Science, Faculty of
Microbiology and Immunology, Department of
DSpace
UBCV
Rosen, Ariel R.
Master of Science - MSc
University of British Columbia
Multiple Sclerosis (MS) is a chronic, neurodegenerative autoimmune disease of the central nervous system (CNS). The aetiology of the disease remains unknown; however, it is thought to be a combination of environmental factors in genetically susceptible individuals. One such environmental factor is infection with Epstein-Barr virus (EBV), a DNA virus of the gammaherpesvirus family that infects over 95% of individuals worldwide. Following primary infection, EBV establishes a life-long latent infection in the host’s B cells. MS does not develop in the absence of EBV exposure, and EBV is necessary, but insufficient on its own, to cause disease. In a mouse model of EBV and MS, latent infection of mice with γ-herpesvirus-68 (γHV68), the murine homologue of EBV, exacerbates the clinical and immunological outcomes of experimental autoimmune encephalomyelitis (EAE), a common mouse model for MS. The compelling evidence supporting EBV infection as a co-factor in the development of MS has sparked interest in the potential of EBV-targeted therapies to treat the disease, for example by preventing or controlling primary EBV infection using a prophylactic vaccine. As such, mice were vaccinated against, and then challenged with, γHV68, which successfully reduced the viral load of γHV68 latent infection. Mice were subsequently induced with EAE. Vaccinated mice displayed an attenuation of viral-enhanced disease, and early in the course of EAE, vaccinated mice displayed significantly reduced CNS immune infiltration compared to unvaccinated mice. This work demonstrates the potential for vaccination against EBV to prevent or ameliorate MS and highlights the urgency and necessity for the development of an EBV vaccine.
VACCINATION AGAINST GAMMAHERPESVIRUS ATTENUATES VIRAL ENHANCED DISEASE IN A MURINE MODEL OF MULTIPLE SCLEROSIS by Ariel R. Rosen B.Sc., The University of British Columbia, 2022 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2024 © Ariel R. Rosen, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Vaccination against gammaherpesvirus attenuates viral enhanced disease in a murine model of multiple sclerosis submitted by Ariel R. Rosen in partial fulfilment of the requirements for the degree of Master of Science in Microbiology and Immunology Examining Committee: Dr. Marc Horwitz, Professor, Dept. of Microbiology and Immunology, UBC Supervisor Dr. Lisa Osborne, Associate Professor, Dept. of Microbiology and Immunology, UBC Supervisory Committee Member Dr. Maria Tokuyama, Assistant Professor, Dept. of Microbiology and Immunology, UBC Additional Examiner Additional Supervisory Committee Members: Dr. Laura Sly, Professor, Dept. of Pediatrics – Div. of Gastroenterology, UBC Supervisory Committee Member iii Abstract Multiple Sclerosis (MS) is a chronic, neurodegenerative autoimmune disease of the central nervous system (CNS). The aetiology of the disease remains unknown; however, it is thought to be a combination of environmental factors in genetically susceptible individuals. One such environmental factor is infection with Epstein-Barr virus (EBV), a DNA virus of the gammaherpesvirus family that infects over 95% of individuals worldwide. Following primary infection, EBV establishes a life-long latent infection in the host’s B cells. MS does not develop in the absence of EBV exposure, and EBV is necessary, but insufficient on its own, to cause disease. In a mouse model of EBV and MS, latent infection of mice with -herpesvirus-68 (HV68), the murine homologue of EBV, exacerbates the clinical and immunological outcomes of experimental autoimmune encephalomyelitis (EAE), a common mouse model for MS. The compelling evidence supporting EBV infection as a co-factor in the development of MS has sparked interest in the potential of EBV-targeted therapies to treat the disease, for example by preventing or controlling primary EBV infection using a prophylactic vaccine. As such, mice were vaccinated against, and then challenged with, HV68, which successfully reduced the viral load of HV68 latent infection. Mice were subsequently induced with EAE. Vaccinated mice displayed an attenuation of viral-enhanced disease, and early in the course of EAE, vaccinated mice displayed significantly reduced CNS immune infiltration compared to unvaccinated mice. This work demonstrates the potential for vaccination against EBV to prevent or ameliorate MS and highlights the urgency and necessity for the development of an EBV vaccine. iv Lay Summary Multiple sclerosis (MS) is a disease of the central nervous system (CNS) wherein the immune system attacks the protective covering of nerves, leading to an array of debilitating symptoms. The cause of MS remains unknown; however, the disease is likely initiated by a combination of genetic and environmental factors. One such environmental trigger is infection with Epstein-Barr virus (EBV). There is currently no cure for MS, highlighting the vital role for preventative interventions. For instance, it remains unknown whether vaccination against EBV could prevent MS development. To answer this question, mice were vaccinated against and then infected with a mouse virus that resembles EBV. Clinical and immunological outcomes of a common mouse model for MS were then assessed. Vaccinated mice displayed improved outcomes compared to unvaccinated mice, highlighting the potential role of vaccination against EBV to reduce the risk of MS and underscoring the urgent need for an EBV vaccine. v Preface All work was conducted at the Life Sciences Institute and the Modified Barrier Facility at the University of British Columbia (UBC). Research was performed under the supervision of Dr. Marc Horwitz. Animal work was conducted in accordance with protocol A21-0228 approved by the Animal Care Committee (ACC) of UBC. The following individuals are acknowledged for their assistance in the studies conducted for this thesis. Dr. Marcia A. Blackman for providing the ACRTA HV68 strain, which was developed by Dr. Ting-Ting Wu; the staff at the Modified Barrier Facility including Angel Chu and Marcos Aguiar for animal husbandry, and Dr. Po-Yan Cheng for veterinary support; Andrew Johnson and Justin Wong for assistance with flow cytometry; Dr. Jessica Allanach for guidance in data analysis and experimental design; Thomas Worthington, Erin Goldberg, Cynthia Chung and Danielle Keith for aid in animal monitoring and tissue processing; Danielle Keith and Michaela Schweitzer for assistance with DNA extraction and PCR; Iryna Shanina for technical support and guidance during all experiments. This work was possible through support from the Canadian Institutes of Health Research, MS Canada and the National Multiple Sclerosis Society. vi Table of Contents Abstract .......................................................................................................................................... iii Lay Summary ................................................................................................................................. iv Preface............................................................................................................................................. v Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................. x List of Abbreviations .................................................................................................................... xii Acknowledgements ..................................................................................................................... xvii CHAPTER 1: INTRODUCTION ................................................................................................... 1 1.1 Multiple Sclerosis ................................................................................................................. 1 1.1.1 Disease epidemiology and risk factors .......................................................................... 2 1.1.2 Disease pathology and treatments ................................................................................. 5 1.1.3 Modelling disease ........................................................................................................ 11 1.2 Epstein-Barr virus ............................................................................................................... 14 1.2.1 EBV biology and lifecycle ............................................................................................ 14 1.2.2 Immune response to EBV ............................................................................................. 18 1.2.3 EBV vaccines ............................................................................................................... 20 1.2.4 EBV and MS ................................................................................................................. 22 1.2.5 -herpesvirus-68 as a model for EBV .......................................................................... 26 1.2.6 Modelling EBV and MS in mice ................................................................................... 29 1.3 Research aims and rationale................................................................................................ 30 vii CHAPTER 2: MATERIALS AND METHODS .......................................................................... 32 2.1 Animals ............................................................................................................................... 32 2.2 Gammaherpesvirus 68 ........................................................................................................ 32 2.2.1 Virion generation and infection of C57Bl/6 mice ........................................................ 32 2.2.2 PCR confirmation of infection ..................................................................................... 33 2.2.3 qPCR determination of viral load ................................................................................ 34 2.3 Experimental autoimmune encephalomyelitis .................................................................... 36 2.4 Tissue collection ................................................................................................................. 37 2.5 Flow cytometric analysis .................................................................................................... 37 2.5.1 Sample processing ....................................................................................................... 37 2.5.2 Antibody and tetramer staining and analysis .............................................................. 38 2.6 Software and statistical analyses ......................................................................................... 39 CHAPTER 3: RESULTS .............................................................................................................. 41 3.1 ACRTA HV68 vaccination reduces HV68 viral load in the spleen ................................ 41 3.2 ACRTA HV68 vaccination attenuates HV68-driven clinical EAE enhancement........... 43 3.2.1 ACRTA HV68 vaccination ameliorates EAE outcomes during a 15-day clinical course .................................................................................................................................... 44 3.2.2 ACRTA HV68 vaccination ameliorates short-term and long-term EAE outcomes .... 46 3.3 Characterization of immune infiltration and polarization of vaccinated EAE mice in the CNS and spleen ......................................................................................................................... 48 3.3.1 Vaccinated mice display reduced CNS immune infiltration 10 d.p.i. compared to unvaccinated HV68-infected mice ....................................................................................... 49 3.3.2 Vaccinated mice display reduced T cell infiltration and elevated ratios of CD8+ to CD4+ T cells in the CNS compared to unvaccinated mice during EAE ............................... 50 viii 3.3.3 Analysis of T regulatory, Th1 and Th17 CD4+ T cells in the spleens and CNS of vaccinated and unvaccinated mice during EAE ................................................................... 56 3.3.4 Analysis of IFN and granzyme B production of CD8+ T cells in the spleens and CNS of vaccinated and unvaccinated mice during EAE ............................................................... 63 3.3.5 Quantification of HV68-specific CD8+ T cells in the spleens and brains of vaccinated and unvaccinated mice before and during EAE ................................................................... 68 3.3.6 Vaccinated mice display increased levels of NK cells in the spleen 10 d.p.i compared to unvaccinated mice............................................................................................................. 71 CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS ..................................................... 75 4.1 Summary and significance of findings ............................................................................... 75 4.2 Limitations and future directions ........................................................................................ 78 REFERENCES ............................................................................................................................. 81 APPENDIX ................................................................................................................................... 99 ix List of Tables Table 2.1: Primer sequences for HV68 ORF50 PCR assay…………………….………………34 Table 2.2: Primer sequences for GAPDH PCR assay……………………………………………34 Table 2.3: Sequences for HV68 ORF50 qPCR assay…………………………………………..35 Table 2.4: Sequences for PTGER2 qPCR assay…………………………………………………35 Table 2.5: MHC Class I tetramers……………………………………………………………….38 Table 2.6: Antibodies for extracellular targets…………………………………………………..39 Table 2.7: Antibodies for intracellular targets…………………………………………………..39 Table 3.1: Spleen %CD4+ and %CD8+ of CD3+ T cells…………………………………………55 Table 3.2: Spinal Cord %CD4+ and %CD8+ of CD3+ T cells……………………………………55 Table 3.3: Brain %CD4+ and %CD8+ of CD3+ T cells…………………………………………..55 Table 3.4: Functional NK cell subtypes in the spleen 10 d.p.i……………………………...…...73 x List of Figures Figure 1.1: Main forms of MS…………………………………………………………………….2 Figure 1.2: Global prevalence of MS…………………………………………………………...