine expressing RHV non-structural proteins as an experimental tool, we demonstrate that T cells are a critical component of immunity to persistent infection.

Vaccination prevented RHV persistence in most animals and transient depletion of CD8 or CD4 T cells undermined protective immunity. We also describe the nature and fate of RHV-specific CD8 T cells induced during persistent infection and reveal early viral load as a key factor underlying their inability to mediate protective immunity in this model. Finally, using an RHV strain containing immune escape mutations within ii dominant CD8 T cell epitopes, we highlight antigen mismatch as an important determinant of RHV vaccine efficacy and reveal correlates of successful immunity to heterologous virus. Overall, our data provide direct evidence that T cells are vital for hepacivirus control and that vaccines that stimulate cellular immunity without parallel antibody induction can be effective in preventing virus persistence.

Furthermore, our studies highlight the ease and utility of rat RHV infection for investigating mechanisms of HCV immunity, persistence, and vaccine protection. iii To my family, friends, and all those without whom this work would not have been possible. My success is truly yours to share. iv Acknowledgements I am forever indebted to my graduate advisors Drs.

Kapoor and Walker who provided endless support and career guidance for me throughout my training. When I entered graduate school four years ago, I had a growing interesting in the science of vaccines and viral diseases but little experience within this field. Thank you both for sharing your knowledge and passion with me and helping me grow as an independent scientist and professional. The opportunity to pursue science with you has been forever life-changing.

From the bottom of my heart, thank you. Additionally, I would like to extend my deepest gratitude to my committee members Drs. Grakoui, Guerau, and Niewiesk for their thoughtful teachings and advice. Your friendly and kindhearted support has meant the world to me these past few years.

I must also acknowledge Dr. Victoria Velazquez for her unwavering encouragement and support during my training. Victoria, your dedication and patience with me has been a cornerstone of my graduate school success. I will forever remember the technical, scientific, and personal help you graciously provided me with during these past several years.

Thank you immensely for your lessons and time. And finally, thank you to my wonderful soon-to-be wife Kelsey for all her love and support and for giving my life meaning outside of science and medicine. You are my world. v Vita Born – Cincinnati, OH………………………………….

Biochemistry, The Ohio State University……………………………………….2010-2013 Medical Scientist Training Program, The Ohio State University………………………2014-present Publications Hartlage AS, Walker CM, Kapoor A. Priming of antiviral CD8 T cells without effector function by a persistently replicating hepatitis C-like virus. Hartlage AS, Srinivasa Murthy S, Kumar A, Trivedi S, Dravid P, Sharma H, Walker CM, Kapoor A. Vaccination to prevent T cell subversion can protect against persistent hepacivirus infection.

Kohnken R, Wen J, Mundy-Bosse B, McConnell K, Keiter A, Grinshpun L, Hartlage AS, Yano M, McNeil B, Chakravarti N, William B, Bradner J, Caligiuri MA, Porcu P, Mishra A. 2018 Diminished microRNA-29b level is associated with BRD4-mediated activation of oncogenes in cutaneous T- cell lymphoma. Trivedi S, Murthy S, Sharma H, Hartlage AS, Kumar A, Gadi S, Simmonds P, Chauhan LV, Scheel TKH, Billerbeck E, Burbelo PD, Rice CM, Lipkin WI, Vandergrift K, Cullen JM, Kapoor A. Viral persistence, liver disease and host response in Hepatitis C-like virus rat model.

Billerbeck, E, Wolfisberg R, Fahnoe U, Xiao JW, Quirk C, Luna JM, Cullen JM, Hartlage AS, Chiriboga L, Ghoshal K, Lipkin WI, Bukh J, Scheel TKH, Kapoor A, Rice CM. Mouse models of acute and chronic hepacivirus infection. Hartlage AS, Cullen JM, Kapoor A. The Strange, Expanding World of Animal Hepaciviruses.

Hartlage AS*, Liu T*, Patton JT, Garman SL, Lozanski G, Zhang X, Habibe K, Losanski G, Lustberg ME, Caligiuri MA, and Baiocchi RA. The Epstein-Barr virus lytic protein BZLF1 as a candidate target antigen for vaccine development. *equal co-authorship Briercheck EL, Trotta RT, Chen L, Hartlage AS, Cole JP, Cole TD, Mao C, Banerjee PP, Hsu HT, Mace EM, Ciarlariello D, Mundy-Bosse BL, Garcia-Cao I, Yu L, Pilarsky R, Yu J, Leone G, Pandolfi PP, Orange JS, and Caligiuri MA. PTEN is a negative regulator of natural killer cell cytolytic function.

vi Fields of Study Major Field: Biomedical Sciences Graduate Program Area of emphasis: Immunology vii Table of Contents Abstract……………………………………………………………………………………………….……vi List of Tables…………………………………………………………………………….ix List of Figures………………………………………………………………………………….x Chapter 1: Introduction – Background and Objectives……………………………………….………1 Chapter 2: Vaccine prevention of hepacivirus persistence………………………………….16 Chapter 3: Antiviral CD8 T cell dysfunction and response to antiviral treatment……………….49 Chapter 4: Vaccine mismatch and CD8 T cell escape……….80 Chapter 5: Identification of a novel Rano-A allele………………………………………………….107 Chapter 6: Final discussion and conclusion………………………………………….122 viii List of Tables Table 2. PCR primer sequences…………………………………………………………………. RHV core class II epitopes……………………………………………………………. RHV primer sequences………………………………………………………………….