…4 Figure 1.3: Immunopathogenesis of MS…………………………………………………………..7 Figure 1.4: Overview of EBV infection………………………………………………………….15 Figure 1.5: HV68-EAE model experimental overview…………………………………………30 Figure 3.1: WT HV68 challenge in mice vaccinated with ACRTA HV68……………………42 Figure 3.2: ACRTA HV68 vaccination experimental overview………………………………..43 Figure 3.3: ACRTA HV68 vaccination attenuates HV68-driven EAE enhancement 15 d.p.i...45 Figure 3.4: HV68 vaccination driven attenuation of EAE is mouse sex independent………….46 Figure 3.5: HV68 vaccination driven attenuation of EAE 24 d.p.i……………………………..47 Figure 3.6: HV68 vaccination driven attenuation of EAE 10 d.p.i……………………………..48 Figure 3.7: Vaccinated mice display reduced CNS immune infiltration 10 d.p.i………………..49 Figure 3.8: Quantification of CD3+ T cells in the spleen and CNS of vaccinated and unvaccinated mice during EAE…………………………………………………………………………………52 Figure 3.9: CD4+ vs. CD8+ T cell gating 10 d.p.i………………………………………………..53 Figure 3.10: CD4+ and CD8+ T cell counts and ratios for vaccinated and unvaccinated mice….54 Figure 3.11: Gating for FOXP3+ and CD25+ CD4+ T cells in the CNS and spleens of vaccinated and unvaccinated mice…………………………………………………………………………...57 Figure 3.12: T regulatory cells in the spleen and CNS of vaccinated and unvaccinated mice…..58 Figure 3.13: IFN+CD4+ Th1 cells in the spleen and CNS of vaccinated and unvaccinated mice……………………………………………………………………………………………....60 xi Figure 3.14: IL-17A+CD4+ Th17 cells in the spleen and CNS of vaccinated and unvaccinated mice………………………………………………………………………………………………62 Figure 3.15: Ratio of Th1 to T regulatory cells in the spleen and CNS for vaccinated and unvaccinated mice………………………………………………………………………………..63 Figure 3.16: IFN+CD8+ Tc1 cells in the spleen and CNS of vaccinated and unvaccinated mice………………………………………………………………………………………………65 Figure 3.17: Ratio of Tc1 to T regulatory cells in the spleen and CNS for vaccinated and unvaccinated mice………………………………………………………………………………..66 Figure 3.18: CD8+ T cell Granzyme B production in the spleen and CNS of vaccinated and unvaccinated mice 10 days post EAE induction…………………………………………………67 Figure 3.19: HV68-specific CD8+ T cells in the spleen and brain of vaccinated and unvaccinated mice………………………………………………………………………………..70 Figure 3.20: NK cells in the spleens of vaccinated and unvaccinated mice 10 d.p.i…………….72 Figure 3.21: NK cells in the brains of vaccinated and unvaccinated mice 10 d.p.i……………...74 Supplemental Figure 1: Spleen general gating scheme…………………………………….……99 Supplemental Figure 2: Spinal cord gating scheme…………………………………….………100 Supplemental Figure 3: Brain gating scheme…………………………………………..………101 Supplemental Figure 4: Spleen gating scheme for HV68 tetramer staining……..……………102 xii List of Abbreviations HV68 -herpesvirus-68 AJ Adherens junction APC Antigen presenting cell AT-EAE Adoptive transfer EAE AUC Area under the curve BART BamHI A-encoded transcript BBB Blood-brain barrier BCR B cell receptor BHK-21 Baby hamster kidney fibroblasts 21 BMI Body mass index cDC Conventional DC CFA Complete Freund’s adjuvant CMV Cytomegalovirus CNS Central Nervous System CRYAB -crystallin B chain CSF Cerebrospinal fluid CT Cycle threshold D.P.I. Day post induction DC Dendritic cell DIS Dissemination in space DIT Dissemination in time DMT Disease modifying therapy xiii dsDNA Double stranded DNA dsRNA Double stranded RNA EAE Experimental autoimmune encephalomyelitis EBER EBV-encoded RNA EBNA1 EBV nuclear antigen 1 EBV Epstein-barr virus EC Epithelial cell ER Endoplasmic reticulum FOXP3 Forkhead box P3 GA Glatiramer acetate GAPDH Glyceraldehyde 3-phosphate dehydrogenase GC Germinal center GlialCAM Glial cell adhesion molecule HERV Human endogenous retrovirus HHV-4 Human herpesvirus 4 HHV-6 Human Herpesvirus-6 HLA Human leukocyte antigen IE Immediate early IFN Interferon IFN Interferon  IgG Immunoglobulin G IgM Immunoglobulin M IL Interleukin xiv IM Infectious mononucleosis IMA Immunomodulatory agent IN Intranasal iNK Immature NK cell IP Intraperitoneal JCV John Cunningham virus KSHV Kaposi’s sarcoma herpesvirus LCMV Lymphocytic choriomeningitis virus LMP1 Latent membrane protein 1 MBP Myelin basic protein MCMV Murine cytomegalovirus MEM Minimal essential media MHC Major histocompatibility complex miRNA Micro RNA mNK Mature NK cell MOG Myelin oligodendrocyte glycoprotein MRI Magnetic resonance imaging MS Multiple Sclerosis NK Natural killer NOD Non-Obese Diabetic NPC Nasopharyngeal carcinoma OCB Oligoclonal band OMA Phorbol 12-myristate 13-acetate xv OriP Origin of plasmid replication PAMP Pathogen associated molecular pattern PCR Polymerase chain reaction pDC Plasmacytoid DC PFU Plaque-forming units PLP Proteolipid protein PPMS Primary progressive MS PTGER2 Prostaglandin E receptor 2 PTx Pertussis toxin qPCR Quantitative polymerase chain reaction RBC Red blood cell rhMOG Recombinant human MOG RRMS Relapsing-remitting MS RTA Replication and transcription activator SD Standard deviation SEM Standard error of the mean SJL/J Swiss Jim Lambert SPMS Secondary progressive MS SQ Subcutaneous TAP Transporter associated with antigen processing Tc1 Cytotoxic T cell (IFN+CD8+) Th1 T helper 1 (IFN+CD4+) Th17 T helper 17 (IL-17A+CD4+) xvi TJ Tight junction TLR Toll-like receptor TMEV Theiler’s murine encephalomyelitis virus TNFR Tumor necrosis factor receptor Treg T regulatory UVB Ultraviolet B radiation VLP Virus-like particle VSV Varicella-zoster virus VV Vaccinia virus WT Wild type xvii Acknowledgements I would like to thank my supervisor Dr. Marc Horwitz for his support throughout this research project, and for providing me the opportunity to be his first ever master’s student. I am also thankful for the feedback and guidance from my thesis committee members, Dr. Laura Sly and Dr. Lisa Osborne. I would also like to acknowledge that this work was performed on the traditional, ancestral, and unceded territory of the xwməθkwəy̓əm (Musqueam) People. This work would not have been possible without the support of the other Horwitz lab members, namely Dr. Jessica Allanach, Dr. Zach Morse, Virginie Jean Baptiste, Miguel Mejias, Naomi Fettig, Erin Goldberg, Tom Worthington, Cynthia Chung and Erin Evans, and undergraduate students Danielle Keith and Michaela Schweitzer. I would also particularly like to acknowledge Iryna Shanina for being an incredible lab manager. I am grateful for the work of the staff and faculty within the M&I department and the LSI, as well as the MBF staff and veterinarians for making this research possible. I have been fortunate to become a part of the MS Clinical Trials (MSCT) team at the Djavad Mowafaghian Centre for Brain Health, working with the Canadian Prospective Cohort Study to Understand Progression in MS. I am grateful to everyone on the MSCT team, particularly Tara Martin, Kyra West and Sara Tipnis, for providing me with the opportunity to work with individuals with MS. I am thankful to all the study participants for their time and dedication to MS research. Finally, I would like to thank my friends and family. The UBC Triathlon Team has been a source of incredible friendships and adventures and has been the best way to balance long hours in the lab. I would like to thank my parents, grandparents, brothers and Paige for their unwavering support. 1 CHAPTER 1: INTRODUCTION 1.1 Multiple Sclerosis Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that impacts approximately 2.8 million individuals worldwide 1. The disease is autoimmune in nature, wherein autoreactive lymphocytes mount aberrant responses against CNS antigens, leading to inflammation, demyelination and neuroaxonal degeneration 2,3. This disruption in neuronal signalling has a heterogenous presentation, producing symptoms such as loss of coordination and balance, sensory and visual disturbances, bladder dysfunction, numbness, weakness, and mood changes 3,4. General patterns of disease progression have allowed several disease subtypes to be recognized (Fig 1.1). Relapsing-remitting MS (RRMS) is the most common form, impacting approximately 85% of individuals with MS 2,3. RRMS is characterized by distinct episodes of neurologic dysfunction (relapses) followed by periods of partial or complete remission 3,5. The frequency and duration of relapses varies widely between individuals with MS 5. One or two decades post initial diagnosis, approximately 80% of individuals with RRMS will develop secondary progressive MS (SPMS), wherein neurologic function worsens over time and disability increases 2. Approximately 10% of individuals with MS do not experience relapses in the early disease course and instead undergo progressive decline from the outset of disease 2. These individuals are diagnosed with primary progressive MS (PPMS) 2. 2 Figure 1.1: Main forms of MS. Three most common clinical forms of MS include relapsing-remitting, secondary progressive, and primary progressive MS. Adapted from GoodRx and created using BioRender. Increasing availability of disease modifying therapies (DMTs) presents a chance to reduce disability and prolong survival for individuals impacted by MS. However, a definitive cure for MS is still lacking, and the cause of the disease remains elusive 1. 1.1.1 Disease epidemiology and risk factors In Canada, rates of MS are among the highest in the world, with a prevalence of around 1 in 400 individuals 6. MS is also the most common non-traumatic disabling neurological condition among young adult Canadians 7. The average age of onset is between the ages of 20-40 years, a time when young adults are starting to build their careers and families 7. MS is therefore associated with significant economic burden on society 7,8. The disease has a major impact on patient’s quality of life and productivity; a qualitative research study of 26 individuals with MS and 11 family members revealed that MS places significant strain on patients and their families, and has a substantial impact on their financial situation 9. 3 There is a significant predominance of MS in females compared to males, with a sex ratio of 2-3 that has been increasing over the last few decades 10,11. There is evidence that sex also impacts the clinical features and prognosis of MS 12. Generally, females experience earlier onset of disease, a slightly lower prevalence of primary progressive disease course and show overall less progression of disability than males 13. The main factors impacting sex bias in autoimmune disease include sex hormones, such as estrogens, incomplete X chromosome inactivation of immune-related genes, expression of X-linked microRNAs, and differential responses to environmental factors such as microbial exposure and diet 14. Though the exact etiology of MS remains unknown, it is considered to result from environmental influences in genetically susceptible individuals 2. The significance of genetic factors in an individual’s susceptibility to MS have been revealed by genetic epidemiological studies 15,16. For instance, first degree relatives of individuals with MS are generally at a 10-25 times greater risk of developing MS than the general population 15. Twin studies have revealed higher concordance in monozygotic twins (~25%) compared to dizygotic twins (~5%) 16,17. Specific variations in the human leukocyte antigen (HLA) genes have been associated with increased MS risk. The strongest genetic risk of MS is associated with the HLA-DRB1*1501 allele, which has a 14-30% frequency in countries with high MS risk 15,18. The incidence and prevalence of MS increases with latitude, with individuals living closer to the equator being at a lower risk of MS than those living in countries at higher latitudes (Fig 1.2) 6,15,19. Sunlight exposure and associated vitamin D status may contribute to the latitudinal variation seen in MS. Increasing latitude is strongly inversely correlated with duration and intensity of ultraviolet B radiation (UVB) from sunlight and vitamin D concentrations, while a lack of UVB exposure and low serum vitamin D levels are associated with an increased risk of MS 4 20. Overall, evidence suggests a protective effect of vitamin D in MS 20. The detailed mechanism by which this occurs remains to be elucidated, however, vitamin D is suspected to modulate immune responses and contribute to the maintenance of immune homeostasis 21. Figure 1.2: Global prevalence of MS. Prevalence of MS per 100,000 people. Acquired from the Atlas of MS 3rd edition Part 1 (2020) 6. Several modifiable risk factors such as smoking and diet been associated with increased MS susceptibility and worsened disease progression 22. For example, the MS risk for cigarette smokers is approximately 50% higher than non-smokers, and active smoking has been associated with faster disease progression and an earlier transition to progressive MS 18,22. Furthermore, the gut microbiota composition in individuals with MS has been reported to be distinct compared to healthy individuals, and the transplantation of MS intestinal microbiota has been shown to 5 exacerbate or induce murine models of MS 23. Obesity in adolescence and young adulthood, measured by elevated body mass index (BMI), increases the risk of MS by approximately two-fold 5,23. Successful management of the modifiable risk factors may lead to better long-term outcomes and improve patients’ quality of life. Accumulating evidence suggests a role for infectious agents, such as viruses, in MS pathogenesis. Several viruses have been proposed to play a role in MS development, including Epstein-Barr virus (EBV), Human Herpesvirus-6 (HHV-6), Varicella-zoster virus (VSV), John Cunningham virus (JCV), and human endogenous retroviruses (HERVs) 24. These viruses are persistent and cause life-long infections 24. The largest body of evidence during the last few years has accumulated around EBV and HHV-6. Evidence for the involvement of HHV-6 includes increased prevalence of HHV-6 DNA, mRNA and protein expression in MS plaques compared to in normal appearing white matter 25. Increased titers and frequencies of anti-HHV-6 immunoglobulin G (IgG) and M (IgM) have been observed in the CSF of MS patients compared to controls 25. Furthermore, a recent study indicated that elevated HHV-6 antibodies both precede and are associated with a higher degree of axonal injury in MS 26. EBV infection displays a near 100% epidemiological association with MS, and is considered to play a leading role in MS pathogenesis 24. The role of EBV in MS is reviewed in section 1.2.4. 1.1.2 Disease pathology and treatments The key indicator for diagnosing MS is the presence of large confluent demyelinating lesions in the white and gray matter of the CNS 27. Diagnosis of MS uses the McDonald Criteria, and requires evidence of lesional dissemination in time (DIT) and space (DIS), such that more than one CNS location is impacted with evolution over time 28,29. The main tools to support diagnosis are magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) analysis 29. MS symptoms 6 vary according to location and severity of lesions 29. In early MS, axons and neurons are mostly preserved 2. However, as the disease progresses, there is increased neuronal, axonal and myelin loss and failure of CNS repair mechanisms, which is reflected by increased disability 2,30. An essential step in the pathogenesis of MS is the infiltration of leukocytes into the CNS via the blood-brain barrier (BBB). In MS, the display of chemokines by endothelial cells of the BBB may be altered, promoting leukocyte entry into this immunologically distinct site 31. Furthermore, under neuroinflammatory conditions, the tight junctions (TJs) and adherens junctions (AJs) that typically seal the BBB are dysregulated, leading to increased permeability and immune infiltration into the CNS 32. Alterations to the BBB can be observed in MS via post-mortem histological analysis or clinically using MRI 32. Both innate and adaptive immunity are involved in the overall process of demyelination and neuroaxonal damage (Fig. 1.3). Acting as professional antigen presenting cells (APCs), dendritic cells (DCs) are important in promoting pro-inflammatory T cell responses in MS 33. Mouse models of MS have revealed that DCs accumulate in the inflamed CNS of mice with both chronic and relapsing-remitting disease 34. Microglial cells, considered the resident macrophages of the nervous system, also contribute to MS inflammation through antigen presentation and secretion of pro-inflammatory cytokines 33. Microglia are further involved in phagocytosis of degraded myelin, which results in heightened expression of myeloperoxidases, which can cause further neuronal damage 33. However, there is also evidence for a potential beneficial role of microglia in MS, as they are capable of secreting anti-inflammatory cytokines depending on the inflammatory milieu of the CNS 35. Similarly, natural killer (NK) cells contribute both effector and regulatory functions in the innate immune system 33,36. A majority of evidence from mouse models points to a regulatory role for NK cells in MS, via mechanisms such as general T cell 7 suppression, inhibition of the differentiation of myelin-reactive T helper 1 (Th1) or 17 (Th17) cells in the CNS, and secretion of neurotrophic factors 36. Figure 1.3: Immunopathogenesis of MS. Summary of several elements contributing to the immunopathogenesis of MS. Pro-inflammatory environment in the CNS results in release of antigens into the periphery, where T and B cell responses are primed. T cell responses are stimulated by dendritic cells presenting CNS antigens. T and B cells cross the blood brain barrier to infiltrate the CNS. B cells encounter their specific antigen and mature to plasma cells which release IgG antibodies. Invading CD8+ T cells may encounter their specific ligands present on MHC class I molecules by glial or neuronal cells. CD4+ T cells may be activated by microglial cells presenting antigens on MHC class II molecules. Microglia and activated T cells secrete inflammatory cytokines. The release of cytokines continues attracting immune cells such as macrophages, overall resulting in the immune-mediated damage of the myelin sheath. Adapted from Hemmer et al. 37. Created using BioRender. 8 Studies investigating the immunopathology of MS have largely focused on adaptive T and B lymphocytes. MS has historically been viewed as a T cell mediated disease, as T cells appear early in lesion formation, and a significant correlation exists between the number of inflammatory T cells and the extent of acute axonal injury within lesions 2,38. Overall, CD8+ T cells outnumber CD4+ T cells in brain lesions of MS patients 38,39. Infiltrating CD8+ T cells express granzyme B and interferon  (IFN), and animal models have identified CD8+ T cells as potent effectors in the disease 39. Myelin-specific CD4+ Th1 (IFN-producing) and Th17 (Interleukin (IL)-17-producing) lymphocytes also play important roles in the pathophysiology of MS, as evidenced by the presence of these pro-inflammatory cells within the brain and active plaques in individuals with MS 30. The relative importance of Th1 versus Th17 cells in MS pathogenesis remains poorly understood, with conflicting studies reporting the prevalence of one cell type over another at different phases of disease 2. Furthermore, some lesional CD4+ T cells display an intermediate phenotype, expressing both IFN and IL-17, and cells of this phenotype are consistently detected in vivo in the inflamed CNS 40,41. T regulatory cells (Tregs) play an important role in the maintenance of peripheral immune tolerance by suppressing autoreactive effector CD4+ T cell responses 42. Significant differences in the number of circulating Tregs in MS patients relative to healthy controls are not frequently reported 42,43. However, Tregs from MS patients have displayed lower suppressive capabilities, such as reduced IL-10 production, which may contribute to disease pathogenesis 43–45. Although T cells have classically been viewed as the major contributors to inflammatory demyelination in MS, a growing body of evidence has revealed an important role of B cells in disease pathogenesis 46. Both antibody-dependent and -independent mechanisms underlie B cell mediated CNS injury in MS 46. Early evidence implicating B cells in MS pathogenesis came from 9 the detection of antibodies bound to myelin fragments within phagocytic cells in CNS tissue of MS patients47,48. Furthermore, elevated immunoglobulin synthesis rates and a typical oligoclonal band (OCB) pattern of immunoglobulins in the CSF are hallmarks of MS 47. Aside from antibody secretion from plasmablasts and plasma cells, B cells may be involved in MS pathogenesis via antigen presentation to T cells, production of pro-inflammatory cytokines and soluble toxic factors contributing neuronal injury, contribution to the formation of meningeal lymphoid aggregates, and by providing a reservoir for latent Epstein-Barr virus (EBV) infection 46. The success of B cell depletion via anti-CD20 therapy in treating MS is a testament to the antibody-independent roles of B cells in MS, as plasmablasts express little or no CD20 47. It remains an open question whether MS is triggered in the periphery or in the CNS 2. In the peripheral model (“outside-in”), autoreactive T cells are activated at peripheral sites due to environmental and/or genetic, CNS-independent, factors 2,52. These T cells traffic to the CNS, where they are reactivated by CNS-resident APCs and release cytokines leading to direct and indirect damage to myelin 52. Alternatively, CNS intrinsic events may trigger disease development (“inside-out”), with the primary pathogenesis being oligodendrocyte injury and myelin destabilization, and the infiltration of autoreactive lymphocytes occurring as a secondary phenomenon 2,52. Various experimental murine models can be utilized to investigate both “outside-in” immune-mediated demyelination and “inside-out” CNS neurodegeneration-driven disease 52. Current treatment strategies for MS are focused on treating acute attacks, alleviating symptoms, and decreasing biologic activity through DMTs 29. The first approved therapies for MS were immunomodulatory agents (IMAs), namely interferons and glatiramer acetate (GA) 29,49. These IMAs have had success in decreasing the inflammatory burden of disease and reducing new 10 lesion activity, though to varying degrees in different individuals 49. Other DMTs such as natalizumab and fingolimod target lymphocyte migration pathways, aiming to prevent the infiltration of autoreactive lymphocytes into the CNS 29. Both drugs have demonstrated the ability to reduce clinical and radiological measures of disease activity, and exhibit beneficial effects quickly after treatment initiation 50. An increased understanding of the role of humoral immunity in MS has led to the rise of B cell depleting therapies, initially with the anti-CD20 monoclonal antibody rituximab, followed by the development of ocrelizumab and ofatumumab 29. Anti-CD20 therapies lead to a rapid and nearly complete depletion of circulating CD20+ B cells, however have a limited ability to deplete B cells within lymphoid tissues 51. Data indicate that following a single course of rituximab treatment, memory B cells remain suppressed for at least 1-2 years, and that reconstituted B cells following anti-CD20 therapy demonstrate decreased pro-inflammatory cytokine production 51. B cell depletion therapy has led to major breakthroughs in the treatment of RRMS, as they have been effective at delaying relapses and slowing the development of new white matter lesions detected by MRI 29,51. Despite advances in RRMS treatment, PPMS has remained notoriously difficult to treat 51. For example, initial PPMS trials investigating interferons, GA and fingolimod all failed to meet their endpoints 51. B cell therapy for PPMS has shown more promising results, and ocrelizumab is currently the only approved DMT for the treatment of PPMS 29,51. Numerous outstanding questions remain regarding anti-CD20 therapies for both RRMS and PPMS, including optimizing strategies for treatment initiation and dosing, as well as determining the effects on long-term disability and adverse effects of prolonged B cell depletion 51. Overall, current treatments approved for MS can 11 reduce relapses, however, neuroaxonal damage and the ensueing physical disability becomes permanent, highlighting the need for a better understanding of the disease 2. 1.1.3 Modelling disease Given the complexity of MS, there is no single animal model that can capture the heterogeneity of the disease and its variety of presentations. However, animal models can still be used as research tools to study disease development and examine therapeutic approaches 53. The most used animal models of MS are (1) experimental autoimmune encephalomyelitis (EAE); (2) viral-induced models with chronic demyelination; and (3) toxin-induced models of demyelination53. EAE is the most studied animal model of MS. The model was first described in 1933 by Thomas Rivers and colleagues, to better understand the inflammatory demyelination resulting from rare complication of a rabies vaccine grown on rabbit spinal cord 54,55. They demonstrated that the encephalomyelitis was not due to rabies itself, but to an autoimmune response stimulated by the spinal cord contaminant of the vaccine 55. Since then, EAE has been induced using an array of methods in a variety of animal species 55. Active EAE is induced by immunization with CNS tissue or myelin peptides, such as myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP) and proteolipid protein (PLP), in complete Freund’s adjuvant (CFA) 55. Pertussis toxin (PTx) is typically co-administered, and promotes EAE by opening up the BBB, facilitating migration of pathogenic T cells into the CNS 56. PTx also has biologic effects that contribute to EAE such as increasing cytokine production by T cells, and enhancing clonal expansion of both neuroantigen-specific Th1 and Th2 cells 56. EAE in mice is characterized by an ascending paralysis beginning at the tail, followed by hindlimb and forelimb paralysis, beginning 9-12 days post immunization 53. EAE can be induced 12 in variety of mouse strains, such as C57Bl/6, Non-Obese Diabetic (NOD), and Swiss Jim Lambert (SJL/J), and the combination of immunizing agent and mouse strain used yields different patterns of disease 53. For instance, immunization of SJL/J mice with PLP139-151 induces a relapsing-remitting disease, while induction with MOG35-55 in C57BL/6 mice typically leads to a chronic presentation of disease 53. The choice of immunizing agent also impacts the immunopathogenesis of the disease. For example, the use of MOG35-55 in C57Bl/6 mice triggers a strong inflammatory response in the CNS, but neglects the function of B cells as MOG APCs in MS pathogenesis 57. However, the use of a more complex immunizing agent such as the entire extracellular domain of recombinant human MOG (rhMOG1-120) promotes the involvement of B cells as APCs in EAE 58. In contrast to active EAE, passive or adoptive transfer EAE (AT-EAE) can be induced in recipient animals by transferring myelin-specific T cells generated by active immunization in donor animals 55. AT-EAE is an important tool to study the effector phase of the disease and bypass EAE induction phase 55. After EAE induction, myelin-specific CD4+ T cells are primed outside the CNS 59. Primed T cells gain access to the CNS via expression of adhesion molecules, chemokine receptors and integrins, which allow them to pass through the BBB, which may already be compromised due to the administration of PTx 59. In the CNS, T cells are reactivated by local APCs presenting myelin antigens, such as microglia 59. Autoreactive T cells then produce pro-inflammatory cytokines, initiating CNS inflammation, as well as chemokines, attracting other immune cells to the site of inflammation (including macrophages, B cells, and CD8+ T cells) 59. Certain T cell subsets are known to be particularly important in EAE pathogenesis. For instance, similar to MS, Th1 and Th17 cells have well-established roles in EAE 57. Interestingly, clinical symptoms of Th1- and Th17- mediated EAE are different; Th1 cells induce classical EAE, 13 whereas Th17 cells tend to induce “atypical” ataxic EAE 53,60. The role of CD4+CD25+FOXP3-expressing (forkhead box P3) Tregs in MS have been extensively studied using the EAE model. Adoptive transfer of Tregs to EAE mice can reduce disease severity, and though the Treg population in the CNS is initially small, it rapidly expands during EAE 53. Although CD8+ T cells play an important role in MS pathogenesis, most EAE models are CD4+ T cell-driven 53. Furthermore, EAE is mainly a disease that impacts the spinal cord white matter, whereas MS is mainly a brain disease 53. As such, although the EAE model has significantly contributed to our knowledge of autoimmunity and neuroinflammation, efforts must be put towards continually improving the model. Viral- and toxin-induced models are also important tools in studying MS in vivo, although these models have been less widely used compared to EAE. Viral infections of the CNS, such as that caused by Theiler’s murine encephalomyelitis virus (TMEV), can induce demyelination 53. TMEV-induced disease represents the “inside-out” model, where axonal damage precedes immune-mediated demyelination by macrophages, CD4+ and CD8+ T cells 53,61. 