RHV class I and II epitopes………………………………………………………. NS5B polymerase mutations associated with Sofosbuvir resistance……………. RHV primer sequences……………………………………………………………. RHV class I and II epitopes………………………………………………………….……106 ix List of Figures Figure 2.

Functional profile of RHV-specific T cells after infection…………………………. Breadth of core-specific CD4+ T cell response……………………………………. Direct tetramer staining of RHV-specific CD8+ T cells after infection…………………39 Figure 2. Immunogenicity of Ad-NSmut and characterization of vaccine-elicited T cell repertoire…………………………………………………………………………………………………40 Figure 2.

T cell immunization confers partial protection against RHV infection…………. Class I epitope evolution in vaccinated rat R558……………………. Vaccine-induced T cell responses after RHV challenge……………. T cell responses in Ad-null vaccinated rats after challenge………………….

Impact of CD8 T cell depletion upon vaccine efficacy…………………. Impact of CD4 T cell depletion upon vaccine efficacy……………………. Identification of novel RHV class I epitopes………………………………. Early expansion of intrahepatic CD8 T cells targeting RHV after infection……………69 Figure 3.

RHV-specific CD8 T cells fail to produce effector cytokines……………………………70 Figure 3. RHV-specific CD4 T cell cytokine responses after infection……………………………72 Figure 3. Phenotypic differentiation of RHV-specific CD8 T cells…………………………………73 Figure 3. Early DAA treatment partially ameliorates RHV-specific CD8 T cell dysfunction….

Evolution of R558 immune escape variants………………………………………. Evolution of RHV polyprotein in vaccinated rat R558………………………………. Stability of R558 escape mutations after passage into naïve rats……………. Vaccine efficacy against wild-type versus escape virus………………………….

T cell responses and viral evolution in infection non-resolvers………………………. RHV-specific T cell responses after clearance or persistence of escape virus. Secondary immunity to escape virus after clearance of wild-type RHV infection. Evolutionary divergence of Rano-AHz………………………………………………….110 xi CHAPTER 1 Introduction – Background and Objectives 1.1 Overview of hepatitis C virus infection and treatment: the need for a vaccine Chronic infections by the hepatitis C virus (HCV), a bloodborne RNA hepacivirus of the Flaviviridae family, are a primary cause of severe progressive liver diseases and a major public health threat.

Approximately 71 million people are infected worldwide and at least 400,000 die annually from HCV-related liver complications, including cirrhosis, end-stage liver disease, and hepatocellular carcinoma(1, 2). Within the United States, HCV is the leading cause of death by an infectious pathogen, exceeding that of HIV and tuberculosis combined, and transmission is increasing due to rising injection drug abuse fueled by an ongoing opioid epidemic(3, 4). For over two decades since the virus’ discovery in 1989 as the primary cause of transfusion- acquired non-A, non-B hepatitis(5), standard-of-care HCV treatment consisted of combination pegylated interferon-a plus ribavirin, a regimen which yielded sustained virologic cure in approximately 50% of adherent patients but was often accompanied by severe, sometimes dangerous, side effects(6, 7). In 2011 the first direct-acting antivirals (DAAs), targeting the HCV protease, were approved for clinical treatment.

These agents elevated treatment success when combined with interferon-based therapy but were still suboptimal and associated with unwanted side effects(8, 9). Subsequent development of newer DAAs, targeting additional viral proteins critical for the HCV lifecycle, has revolutionized HCV therapy, allowing for an all-oral, interferon- free regimen that safely achieves cure in >95% of treated individuals after only 8 weeks of therapy(10, 11). 1 Because of the remarkable efficacy and safety profile of DAA treatment, the World Health Organization has now set a target for HCV elimination by 2030(1). Indeed, progression of HCV- associated liver complications often takes years to decades to develop(2) and termination of HCV infection via therapy substantially reduces the risk of liver-related mortality and disease(12, 13).

Thus, intervening with these agents represents a conceptually plausible strategy to reduce, and possibly even eradicate, HCV-related disease(14). However, significant barriers to HCV treatment remain that are likely to hinder global elimination efforts. These include the high cost of antiviral therapy, risk for DAA resistance, insufficient infection surveillance programs, and poor treatment adherence within difficult-to-treat patient groups(11, 15). Additionally, individuals cured by DAA therapy remain immunologically susceptible to HCV reinfection(16, 17), which will likely complicate elimination of the virus from high-risk populations such as injection drug users.

Thus, there is a growing belief that a preventive vaccine, effective in HCV-naïve and/or DAA-cured individuals, will ultimately be needed to help curb HCV transmission and achieve global elimination goals(11, 14, 18). Since acute HCV is rarely symptomatic and disease development is only observed in the chronic phase of infection, an effective HCV vaccine would need only to reduce or eliminate progression to lifelong HCV persistence. However, despite over thirty years of sustained research efforts, a vaccine is still unavailable and development is progressing at a disappointingly slow pace.2 Feasibility of an HCV vaccine Several lines of evidence highlight the feasibility of an HCV vaccine. First, during the acute of phase of infection, initial control of HCV viremia coincides with the appearance of virus-specific B and T cell responses in blood(19-21).

Long-term maintenance of these responses, particularly CD4 T cell help, is strongly correlated with spontaneous resolution of infection(22-25), which occurs in approximately 30% of HCV-naïve individuals. Second, natural clearance of HCV 2 infection generates long-lived immunity that significantly reduces the risk of HCV chronicity upon re-infection, including occasionally against heterologous strains(26, 27). Indeed, prospective studies of injection drug users demonstrated a 60% reduction in rate of HCV persistence during secondary infection of those that resolved prior infection(28, 29).