30-40 days post TMEV infection, susceptible mice develop late chronic demyelinating disease with extensive lesions of the white matter and cell infiltrates into the spinal cord 61. The disease ultimately leads to progressive spinal cord atrophy, which is reflected by neurological deterioration, including loss of motor coordination, hind limb paralysis, spasticity, and ataxia 61. TMEV induces an inflammatory disease only in mice, and not in other rodent species or in primates 53. While EAE and viral-induced models are useful to study the autoimmune origin of MS, toxic demyelination models are useful to study the processes of de- and re-myelination 53. For example, cuprizone is a copper chelating agent which causes oligodendrocyte cell death and subsequent demyelination and 14 astrocytes and microglia 53. Despite the experimental use of cuprizone, the specific mechanism of action underlying oligodendrocyte damage is not fully understood 53. 1.2 Epstein-Barr virus Epstein-Barr virus (EBV), also known as human herpesvirus 4 (HHV-4), is a double- stranded DNA virus of the gammaherpesvirus family 62. It is a highly successful pathogen that infects up to 95% of the world’s population and establishes a life-long latency in the human host 62,63. EBV was discovered in 1964 by Anthony Epstein and Yvonne Barr via electron microscopy of cells cultured from an African Burkitt’s lymphoma biopsy, making it the first human tumorigenic virus to be characterized 63. Since its discovery, it has been implicated in an array of diseases and illnesses, including infectious mononucleosis (IM), oral diseases, autoimmune diseases and various malignancies including B cell, NK cell and T cell lymphomas, epithelial carcinomas, and gastric cancer 63. Although EBV molecular biology and EBV-related diseases have been investigated thoroughly for 60 years, many questions regarding the precise role of EBV in promoting these diseases remain unanswered. 1.2.1 EBV biology and lifecycle The stages of the EBV lifecycle include the primary infection, the establishment of latency, and the reactivation or lytic stage to produce new virions (Fig. 1.4) 63. Typically, primary infection is asymptomatic and occurs during childhood 64. Infection during adolescence and young adulthood may lead to IM, also called the “kissing disease”, as saliva is the major transmission route for the virus 63. Evidence also suggests that EBV can be transferred via organ transplantation, blood transfusion, and semen during sexual contact 65. Following primary infection, EBV infects 15 epithelial cells (ECs) in the oropharynx where it can replicate and subsequently infect B cells trafficking to the oral cavity 62. Figure 1.4: Overview of EBV infection. (1) EBV infects epithelial cells in the oropharynx via attachment of EBV gH/gL to epithelial cell Ephrin A2. (2) Infection of naïve B cell via attachment of gH/gL/gp42 to MHC class II and gp350 to CD21. (3) Establishment of latency III and trafficking to germinal centers. (4) Establishment of latency II in memory B cells. (5) Latency I and 0 in memory B cell reservoir. (6) Reactivation and lytic replication in plasma cells leading to viral shedding. EBV attachment and entry into target cells is mediated by different mechanisms depending on the cell type. During B cell infection, EBV envelope glycoprotein gp350/220 binds to the B- cell complement receptor CD21 62,65. EBV attachment to ECs, which lack CD21, is less well 16 understood, but likely involves the binding of EBV glycoproteins gH and gL to receptors on the surface of ECs such as Ephrin A2 65. Following attachment, gH, gL and gp42 participate in viral entry. In B cells, entry involves binding of gp42 to major histocompatibility complex (MHC) class II molecules, whereas in ECs, entry is mediated by the gH/gL complex 62,65. Following attachment and entry to B cells, EBV virions are endocytosed and the viral membrane fuses with the endosomal membrane, an event mediated by gH/gL and gB 62. Once B cell infection has occurred, EBV can establish latency. The viral genome circularizes through the terminal repeats, and is retained as an extrachromosomal episome wrapped in nucleosomes, characteristic of herpesvirus latency 62. During cell division, the viral genome is replicated by the host DNA polymerase, starting at the origin of plasmid replication (OriP), and partitioned into the daughter cells 62. The EBV genome is composed of linear, double-stranded DNA (dsDNA) with a relatively large genome size of ~172 kpb, that encodes for more than 85 genes 64. Most proteins encoded by the EBV genome are involved in nucleotide metabolism, to maintain the replication of the viral DNA, and to build viral structural components 64. The EBV genome also contains several genes essential for latency 64. For example, EBV nuclear antigen 1 (EBNA1) is required for viral genome maintenance during latency by binding directly to OriP to ensure the dsDNA EBV genome is replicated as the host cell multiplies 62,63. EBNA1 can also enhance cell survival by inhibiting apoptosis, and it interacts with viral promotors to contribute to the transcriptional regulation of the EBNAs and latent membrane protein 1 (LMP1) 63. LMP1 is the principal viral oncoprotein implicated in several patterns of EBV latency 66. Acting as a constitutively active member of the tumor necrosis factor receptor (TNFR) family, LMP1 significantly contributes to the sustained cellular proliferation and survival observed in EBV-associated malignancies 66. Several viral non- 17 coding RNAs also play a role in maintenance of viral latency. EBV-encoded RNAs (EBERs) are expressed throughout all different latency states and contribute to B cell transformation and maintenance of latency, though they are not essential 62. BamHI A-encoded transcripts (BARTs) give rise to 44 micro RNAs (miRNAs), the majority of which regulate host transcripts 62. Four patterns of EBV latency exist and are defined based on differential latency gene expression programs. Latency 0 generally occurs in circulating memory B cells in healthy people, and displays expression of EBERs and BARTs only 62,63. Latency I is the next most restricted pattern during which EBNA1 is the only viral protein expressed, in conjunction with EBERs and BARTs 62,63. Latency II, also referred to as the default program, is characterized by the expression of EBNA1, LMP1, LMP2, EBERs and high levels of BART expression 62,63. The most complex and well-characterized program is latency III, also known as the growth program 63. Latency III displays expression of three integral LMPs (LMP1, 2A and 2B), six nuclear antigens (EBNA1, 2, 3A, 3B, 3C and EBNA-LP) and multiple non-coding RNAs, EBERs, BHRF1 microRNAs and BARTs 62. It is important to note that subtypes with variations of latency pattern expression can also exist. Two main models exist to explain how EBV establishes a latent infection in memory B cells, a specialized and infrequent cell type: the germinal center (GC) model and the direct infection model 67. The GC model proposes that EBV persists by infecting naïve B cells and ultimately driving B cells to differentiate into a memory phenotype via the expression of viral genes that resemble the GC-dependent maturation process 67. This process involves new latently infected B cells passing through a series of differentiation stages, which can be associated to the different viral latency gene expression programs 67. The direct infection model proposes that EBV 18 directly infects memory B cells. However, unlike the GC model, the direct infection model does not account for the four latency programs of EBV infection 67. In certain instances, such as stimulation of the B cell receptor (BCR) and various stress-related signals, EBV can reactivate from latency into the lytic cycle, leading to the production and shedding of infectious virus 68,69. EBV lytic replication is initiated by the activation of one or both of its immediate early (IE) promoters, Zp and Rp, which leads to the production of two IE proteins, Z (also called Zta, ZEBRA or EB1, the product of the BZLF1 gene) and R (also called Rta or EB2, the product of the BRFL1 gene), respectively 69. An ordered cascade of viral gene expression then ensues, starting with early lytic gene expression and followed by viral genome replication and late gene expression 70,71. Viral DNA is replicated as large, complete molecules, which are then cleaved and packaged into viral progeny to be released and infect neighbouring cells 70. 1.2.2 Immune response to EBV Both the innate and adaptive immune systems play roles in protective immunity against EBV. Toll-like receptors 3 (TLR3) and 9 (TLR9) have been implicated in sensing of EBV pathogen-associated molecular patterns (PAMPs), which engages conventional and plasmacytoid dendritic cells (cDCs and pDCs), respectively 73. For example, double stranded RNA (dsRNA) structures of EBERs can stimulate cDCs via TLR3 73. Further, B cells, the primary target of EBV, also express TLR3 and TLR9 73. However, once infection has occurred, EBV can down-regulate B cell TLR9 signalling via LMP-1 during latent infection, and BGLF5 during lytic replication, ultimately protecting itself from cell-death 73. Further, NK cells and monocytes have also been implicated in innate immune control of EBV infection 73. In response to EBV infection, DCs produce antiviral type I interferons (IFN), stimulating other innate immune cells to limit viral replication and promote the development of adaptive 19 immune responses 73. Both B and T cells are part of the adaptive immune response to EBV 62. B cell activation results in increased production of IgM, IgG and IgA, and antibodies against both latent and lytic viral antigens are detected during IM 62. Furthermore, IM is characterized by peripheral CD8+T cell lymphocytosis, including large clonal and oligoclonal populations of CD8+ T cells, which may comprise up to 30% of the entire CD8+ T cell population 72. Evidence suggests that the CD8+ T cells play a protective role during primary infection, however the extent of expansion of activated cells may contribute to the clinical features of IM 72. A period of CD8+ T cell downregulation occurs following primary infection, and most EBV-specific CD8+ T cells do not survive to mediate protection during the persistent phase of EBV infection 72. The overall CD4+ T cell compartment expands relatively little during primary EBV infection, however evidence suggests that a small population of activated CD4+ T cells are reactive to EBV during primary infection 72. EBV employs several strategies to evade host adaptive immune responses. For example, EBV BDLF3 inhibits MHC class I and II presentation by ubiquitination-induced internalization, and BILF1 promotes the degradation of HLA-A and HLA-B 62. Furthermore, EBV BNLF2A prevents the transporter associated with antigen processing (TAP), which hinders peptide translocation from the cytoplasm to the endoplasmic reticulum (ER) 62. Immunomodulatory functions have been ascribed to several EBV latent proteins 74. For example, EBNA1 contains a long glycine-alanine repeat that inhibits translation as well as proteasomal degradation, to ensure sufficient EBNA1 levels to maintain the viral genome while decreasing protein turnover to minimize antigen presentation to CD8+ T cells 74. Overall, EBV employs a wide range of strategies to evade host immunity during the lytic and latent phases of replications. These evasion mechanisms likely contribute to the development of EBV-associated diseases 74. 20 1.2.3 EBV vaccines After almost 30 years of trials, there are no prophylactic or therapeutic EBV vaccines currently approved for widespread use. Due to the tumorigenicity and life-long infection of EBV, vaccination with live attenuated virus is not sensible 75,76. As such, EBV vaccine studies have encompassed subunit vaccines, epitope vaccines, DNA vaccines, viral vector vaccines, virus-like-particles (VLPs), or dendritic cell vaccines 76. Most previous vaccine studies have focused on gp350, as it is the most abundant glycoprotein on the EBV envelope 76. For example, the first clinical trial of an EBV vaccine was reported in 1995, using gp350 expressed in a vaccinia virus (VV) 75,77. A single dose of the vaccine was given to (1) EBV and VV-seropositive adults, (2) EBV-positive, non-VV-exposed juveniles, and (3) EBV- and VV-naïve infants 77. The outcomes were analyzed 16 months after vaccine administration 77. In adults, the VV did not multiply due to the anti-VV immunity, and the vaccine had no effect 78. In juveniles, anti-EBV neutralizing antibodies increased, and a subset of the vaccinated infants remained protected from infection 78. This initial clinical study was not pursued further. Since then, vaccines comprising gp350 combined with various adjuvants have been investigated 76. For example, immunization of rabbits with monomeric gp350 in adjuvant elicited EBV-neutralizing antibodies, and studies in non-human primates using gp350-based vaccines have shown protection against EBV-induced lymphoma and replication 79. Notably, from 2001 to 2003, Sokal et al. conducted a phase II, double-blind, placebo control randomized trial to evaluate the safety and immunogenicity of a gp350 monomer in alum/monophosphoryl lipid A adjuvant (NCT00430534) 80,81. 181 EBV-seronegative young adults received 3 doses of either vaccine or placebo, and vaccine efficacy was assessed over 18 months 80. One month after receipt of the final dose of the vaccine, 98.7% of vaccinated subjects showed seroconversion to anti-gp350 antibodies, 21 and these antibodies remained present for a period of over 18 months 80. Although this humoral response was not sufficient to prevent EBV infection, the vaccine reduced rates of IM by 78%, demonstrating an ability to attenuate the severity of disease associated with EBV-infection 78,80. More recently, in 2022, a phase I clinical trial evaluating the safety and immunogenicity of an adjuvanted gp350-ferritin nanoparticle vaccine was launched (NCT04645147) 82. In addition to gp350, other glycoproteins are targeted by neutralizing antibodies in the humoral response to EBV, and these antigens have been the focus of several vaccine studies. For instance, a core fusion apparatus made up of gB trimers and gH/gL heterodimers is essential for the membrane fusion process 76. Neutralizing titers of sera from rabbits immunized with trimeric or monomeric gH/gL, trimeric gB and tetrameric gp350 were higher than those elicited by monomeric gp350 83. Recently, a phase I clinical trial has been launched by Moderna to evaluate an mRNA vaccine including four mRNA encoding gH, gL, gp42 and gp220 (NCT05164094) 84. Overall, combining glycoprotein antigens is an attractive approach for EBV vaccine development. Although the ideal prophylactic vaccine would provide sterilizing immunity against EBV, a vaccine that does not completely prevent EBV infection, but can still reduce EBV-associated diseases, may be a more realistic solution that merits continued investigation. Therapeutic vaccine strategies have also been explored to boost existing antiviral T cell responses or induce novel antiviral responses 85. The initial therapeutic vaccine trials in nasopharyngeal carcinoma (NPC) patients used autologous monocyte-derived DCs loaded with LMP-2 CD8+ T cell epitope peptides 85,86 . In this trial, 9 out of 18 vaccinated patients exhibited an increase in LMP-2-specific T cells, and 2 individuals displayed partial clinical responses 86. Non-cell based therapeutic vaccines using recombinant viral vectors expressing EBNA1, LMP1 and/or LMP2 have also undergone clinical trials in NPC patients, some showing modest clinical 22 activity 85. It is important to note that many of the patients undergoing clinical trials for therapeutic EBV vaccines had previously received cytotoxic chemotherapy, which would likely impair their response to vaccines 85. The development of an EBV vaccine presents several challenges. Vaccination at a young age and subsequent waning immunity may delay primary EBV infection until an age when the risk of IM is higher 78. To overcome this issue, vaccination of individuals at higher risk for EBV-induced disease as opposed to universal childhood vaccination may be a more reasonable approach 87. Furthermore, the seroprevalence of EBV in 95% of the world’s population makes finding candidates for prophylactic clinical trials difficult. A lack of efficient animal models to study EBV, a human-tropic virus, has hindered the development of both prophylactic and therapeutic vaccines 78. Finally, if the objective of an anti-EBV vaccine is to limit the occurrence of disease associated with the virus, the low incidence of these diseases and the time they take to appear complicates clinical trials capable of providing vaccine efficacy 78. 1.2.4 EBV and MS The first evidence for a role of EBV in MS was reported in 1979, when Fraser and colleagues observed that blood lymphocytes from individuals with clinically active MS had an increased tendency to transform spontaneously on in vitro culture compared to cells from those with clinically inactive disease or from healthy donors 88,89. The transformed cells had characteristics of B lymphocytes and carried EBV antigens 88. In 1981, the link between EBV and MS was again proposed, due to the overlap in cases between IM and MS 90. Since then, meta-analyses have consistently shown that a history of IM significantly increases the risk of MS, and have established that the risk of MS is increased more than 2-fold following IM 91–93. Beyond the association between MS and IM, epidemiological, virological and immunological evidence has 23 accumulated to support the role of EBV in the aetiology of MS 94. Nearly all patients with MS are EBV-seropositive, whereas the risk of MS in seronegative individuals is extremely low 23,94–96. The risk of developing MS among seemingly healthy young adults is strongly related to serum titers of anti-EBNA IgG antibodies 85,97–99. Notably, in 2022, a 20-year longitudinal analysis of over 10 million young adults on active duty in the US military revealed that the risk of MS increases 32-fold after infection with EBV, but not after infection with other viruses, such as the similarly transmitted cytomegalovirus (CMV) 96. The same study demonstrated that EBV seroconversion preceded increased serum levels of neurofilament light chain, a biomarker for neuroaxonal degeneration, in individuals with MS 96. This is the largest and most definitive study suggesting that EBV is necessary, but insufficient on its own, for the development of MS. Despite EBV infection being necessary for the initiation of MS, elucidating the mechanisms linking the virus to CNS inflammation and neurodegeneration remains challenging. Several hypotheses delineating this mechanism have been proposed. The Pender hypothesis, stipulated by Dr. M Pender in 2003, proposes that a genetically determined defect in the elimination of EBV-infected B cells by cytotoxic CD8+ T cells might predispose the development of MS by allowing EBV-infected autoreactive B cells to accumulate in the CNS 100–104. EBV-infected autoreactive B cells may seed in the target organ, producing pathogenic autoantibodies and providing costimulatory survival signal to autoreactive T cells, which would otherwise die in the target organ 89. This hypothesis may be supported by reports of the accumulation of EBV-infected B cells and plasma cells in the brain 105–107. For example, in 2007, Serafini et al. investigated expression of EBV markers in post-mortem brain tissues from MS cases of varying clinical courses, and found that the accumulation of EBV-infected B cells and plasma cells in the meninges and perivascular compartment of white matter lesions is a common feature of MS 105. 24 However, two studies preceding Serafini et al. had failed to detect EBV in the MS brain using in situ hybridization for EBER and immunohistochemistry for viral proteins 108,109. Furthermore, several groups have been unable to reproduce Serafini and colleagues’ findings, even when using specimens from the same tissue bank 110–112. Some of these conflicting findings may be due to technical challenges of detecting low-levels of EBV gene expression in migratory B cells in the CNS of individuals with MS or in post-mortem samples 94. Nevertheless, should the Pender hypothesis be true, then EBV-specific T cell therapy that kills EBV-infected B cells in the CNS may prevent disease progression and lead to clinical improvement 104. A recent phase I trial assessing the feasibility and safety of treating progressive MS patients with autologous EBV-specific therapy demonstrated clinical improvement in 7 out of 10 treated patients, with 6 experiencing both symptomatic and neurological improvement, together with a reduction in fatigue, improved quality of life, and in 3 patients, reduced intrathecal IgG production (ACTRN12615000422527) 104. This work sets the stage for future clinical trials using EBV-targeted T cells in MS. Another hypothesis to mechanistically explain the role of EBV in MS is the EBV bystander damage hypothesis, wherein immune attack on the CNS in MS is primarily directed against EBV, which results in bystander damage in the CNS 89,105. This hypothesis is also dependent on the presence of EBV-infected B cells in the CNS of individuals with MS. Furthermore, this hypothesis does not explain evidence for a primary role of autoimmunity in the development of MS, although sensitization to CNS antigens released after virus-targeted bystander damage could result in secondary autoimmune responses 89. Overall, EBV-targeted bystander damage may contribute to the disease process in MS, but may not constitute the main role of EBV in the pathogenesis of MS 89. 25 EBV may also be involved in the development of MS via the rescue of autoreactive and inflammatory B cells 94. EBV immortalization of B cells bypasses many of the requirements for T cells help through the expression of CD40-like receptor LMP1 and BCR-like receptor LMP2, and these ligand-independent receptors may be able to rescue autoreactive B cells 94. EBV-infected B cells also express mature BCR and IgG without necessarily passing through GC selection 94. It remains unknown whether these B cells act as APCs or are antibody-producing, and this avenue merits further investigation 94. Molecular mimicry was first proposed as a mechanism for the initiation of autoimmune disease in the early 1980s and occurs when peptides from viral or bacterial proteins share sequence or structural similarities with self-peptide 113. These similarities can stimulate autoreactive immune responses and cause the production of self-reactive antibodies 113. Early studies demonstrated that patient-derived T cells autoreactive to MBP cross-reacted to a wide range of viral peptides, including peptides from EBV 94,114. Subsequent studies characterized an epitope derived from BALF5, the EBV DNA polymerase, that showed strong agonistic activity in MBP-specific T cells 115,116. Furthermore, evidence suggests that EBNA1-specific CD4+ Th1 cells are selectively expanded in MS, and that some of these cells can cross-recognize MS-associated myelin antigens such as MBP, PLP, MOG and 2’,3’-cyclic nucleotide 3’ phosphodiesterase 116. Similar cross-reactivities with EBNA1 have been found for immune cells autoreactive to anoctamin 2 117, glial cell adhesion molecule (GlialCAM)118, and -crystallin B chain (CRYAB) 94,119,120. Autoreactive antibodies in MS can also cross-react with viral proteins, especially EBNA1, and higher levels of antibodies to EBNA1 are typically observed in the serum and CSF of patients with MS 94,118. Overall, detailed mechanisms delineating the specific role of cross-reactive immune cells in MS pathogenesis are not well-established. Furthermore, an improved understanding of the role of 26 autoantibodies in disease is necessary in the design of vaccinations against EBV, to avoid the exacerbation of cross-reactive responses, such as to EBNA-1 85. Considerable efforts have been made towards understanding the mechanisms by which EBV is involved in the development of MS, however, these mechanisms are not yet fully understood. Elucidating these mechanisms will contribute to testing whether EBV-directed treatments can benefit patients with MS. 1.2.5 -herpesvirus-68 as a model for EBV Efforts to characterize the role of EBV in MS and other diseases has been limited due to a lack of suitable mouse models, as EBV primarily infects humans. Some animal species are susceptible to EBV infection, such as cotton-top tamarins, rhesus macaques, and tree shrews, however, these species do not necessarily generate immune responses to infection that are akin to humans 121,122. As such, considerable interest has focused on the development of small animal model systems to investigate the pathogenesis and immunology of EBV infection 123. One such model system involves infection of laboratory mice with murine -herpesvirus-68 (HV68 or MHV68) 123. HV68 is a naturally occurring virus that was originally isolated from Slovakian bank voles and is endemic in European wood mice 124. The HV68 genome contains 118,327 bp of unique sequence that is estimated to encode at least 80 genes, and is largely colinear with the genomes of EBV and Kaposi’s sarcoma herpesvirus (KSHV) 125. Following infection of mice by various routes, including intraperitoneal (IP) and intranasal (IN), HV68 undergoes acute replication at the primary site of inoculation and subsequently establishes a latent infection in several cell types, primarily B cells, but also macrophages, DCs and ECs 126. IN infection results in an acute self-limiting infection of lung epithelial cells, which is followed by a persistent, latent infection of the 27 lungs and lymphoid organs 127. Regardless of the route of infection, the spleen is the major site of latently infected cells, and acute virus replication is cleared from the spleen by 14-16 days post infection 128. The frequency of latently infected cells is independent of the inoculating dose of the virus; 0.1 to 1 x 106 plaque-forming units (PFU) of HV68 administered IP or 4 to 40 x 105 PFU administered IN resulted in the same levels of splenic latent infection 128,129. HV68 ORF73 encodes a homologue of the KSHV latency-associated nuclear antigen, which functions similarly to EBV’s EBNA1 protein 128. As such, the HV68 LANA homologue (mLANA) is required for the maintenance of viral episomes during latency 126,130,131. For instance, in animals infected with a mutated strain of HV68 lacking ORF73, latency is severely deficient in splenocytes, as evidenced by infectious center reactivation assays and by in situ hybridization detection of latent virus 131. Similarities exist between the immune response to HV68 in mice and the response to EBV in humans 123. For instance, during HV68 infection, CD8+ T cells are essential for the resolution of acute infection and the long-term control of persistent infection 128. Depletion of CD8+ T cells results in an exacerbated infection in the lung and spleen, evidenced by elevated viral titres and an increased number of infected splenocytes around germinal centers 132. In contrast, CD4+ T cells contribute little to recovery from acute HV68 infection, but do play a role in the lymphoproliferation seen in the spleen (splenomegaly) during primary HV68 infection 132. Furthermore, CD8+ T cell control of latent HV68 infection is progressively lost in the absence of CD4+ T cells, reflecting the helper function of CD4+ T cells to continuously produce a cytokine-rich environment within responding lymphoid tissues 133. The antibody response to HV68 is slow to develop, with low levels of virus-specific IgG or neutralizing immunoglobulin in the serum, that drastically increase two weeks post-infection 123. Maintenance of high levels of serum-neutralizing 28 antibody may play a role in limiting persistent infection 123. HV68 infection induces high levels of IFN- production in the spleens and lymph nodes of mice, with IFN- levels peaking just prior to viral clearance from the lungs 134. However, HV68 infection in mice deficient in IFN- does not differ significantly from infection in wild-type mice, indicating that IFN- may not be essential for regulating cell recruitment or proliferation during acute HV68 infection135. Latency-deficient recombinant strains of HV68 have been developed and serve as a useful experimental tool to better understand gammaherpesvirus infection biology and explore novel vaccine strategies. An example of a latency-deficient recombinant strain is ACRTA HV68, constructed by Jia and colleagues 130. ACRTA HV68 was created by replacing a cluster of latency-associated genes, including ORF72, M11 and ORF73, with an ectopic copy of RTA (replication and transcription activator), which dictates the virus to lytic replication 130. For the first 3 days post IN infection of mice, ACRTA HV68 replicates similarly and generates comparable titres to wild type (WT) HV68 130. However, 3 days post infection, viral titres of ACRTA decline, whereas WT replication continues until day 7, resulting in a nearly 100-fold difference in the peak titers between ACRTA and WT HV68 infection 130. Furthermore, ACRTA HV68 infection does not induce splenomegaly, and results from infectious center and limiting dilution assays, as well as real-time quantitative polymerase chain reaction (RT-qPCR), indicate that ACRTA fails to establish latency in the spleen 130. Notably, ACRTA HV68 protects mice against WT HV68 challenge, after IN vaccination and either IN or IP WT challenge, as evidenced by vaccinated mice having viral titers below the limit of detection after challenge infection 130. As such, the ACRTA HV68 is an important experimental tool to investigate HV68 vaccination. 29 1.2.6 Modelling EBV and MS in mice Overall, HV68 is an important model to study EBV infection and EBV-related diseases in the laboratory. For over a decade, the Horwitz lab has employed HV68 to investigate the role of EBV in MS in a murine model. Mice are infected with HV68 and then recover for 5 weeks to enable complete clearance of acute infection and establishment of viral latency 136. Mice are then actively induced with EAE (Fig. 1.5) 136. Overall, latent infection with HV68 enhances EAE symptoms without CNS infection or increased viral reactivation. HV68-EAE mice present with an earlier onset of EAE, worse EAE scores and increased mortality rates compared to naïve-EAE mice 136. HV68-EAE mice also display atypical EAE symptoms including loss of balance, ataxia and hunched posture 136. Virally induced exacerbation of EAE is specific to latent HV68, as mice previously infected with lymphocytic choriomeningitis virus (LCMV) or murine cytomegalovirus (MCMV) develop EAE with the same clinical course as naïve-EAE mice 136. Enhancement of EAE by HV68 is also specific to latent HV68 infection, as mice infected with ACRTA HV68, a latency-deficient strain, have a less severe EAE course and lower amounts of T cell CNS infiltration compared to HV68-infected mice 137. HV68-EAE mice present with increased numbers of CD3+ T cells in the CNS compared to naïve-EAE mice 136. Specifically, HV68-EAE mice display increased percentages of CD8+ T cells in the CNS, including within the brain, which is typically minimally infiltrated compared to the spinal cord in naïve-EAE mice 136. CD8+ T cells infiltrating the CNS of HV68-EAE mice produce granzyme B, and a percentage of CD8+ T cells in the brain and spinal cord are specific to two predominant HV68 viral epitopes (p56 and p79) 136. CNS-infiltrating CD4+ T cells in HV68-EAE mice are heavily Th1-scewed compared to naïve-EAE mice, wherein an increased proportion 30 of CD4+T cells produce IFN in both the CNS and the periphery, with a suppression of IL-17 production 136. Furthermore, HV68-enhanced T cell activation is accompanied by a decrease in FOXP3+CD4+ T regulatory cells in the CNS and the periphery, which may contribute to increased susceptibility to autoimmunity 137. Overall, the HV68-EAE model displays immunopathological features that are reminiscent of human MS, indicating that EBV may contribute similarly to the development of MS 136. Figure 1.5: HV68-EAE model experimental overview. Mice are infected with HV68 and left for 5-weeks for the establishment of viral latency. Active EAE is then induced using MOG35-55 peptide emulsified in complete Freund’s adjuvant with simultaneous administration of pertussis toxin. Clinical EAE outcomes are then monitored. 1.3 Research aims and rationale Despite the substantial progress made towards understanding the role of EBV in MS, whether prophylactic vaccination against EBV could prevent the development of MS remains an unknown, important question. Furthermore, it remains to be determined whether a vaccine inducing sterilizing immunity to EBV is required to prevent MS, or if non-sterilizing immunity that protects against IM and reduces immune responses to EBNA1 may be sufficient to reduce the incidence of MS. Assessing these questions in human clinical trials is exceedingly difficult, due to 31 the challenges in the development of an EBV vaccine. Moreover, even if an EBV vaccine, sterilizing or non-sterilizing, was successfully developed and approved for widespread use, evaluating its efficacy at hindering MS development would pose significant challenges. The disease only occurs in a small subset of individuals, and if vaccination occurred during childhood, the success of the vaccine at preventing MS development would have to be evaluated for at least 20-40 years. As such, leveraging laboratory animal models to answer these questions is essential. This work aims to evaluate whether vaccination against HV68 mitigates HV68-induced exacerbation of EAE. I hypothesized that vaccination with ACRTA HV68 and subsequent challenge with WT HV68 would prevent or reduce the establishment of viral latency, which would in turn ameliorate clinical and immunological outcomes compared to unvaccinated HV68-EAE mice. This hypothesis was addressed via the following aims: Aim 1 – Examine viral load of vaccinated mice to confirm capability of ACRTA HV68 to be used as a vaccine for WT HV68 infection Aim 2 – Investigate clinical EAE outcomes of ACRTA HV68 vaccinated, WT challenged mice compared to unvaccinated, WT HV68 infected mice Aim 3 – Analyze immune infiltration and polarization in the spleen and CNS of ACRTA HV68 vaccinated, WT challenged mice compared to unvaccinated, WT HV68 mice Collectively, the results of this work help inform the potential for EBV vaccination to be used as a preventative intervention for MS incidence and progression. Further, it improves our understanding as to whether non-sterilizing immunity that reduces the likelihood of an aberrant immune response to EBV, but does not prevent infection completely, can be an effective strategy to reduce the risk of MS. 32 CHAPTER 2: MATERIALS AND METHODS 2.1 Animals Animal work was approved by the Animal Care Committee at the University of British Columbia, under regulation of the Canadian Council of Animal Care, under protocol A21-0228. Adult female or male C57Bl/6 (JAX #000664) mice, originally sourced from the Jackson laboratory, were bred at the Modified Barrier Facility and the Center for Disease Modelling and began experiments at 6 to 10 weeks old. A mix of male and female mice were used for each experiment (total between all experiments 61.5% male, 38.5% female). Mice were housed in groups up to four animals per cage, fed ad libitum with standard irradiated chow and given free access to autoclaved acidified water. All housing rooms were set to 22-25oC with a humidity range of 50-70%, and maintained on a 14.5-9.5 hour light-dark cycle with sunrise at 5:40 am at sunset at 8:00 pm. 2.2 Gammaherpesvirus 68 2.2.1 Virion generation and infection of C57Bl/6 mice Wild type HV68 (WUMS strain, purchased from the American Type Culture Collection, ATCC #VR-1465), and ACRTA-HV68 (developed by Dr. Ting-Ting Wu and gifted by Dr. Marcia A Blackman130) were propagated on baby hamster kidney fibroblasts (BHK-21, ATCC, #CCL-10). Virion production and quantification of viral titers was adapted from 138. Briefly, HV68 was produced by infecting BHK-21 cells at 50-70% confluency. Two days later, virion-containing supernatant was collected and concentrated by ultracentrifugation. Viral stocks were concentrated using a Vero cell-based plaque assay. C5Bl/6 mice (6-9 weeks old for ACRTA-HV68, 10-12 weeks old for WT HV68) were infected IP with 104 PFU in 200L of minimal essential media (MEM) or with blank MEM for uninfected controls. 33 2.2.2 PCR confirmation of infection Genomic DNA was isolated from 4x106 mouse splenocytes (Red blood cell (RBC)-lysed) using the PureLinkTM Genomic DNA Minikit (Invitrogen, #K1820), according to the manufacturer’s instructions, quantified using a Nanodrop and stored at -80oC. Infection of splenocytes at EAE endpoint was confirmed by nested polymerase chain reaction (PCR) for the ORF50 viral gene, using the primer sequences in Tables 2.1. The first PCR reaction contained 6 L of 10X DreamTaq Green Buffer (ThermoFisher, #B71), 0.5 M of both the outer forward and reverse primers, 0.2 mM of each dNTP (Thermofisher, #10297117 ), 0.6 U of DreamTaq DNA Polymerase (ThermoFisher, EP0702), 20 L of genomic DNA and up to 60 L of dH2O. Genomic DNA was substituted by dH2O for negative controls, and by a HV68 ORF50 gBlock for positive controls. PCR reactions were completed on a T100 ThermalCycler (BioRad) at the following cycling parameters: 2 min at 95oC, followed by 34X [30s at 95oC, 30s at 57.5oC, 1 min at 72oC], and 5 min at 72oC. For the second (nested) PCR reaction, 5L of the corresponding first PCR products was combined the same concentrations of products as the outer reaction, scaled down to a total reaction volume of 20L. Cycling parameters were 2 min at 95oC, followed by 25X [30s at 95oC, 30s at 58.6oC, 1 min at 72oC], and 5 min at 72oC. A quality control reaction for each genomic DNA sample was performed using 0.4 M per reaction of housekeeping glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene primers (Table 2.2). PCR reaction products (107bp band for ORF50 and 248 bp band for GAPDH) were electrophoresed through a 2% TAE-agarose gel stained with 1X SYBER Safe DNA gel stain alongside a 100bp DNA ladder. 34 Component Sequence (5’ to 3’) Outer forward primer ATC AGC ACG CCA TCA ACA TC Outer reverse primer TTA GTT GTG GTT GAA CGG CG Inner forward primer TTC CGC CAC AGA AGA GTT CC Inner reverse primer ATC CGC CCA TAG CTG AGT TG Table 2.1: Primer sequences for HV68 ORF50 PCR assay Component Sequence (5’ to 3’) Forward primer GTG TTC CTA CCC CCA ATG TGT Reverse primer ATT GTC ATA CCA GGA AAT GAG CTT Table 2.2: Primer sequences for GAPDH PCR assay 2.2.3 qPCR determination of viral load Isolated genomic DNA from 4x106 splenocytes (as above) was used to assess HV68 viral load. qPCR reactions of 20L comprised of 150-300ng genomic DNA, 0.4M of both forward and reverse primers, 0.2M probe, 0.1L ROX Dye Solution, and 1X QuantiNova Probe PCR master mix (Qiagen, #208254, Table 2.3). Standard curves were obtained by serial dilutions of HV68 ORF50 and mouse prostaglandin E receptor 2 (PTGER2) gBlocks (ORF50: 1x105-1x10-3; PTGER2: 5x107-5x100) amplified in parallel (Table 2.4). Standard curve and genomic DNA reactions were each amplified in duplicate. Cycling parameters were 2 min at 95oC, followed by 50X[5 sec at 95oC, 5 sec at 60oC] and reactions were completed in the QuantStudio 3 Real-Time PCR System (Applied Biosystems, #A28567). Sample copies per reaction were determined by interpolation of the standard curve using duplicate-average cycle threshold values (CT), which were then used to calculate viral load. 35 Component Sequence (5’ to 3’) Forward primer TGG ACT TTG ACA GCC CAG TA Reverse primer TCC CTT GAG GCA AAT GAT TC Probe /56-FAM/TGACAGTGC/ZEN/CTATGGCCAAGTCTTG/3 IABkFQ/ gBlock AAGACGCTTACCCAATACACAAGGACATTGAGTCC AGTAGATAAATCAAAGTCTGCAGCTGTTTCTAACTCT AGTGAATACTTTAAGAATGTTTTAAAGTTGTACATATAA GAAAAGTGTTTATTGTCCAACATAGAAGATATTTTTCT CAAGGCTTCCTCGTCTACAGAGGTCAGGAAACATG CACACCATTCATGTACAGGCTGATCTTTGTCATATGG GATAGACAAAATCTCTGGACTTTGACAGCCCAGTAA ATTGACAGTGCCTATGGCCAAGTCTTGTAACAAGTTT GAATCATTTGCCTCAAGGGATAGCTTCATGTAGTCGA GTATGCGCTGAAATTTGGAGGTAGTGGACAACCTCTC CAATAGTGTTAGGTGTCTGATAATTAGTTCCCTCAAGT GGGGCAACAAAAATGGCTCTGTATCCAAGTTGTGGC GCAGTGTCTGACAAATTTTACACAGCACCTGAAGCTT ACACAAACAGGCACTTACTCT Table 2.3: Sequences for HV68 ORF50 qPCR assay Component Sequence (5’ to 3’) Forward primer TAC CTT CAG CTG TAC GCC AC Reverse primer GCC AGG AGA ATG AGG TGG TC Probe /56-FAM/CCTGCTGCT/ZEN/TATCGTGGCTG/3IABkFQ/ gBlock ACAGGCGCCACTTATCGCGCCGCGGGGGTCTGGC GGTGCTGCCTGTCATCTATGGGGCCTCCTTGCTCTTC TGTTCCCTGCCGCTGCTCAACTACGGGGAGTACGTC CAGTACTGTCCTGGGACATGGTGCTTTATCCGGCAC GGGAGGACTGCATACCTTCAGCTGTACGCCACGATG CTCCTGCTGCTTATCGTGGCTGTGCTCGCCTGCAAC ATCAGCGTTATCCTCAACCTCATTCGCATGCACCGTC GGAGCAGAAGAAGCCGCTGCGGATTGTCTGGCAGT AGCCTAAGAGGCCCTGGATCTCGCAGGAGAGGAGA GAGGACTTCGATGGCAGAGGAGACGGACCACCTCAT TCTCCTGGCCATTATGACCATCACCTTCGCCATATGCT CCTTGCCTTTCACAGTAAGTCACCTTTGTTTCCACAGC CCCGCAAGCAGCCCGCGCAGCCTGCTCAAACTCCT CTCACCCCACCCTTTCCACCTTTAGGCACAG Table 2.4: Sequences for PTGER2 qPCR assay 36 2.3 Experimental autoimmune encephalomyelitis C57Bl/6 mice were actively induced with EAE on day 0 by subcutaneous (SQ) injection of 100 L complete Freund’s adjuvant containing 400 g desiccated M. Tuberculosis H37Ra (DIFCO, #231141) in incomplete Freund’s adjuvant (DIFCO, #263910) emulsified with 200 μg MOG35-55 peptide (GenScript, RP10245). On days 0 and 2 post-immunisation, mice received an intraperitoneal injection of 200 ng pertussis toxin in 200 L PBS (List Biological Laboratories, #179A). The mice were monitored and scored daily for EAE symptoms, based on the following 16-point scale: 0–2 for tail paralysis with 0 for no symptoms, 1 for partial loss of tail tone, and 2 for loss of tail tone (limp tail); 0–3 for each of the hindlimbs and forelimbs with 0 assigned for no symptoms, 1 for weakness or altered gait, 2 for paresis (limb slips through bars on the cage on almost every step) and 3 for full paralysis; 0–2 for righting reflex with 0 assigned for normal righting reflex, 1 for delayed righting, and 2 for loss of righting reflex. The scale therefore ranges from 0 (no symptoms) to 16 (fully quadriplegic mouse with limp tail and loss of righting reflex). EAE experiments were performed in a biosafety level 2 facility. Clinical outcome assessors of EAE mice were blinded to vaccination and infection groups. Onset day was defined by the occurrence of a score of at least 1 for two consecutive days. Cumulative EAE scores were calculated by summing daily clinical scores from the day of onset until endpoint. Mice exhibiting signs of illness and/or weight loss were provided supportive care including SQ fluids, cage heating, wet food and nutritional gel. Humane endpoint was defined by a clinical health score of greater than 4. For clinical score versus day post EAE induction curves, scores for mice that did not reach experimental endpoint due to reaching humane EAE endpoint were kept consistent for the days between humane and experimental endpoints. 37 2.4 Tissue collection 100 L of blood was collected via saphenous vein blood sampling 4-weeks post ACRTA-HV68 vaccination before WT-HV68 infection, and again at 5-weeks post WT-HV68 infection, before EAE induction. Blood was centrifuged (13 min 8000 rpm), serum was collected and stored at -80oC for further analysis. Mice were euthanized at experimental endpoint by isoflurane overdose. Blood was collected via cardiac puncture, followed by perfusion with 15mL sterile PBS. Brains, spinal cords and spleens were dissected whole and kept in cold, sterile PBS for further processing. The presence of intact CFA emulsion was confirmed by examining the skin at the injection site. Serum was extracted from untreated blood samples by centrifugation (as above). 2.5 Flow cytometric analysis 2.5.1 Sample processing Brains, spinal cords and spleens were kept in sterile PBS on ice, then immediately processed into single cell suspensions by passing the tissue through a 70-m cell strainer with a syringe insert. Splenocyte suspensions were incubated in red blood cell lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2-EDTA) for 10 min on ice. Samples (2x106 splenocytes, half isolated brain suspension, or total isolated CNS suspension) analyzed for cytokine expression were incubated at 37o for 4 hours in 200 L of stimulation media, containing 10% FBS, 10 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, #P1585), 500ng/mL ionomycin (Thermo Fisher, #I24222) and 1 μL/mL GolgiPlugTM Protein Transport Inhibitor (containing Brefeldin A, BD Biosciences, #555029). Cell suspensions were washed between each step with sterile PBS containing 2% FBS (FACS buffer). 38 2.5.2 Antibody and tetramer staining and analysis Cells were stained in 1X eBioscienceTM Fixable Viability Dye eFluorTM 506 (Thermo Fisher, 65-0866-14) for 20 min at 4°C in PBS. Cells were then washed in FACS buffer, and preincubated with mouse Fc block in FACS buffer for 10 min at RT. For detection of epitope specific CD8+ T cells, MHC class I tetramers, provided by the NIH Tetramer Core Facility at Emory Univeristy, were incubated with the cell suspensions for 1 hour at RT at a 1:400 or 1:300 dilution in FACS buffer (Table 2.5). Cells were washed again and incubated with fluorochrome- labelled antibodies specific to extracellular markers (Table 2.6) in FACS buffer for 30 minutes at 4°C. Cells were washed and then treated with transcription factor fixation and permeabilization reagent (Thermo Fisher, #00-5521-00) for 30 min at 4°C, followed by a wash with permeabilization buffer (Thermo fisher, #00-8333-56). Cells were incubated with fluorochrome-labelled antibodies specific to intracellular markers (Table 2.7) in permeabilization buffer for 30 min at RT. Cells were washed with permeabilization and FACS buffer and resuspended in FACS buffer with 2mM EDTA for acquisition. Stained cell suspensions were collected on an Attune NxT flow cytometer (Thermo Fisher) or CytoFLEX (Beckman Coulter) and analyzed using FlowJoTM v10.10 (BD Life Sciences). Antigen Peptide Sequence Allele Format [Stock] mg/mL [Staining] g/mL HV68 p56 AGPHNDMEI H-2Db APC 1.47 4.9 HV68 p79 TSINFVKI H-2Kb PE 1.33 3.7 Table 2.5: MHC Class I tetramers 39 Target Antigen Clone Source Identifier CD45 30-F11 eBioscience/ThermoFisher 47-0451-82 CD19 eBio1D3 (1D3) eBioscience/ThermoFisher 78-0193-82 CD3e eBio500A2 (500A2) eBioscience/ThermoFisher 48-0033-82 CD8a 53-6.7 eBioscience/ThermoFisher 63-0081-80 CD25 PC61.5 eBioscience/ThermoFisher 25-0251-82 CD4 RM4-5 eBioscience/ThermoFisher 12-0042-82 CD4 RM4-5 eBioscience/ThermoFisher 11-0042-85 CD11b M1/70 eBioscience/ThermoFisher 61-0112-82 CD44 IM7 eBioscience/ThermoFisher 67-0441-82 CD44 IM7 BioLegend 1033012 Fc receptor (CD16/CD32) 2.4G2 BD Biosciences 553142 Table 2.6: Antibodies for extracellular targets Target Antigen Clone Source Identifier FOXP3 FJK-16S eBioscience/ThermoFisher 11-5773-82 Granzyme B NGZB eBioscience/ThermoFisher 25-8898-80 IFN XMG1.2 eBioscience/ThermoFisher 45-7311-82 IL-17A eBio17B7 eBioscience/ThermoFisher 407-7177-82 Table 2.7: Antibodies for intracellular targets 2.6 Software and statistical analyses Graphical illustrations were created in BioRender. Flow cytometric, clinical and qPCR data were assembled in Microsoft Excel and then graphed and statistically analyzed using GraphPad Prism 10.1.1. Graphs present mean with standard error of the mean (SEM), unless otherwise stated in figure legend. For each group of data, the Kolmogorov-Smirnov normality test or Shapiro-Wilk test (for small N number) were used to assess normality. For data with two groups, normally distributed data was analyzed via unpaired t-tests with Welch’s correlation, and non-normally distributed data with Mann-Whitney tests. For data with 2 or more groups, normally distributed data was analyzed with Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple 40 comparisons tests and non-normally distributed data with Kruskal-Wallis with Dunn’s multiple comparisons tests. EAE clinical curves were analyzed via ordinary two-way ANOVA with Tukey’s multiple comparisons test, or by Welch’s t tests comparing the area under the curve (AUC) of each experimental group. Specific statistical tests and number of replicates per group are indicated in figure legends. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05. 41 CHAPTER 3: RESULTS 3.1 ACRTA HV68 vaccination reduces HV68 viral load in the spleen To confirm the ability of ACRTA HV68 vaccination to protect mice against WT HV68 challenge 130, mice were vaccinated intraperitoneally with 104 PFU of ACRTA HV68, or with a blank vehicle control. Four weeks later, mice were challenged with an IP injection of 104 PFU WT HV68 or a vehicle control and left for at least 5-weeks for the establishment of viral latency. At experimental endpoint, splenic DNA was extracted for analysis of viral presence via PCR and qPCR. Nested PCR for HV68 ORF50 demonstrates that, at administration route, dose and timeline used, ACRTA HV68 is not a sterilizing vaccine for HV68, as viral DNA is detected in the spleens of both vaccinated and unvaccinated, infected mice (Fig. 3.1A). However, the viral load of HV68 is significantly higher in the spleens of unvaccinated/HV68-infected mice compared to vaccinated mice, and vaccinated mice display no differences in viral load compared to naïve mice (Fig 3.1B-C). Therefore, vaccination with ACRTA HV68 produces non-sterilizing immunity against HV68. It is important to note that the viral loads observed in these experiments are lower than previously published work from our group and others 130,139. 42 Figure 3.1: WT HV68 viral load in mice unvaccinated mice and mice vaccinated with ACRTA HV68. Genomic DNA was extracted from 4x106 splenocytes to assess viral presence in the spleen of vaccinated and unvaccinated mice using PCR and qPCR. (A) PCR reactions to detect HV68 ORF50 (top) or mouse GAPDH control gene (bottom) in samples 15 days post EAE induction (11 weeks post ACRTA vaccination and 7 weeks post WT HV68 infection). Top lanes 4, 8, 16, 25 and bottom lanes 2,5,14,23 contain an 100bp DNA ladder. Top lanes 1-3 contain negative controls with water instead of DNA. Top lanes 5-7 and bottom lanes 3-4 contain uninfected/unvaccinated samples. Top lanes 9-15 and bottom lanes 6-12 contain vaccinated/infected samples. Top lanes 17-24 and bottom lanes 15-22 contain unvaccinated/infected samples. Lane 26 contains a positive control plasmid with the HV68 ORF50 gene. qPCR reactions detecting copies of (B) mouse PTGER2 and (C) HV68 ORF50 in 108 splenocytes 9-12 weeks post ACRTA HV68 vaccination and 5-8 weeks post WT HV68 infection. n = 8-12 per group. Data shown as mean with SEM and analyzed via Kruskal-Wallis with Dunn’s multiple comparisons tests. **p<0.01. 43 3.2 ACRTA HV68 vaccination attenuates HV68-driven clinical EAE enhancement To assess the impact of vaccination against HV68 prior to WT challenge on clinical EAE outcomes, mice from each of the vaccination/infection combination groups were actively induced with EAE and monitored daily for 10, 15, or 24 days, prior to experimental endpoint (Fig. 3.2). Vaccination against HV68 significantly attenuated the enhanced disease observed in the HV68-EAE model throughout all 3 clinical time courses assessed. Figure 3.2: ACRTA HV68 vaccination experimental overview. Mice were vaccinated IP with 104 PFU ACRTA HV68 or mock vaccinated with a vehicle control. After 4-weeks, serum was collected via saphenous bleeds, and mice were infected IP with 104 PFU WT HV68 or mock infected with a blank vehicle control, resulting in 3 experimental groups: vaccinated/infected, unvaccinated/infected, and unvaccinated/uninfected (naïve). After 5-weeks, serum was collected, and mice were actively induced with EAE and monitored for 10, 15 or 24 days, before experimental endpoint. Created using BioRender. 44 3.2.1 ACRTA HV68 vaccination ameliorates EAE outcomes during a 15-day clinical course Mice from each of the vaccination and infection groups were induced with EAE and monitored daily for 15-days. As previously established 136, mice infected latently with HV68 displayed an exacerbated EAE clinical course, as exemplified by higher clinical scores, compared to uninfected EAE mice (Fig. 3.3A). However, vaccination against HV68 prior to WT infection attenuated HV68-driven EAE enhancement (Fig. 3.3A). Overall, the disease course of vaccinated mice was milder compared to both groups of unvaccinated mice. Vaccinated mice had significantly lower clinical scores compared to HV68-infected mice on days 11-14 of the disease course. Clinical symptoms of EAE began significantly later for vaccinated mice compared to HV68-infected mice, with average EAE onset of 11 and 9 days, respectively (Fig. 3.3C). Vaccinated mice also displayed significantly reduced cumulative EAE scores compared to HV68-infected mice (Fig. 3.3D) and exhibited less weight loss throughout the experiment (Fig. 3.3B). EAE severity did not correlate with viral load for vaccinated or unvaccinated infected mice (Fig. 3.3E). Furthermore, no differences in clinical EAE outcomes were observed between male and female mice (Fig. 3.4A-B). 45 Figure 3.3: ACRTA HV68 vaccination attenuates HV68-driven EAE enhancement 15 d.p.i. (A) Clinical EAE scores over 15 days of EAE. Analyzed via two-way ANOVA with Tukey’s multiple comparisons tests. Asterisks indicate significant difference between vaccinated/infected and unvaccinated/uninfected mice. (B) Weight loss over 13 days of EAE. (C) Day of EAE onset post induction and (D) cumulative EAE scores, analyzed via Kruskal-Wallis with Dunn’s multiple comparisons tests. All clinical data reported as mean with SEM, n = 11-13 mice per group pooled from two independent experiments. (E) Correlation between copies HV68 and cumulative EAE score, analyzed by simple linear regression (solid line). n = 6-8 mice per group. **p<0.01, and *p<0.05. 46 Figure 3.4: HV68 vaccination driven attenuation of EAE is mouse sex independent. (A) Clinical EAE scores over 15 days of EAE, separated by sex. (B) Day of EAE onset post induction for female (F) and male (M) mice. 3.2.2 ACRTA HV68 vaccination ameliorates short-term and long-term EAE outcomes EAE outcomes were subsequently assessed over a clinical course extending beyond 15-days, spanning up to 24 days post induction (d.p.i.). Vaccinated mice displayed a significantly attenuated disease course over 24-days of EAE, had lower cumulative EAE scores, and showed clinical symptoms significantly later compared to unvaccinated, HV68-infected mice (Fig. 3.5A, C-D). From EAE day 17 to 24, clinical scores of mice from both unvaccinated groups remained stable, and the two groups presented with similar average scores each day. However, past 15 d.p.i, vaccinated mice retained milder clinical scores compared to both unvaccinated groups and displayed a clinical phenotype that resembled a mild relapsing and remitting pattern. Furthermore, 100% of vaccinated mice survived until experiment endpoint, however 50% of mice from both unvaccinated groups reached humane endpoint prior to the end of the experiment (Fig. 3.5B). 47 Figure 3.5: HV68 vaccination driven attenuation of EAE 24 d.p.i. (A) Clinical EAE scores over 24 days of EAE. Analyzed via Welch’s t tests comparing the area under the curve (AUC) of each experimental group. (B) Survival curves over 24 days of EAE, representative of mice that reached humane endpoint or survived until experimental endpoint. (C) Day of EAE onset post induction, analyzed by Kruskal-Wallis with Dunn’s multiple comparisons tests. (D) Cumulative EAE scores over 24 days. All data represent mean with SEM. n = 6-8 per group. *p<0.05. Additionally, analyses were conducted 10 d.p.i, since during the 15-day clinical course, the 3 experimental groups became clinically distinguishable at day 10. Ten-days post EAE induction, the vaccinated mice did not yet display any clinical EAE symptoms, while 2/3 and 1/2 of 48 unvaccinated/HV68-infected and naïve mice displayed symptoms, respectively (Fig. 3.6A, C). Vaccinated mice also exhibited less weight loss compared to HV68-EAE mice over ten days of EAE (Fig. 3.6B). Figure 3.6: HV68 vaccination driven attenuation of EAE 10 d.p.i. (A) Clinical EAE scores and (B) percent weight change over 10 days of EAE. (C) Cumulative EAE scores. 3.3 Characterization of immune infiltration and polarization of vaccinated EAE mice in the CNS and spleen To investigate immune infiltration and polarization of ACRTA HV68 vaccinated/WT challenged mice compared to unvaccinated/WT HV68 mice, the brain, spinal cord, and spleen were collected after perfusion and analyzed via flow cytometry at 10, 15, and 24 d.p.i (Appendix 1-4). 49 3.3.1 Vaccinated mice display reduced CNS immune infiltration 10 d.p.i. compared to unvaccinated HV68-infected mice Clinical analyses revealed 10 d.p.i. as an important time point when vaccinated and unvaccinated mice become clinically distinguishable. As such, immune infiltration into the CNS was analyzed at this time point. Overall, after 10 days of EAE, a significantly lower percentage, and lower counts, of CD45hi+ cells are observed in the spinal cords of vaccinated mice compared to unvaccinated/infected mice (Fig. 3.7A-C). In the brain, decreased percentages and counts of CD45hi+ cells are also observed for vaccinated mice compared to unvaccinated/infected mice (Fig. 3.7D-E) though these differences are not as pronounced as in the spinal cord. Figure 3.7: Vaccinated mice display reduced CNS immune infiltration 10 d.p.i. (A) Percentage and (B) total count of CD45hi+ cells in the spinal cord 10 d.p.i. Analyzed via Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparison tests. (C) Representative flow cytometric ploys plots indicating the mean percentage per group of all live cells in the spinal cord that are CD45hi+. (D) Percentage and (E) counts of CD45hi+ cells in the brain 10 d.p.i. Data represents mean with SEM. n = 6 mice per group. *p<0.05. 50 3.3.2 Vaccinated mice display reduced T cell infiltration and elevated ratios of CD8+ to CD4+ T cells in the CNS compared to unvaccinated mice during EAE To further characterize the immune response to ACRTA HV68 vaccination during EAE, proportions of CD3+ T cells in the spleen and CNS were measured at all EAE time points assessed (Fig. 3.8A). In the spinal cord, elevated proportions of T cells are observed for unvaccinated/HV68 infected mice compared to the other two experimental groups. At 10 and 15 d.p.i, vaccinated mice have a significantly lower abundance of T cells infiltrating the spinal cord compared to unvaccinated/HV68 infected mice (Fig. 3.8D,F). In the brain, an increased percentage of T cell infiltrates for unvaccinated/HV68 infected mice is observed at 15 d.p.i., but not at 10 d.p.i (Fig. 3.8E,G). Furthermore, 24 d.p.i, vaccinated mice display the highest proportion of CD3+ T cells in the brain compared to the two groups of unvaccinated mice. Vaccinated mice also display slightly decreased counts of CD3+ T cells in the brain 10 d.p.i. compared to unvaccinated/HV68 infected mice, though by 15 d.p.i., these two groups display comparable counts of T cells in the brain. Although CD4+ T cells are the primary T cell type infiltrating the CNS in classical models of EAE 55, previous data from our lab has demonstrated that in the presence of latent HV68, increased percentages of CD8+ T cells infiltrate the CNS compared to uninfected EAE mice 136. As such, the distribution of CD8+ and CD4+ T cells was assessed in the spleen and CNS for all experimental groups across different EAE time points (Fig. 3.9). At 10 and 15 d.p.i, there is a significantly higher ratio of CD8+ to CD4+ T cells in the spleen of unvaccinated/infected mice compared to the other two groups, while the latter two groups display no significant differences between their splenic CD8+:CD4+ T cell ratios (Fig. 3.10A, same trend observed for day 24). Interestingly, in the CNS 10 d.p.i., there is a higher ratio of CD8+:CD4+ T cells in the vaccinated 51 mice compared to both other experimental groups (Fig. 3.10B-C). By 15 d.p.i., a shift is observed wherein the CD8+:CD4+ T cell ratio is highest in the unvaccinated/infected group compared to the other two groups, in both the brain and the spinal cord. At 24 d.p.i., the CD8+:CD4+ T cell ratio is once again highest in the vaccinated/infected group compared to the other two groups, similar to what is seen on day 10. Changes in the CD8+:CD4+ T cell ratios are also reflected by the percentages (Tables 3.1-3.3) and counts (Fig. 3.10D,E) of CD8+ and CD4+ T cells in the different experimental groups at each of the EAE time points assessed. 52 Figure 3.8: Quantification of CD3+ T cells in the spleen and CNS of vaccinated and unvaccinated mice during EAE. Percentage of CD45(hi)+ cells that are CD3+ 10, 15 and 24 d.p.i. in the (A) spleen, (B) spinal cord, and (C) brain. Total counts of CD3+ T cells 10 d.p.i. in the (D) spinal cord, and (E) brain, analyzed via Kruskal-Wallis with Dunn’s multiple comparisons tests. Total counts of CD3+ T cells 15 d.p.i. in the (F) spinal cord, and (G) brain, analyzed via Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparison tests. Data shown as mean with SEM. n = 3-11 per group. **p<0.01, and *p<0.05. 53 Figure 3.9: CD4+ vs. CD8+ T cell gating 10 d.p.i. Representative flow cytometric plots for CD4+ vs. CD8+ cells in the spleen (top), spinal cord (middle) and brain (bottom) of (A) Vaccinated/Infected, (B) Unvaccinated/Infected, and (C) Unvaccinated/Uninfected mice. Populations were gated on live CD45+CD11b-CD3+ T cells. Mean percentage and standard deviation for each population is indicated. 54 Figure 3.10: CD4+ and CD8+ T cell counts and ratios for vaccinated and unvaccinated mice. Ratio of CD8+ to CD4+ T cells in the (A) spleens, (B) spinal cords, and (C) brains of vaccinated/infected, unvaccinated/infected, and naïve mice 10, 15 and 24 d.p.i. Counts of CD4+ and CD8+ T cells the (D) spinal cords, and (E) brains of mice 10 and 15 d.p.i. Analyzed via Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparison tests or via Kruskal-Wallis with Dunn’s multiple comparisons tests. n = 3-11 per group. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05. 55 Table 3.1: Spleen %CD4+ and %CD8+ of CD3+ T cells. Mean with standard deviation. Table 3.2: Spinal Cord %CD4+ and %CD8+ of CD3+ T cells. Mean with standard deviation. Table 3.3: Brain %CD4+ and %CD8+ of CD3+ T cells. Mean with standard deviation. 56 3.3.3 Analysis of T regulatory, Th1 and Th17 CD4+ T cells in the spleens and CNS of vaccinated and unvaccinated mice during EAE To further characterize the activity of CD4+ T cells in the CNS and spleen, staining for CD25+FOXP3+ Tregs, and T cells expressing CD44, IFN and IL-17A, was performed. Previous findings from our group have demonstrated that the frequencies of FOXP3+ Tregs are decreased in the spleens and CNS of HV68/EAE mice compared to both ACRTA HV68 infected and naïve EAE mice 137. Diminished CD4+CD25+ Treg activity has also been observed in splenocytes isolated from HV68 infected mice 140. In the spleen on 10 and 15 d.p.i., ACRTA HV68 vaccinated mice display significantly higher percentages of FOXP3+ Tregs compared to unvaccinated/HV68 infected mice, and comparable levels to naïve mice (Fig 3.11, Fig. 3.12A). In the CNS, vaccinated mice display slightly elevated levels of FOXP3+ Tregs compared to unvaccinated/HV68 infected mice over the three EAE timepoints assessed (Fig. 3.12B-C). In the spleen, activated CD25+FOXP3+ Tregs make up less than 0.5% of all CD4+T cells for all groups at all EAE time points assessed (Fig. 3.12D). In the spinal cord, 10 days post EAE induction, there are significantly higher levels of CD25+FOXP3+ Tregs in the unvaccinated/HV68 infected group compared to the vaccinated group (Fig. 3.12E). However, from 10 to 15 to 24 d.p.i., levels of CD25+FOXP3+ Tregs in the spinal cords of vaccinated mice increase in a stepwise manner, with significantly higher levels on day 24 than day 10. Similar to the spinal cord, unvaccinated/HV68 infected mice display elevated levels of CD25+FOXP3+ Tregs in the brain compared to the vaccinated mice 10 d.p.i. (Fig. 3.12F). Proportions of CD25+FOXP3+ Tregs increase significantly from 10 to 15 d.p.i. in the brains of vaccinated mice, though do not continue to increase from 15 to 24 d.p.i. 57 Figure 3.11: Gating for FOXP3+ and CD25+ CD4+ T cells in the CNS and spleens of vaccinated and unvaccinated mice. Representative flow cytometric plots gating of FOXP3+ vs. CD25+ cells in the spleen (top), spinal cord (middle) and brain (bottom) of (A) Vaccinated/Infected, (B) Unvaccinated/Infected, and (C) Unvaccinated/Uninfected mice. Populations were gated on live CD45+CD11b-CD3+CD4+ T cells. Mean percentage and standard deviation for each population is indicated. 58 Figure 3.12: T regulatory cells in the spleen and CNS of vaccinated and unvaccinated mice. Top: percentages of FOXP3+CD25- Tregs in the (A) spleen, (B) spinal cord, and (C) brain 10, 15 and 24 d.p.i. Bottom: percentages of FOXP3+CD25+ Tregs in the (D) spleen, (E) spinal cord, and (F) brain 10, 15 and 24 d.p.i. Analyzed via Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparison tests or via Kruskal-Wallis with Dunn’s multiple comparisons tests. Mean with SEM indicated. n = 3-11 per group. **p<0.01 and *p<0.05. 59 Due to the important role played by Th1 and Th17 cells in the immunopathogenesis of MS and EAE, these cell types were investigated in ACRTA HV68 vaccination. In the spleen, proportions of CD4+ T cells that produce IFN (Th1 cells) are not significantly different between experimental groups, however levels are elevated for all groups on days 10 and 15 post EAE induction compared to day 24 (Fig. 3.13A, D). In the CNS, elevated percentages of Th1 cells are observed for all experimental groups 15 d.p.i. compared to days 10 and 24, reflecting the elevated clinical EAE scores at day 15 (Fig. 3.13B-C). Ten days post EAE induction, vaccinated mice display a significantly fewer abundance of Th1 cells in the spinal cord compared to unvaccinated/HV68 infected mice (Fig. 3.13E), whereas in the brain, no significant differences are observed between groups (Fig. 3.13F). By 15 d.p.i., the significant difference between counts of Th1 cells in the spinal cords is no longer observed, as counts increased by 400-fold and 20-fold for the vaccinated/infected and unvaccinated/infected groups, respectively. The abundance of Th1 cells in the brain also increases from 10 to 15 d.p.i. for all experimental groups. 60 Figure 3.13: IFN+CD4+ Th1 cells in the spleen and CNS of vaccinated and unvaccinated mice. Top: percentage of CD4+ T cells that produce IFN in the (A) spleen, (B) spinal cord, and (C) brain 10, 15 and 24 d.p.i. Bottom: total IFN+CD4+T cells counts in the (D) spleen, (E) spinal cord, and (F) brain 10, 15 and 24 d.p.i. Analyzed via Kruskal-Wallis with Dunn’s multiple comparisons tests. Mean with SEM indicated. n = 3-11 per group. **p<0.01 and *p<0.05. 61 Additionally, in the spleen, proportions of Th17 cells are significantly higher for vaccinated mice compared to unvaccinated mice 10 d.p.i, however the percentage of CD4+ T cells that produce IL-17A decreases by 15 and 24 d.p.i. for all groups (Fig. 3.14A). The total abundance of Th17 cells in the spleen follows a similar trend (Fig. 3.14D). In the spinal cord, the percentage of Th17 cells is slightly elevated for unvaccinated/infected mice compared to infected mice at all 3 EAE timepoints assessed, and the same trend is observed in the brain 15 and 24 d.p.i. (Fig. 3.14B-C). Ten days post EAE induction, there is a significantly lower total Th17 cell count in the spleen of vaccinated mice compared to unvaccinated mice, though all groups display a similar abundance of Th17 cells in the spinal cord by 15 d.p.i (Fig. 3.14E). In the brain, total counts of Th17 cells does not differ between groups (Fig. 3.14F). In the spleen, the proportional number of effector IFN+CD4+ Th1 cells compared to Tregs is comparable between the three experimental groups, at the different EAE timepoints assessed (Fig. 3.215A). In the spinal cord, the ratio of effector Th1 cells to Tregs is mildly elevated for the unvaccinated/infected mice compared to the vaccinated mice, reflecting the slightly elevated abundance of Th1 cells and reduced frequency of FOXP3+ Tregs seen across EAE timepoints for the unvaccinated/infected mice (Fig. 3.15B). In the brain, the ratio of Th1 to Treg cells is somewhat higher for the vaccinated mice compared to the other two groups on day 10 post EAE induction (Fig. 3.15C). However, by days 15 and 24, the Th1 to Treg ratios are more comparable between groups, with the unvaccinated/infected mice displaying slightly higher ratios compared to the vaccinated mice 15 d.p.i. 62 Figure 3.14: IL-17A+CD4+ Th17 cells in the spleen and CNS of vaccinated and unvaccinated mice. Top: percentage of CD4+ T cells that produce IL-17A in the (A) spleen, (B) spinal cord, and (C) brain 10, 15 and 24 d.p.i. Bottom: total IL-17A+CD4+T cells counts in the (D) spleen, (E) spinal cord, and (F) brain 10, 15 and 24 d.p.i. Analyzed via Kruskal-Wallis with Dunn’s multiple comparisons tests. Mean with SEM indicated. n = 3-11 per group. **p<0.01 and *p<0.05. 63 Figure 3.15: Ratio of Th1 to T regulatory cells in the spleen and CNS for vaccinated and unvaccinated mice. Ratio of IFN+CD4+ T cells to FOXP3+CD4+ T cells in the (A) spleen, (B), spinal cord, and (C) brain for vaccinated/infected, unvaccinated/infected and unvaccinated/uninfected mice 10, 15 and 24 d.p.i. Samples with total count of 0 cells for Th1 or Treg populations were excluded. n = 2-11 per group. Mean with SEM indicated. 3.3.4 Analysis of IFN and granzyme B production of CD8+ T cells in the spleens and CNS of vaccinated and unvaccinated mice during EAE EAE mice latently infected with HV68 typically present with higher levels of CD8+Granzyme B+ T cells in both the brain and the spinal cord compared to uninfected mice 136. As such, we sought to determine whether ACTRA HV68 vaccination protects EAE mice from the HV68-associated increase in cytotoxic CD8+ T cells in the CNS. CD8+ T cells were assessed for production of IFN- and granzyme B. In the spleen, percentages of CD8+ T cells that produce IFN (Tc1 cells) are lowest 10 days post EAE induction for all three experimental groups compared to the respective percentages on 64 days 15 and 24 (Fig. 3.16A). Counts of Tc1 cells in the spleen demonstrate that 10 d.p.i, unvaccinated/infected mice have the highest abundance of Tc1 cells compared to the other two experimental groups (Fig. 3.16D). However, the inverse is true for 15 d.p.i, wherein the unvaccinated/infected group displays the lowest counts of splenic Tc1 cells compared to the other experimental groups. Interestingly, 10, 15 and 25 d.p.i., percentages of Tc1 cells in the brain trend towards being higher in the vaccinated group compared to the two unvaccinated groups (Fig. 3.16D). However, the number of Tc1 cells in the brains of vaccinated mice remains consistent from 10 to 15 d.p.i. (Fig. 3.16F). In contrast, the abundance of Tc1 cells infiltrating the brains of both groups of unvaccinated mice significantly increases between 10 and 15 d.p.i. In the spinal cord, the percentage of CD8+ cells that produce IFN is mildly higher for the vaccinated mice compared to the other groups at all three EAE timepoints assessed, however this trend is not as pronounced in the spinal cord compared to the brain (Fig. 3.16B). Furthermore, the abundance of Tc1 cells significantly increases from 10 to 15 d.p.i. for all experimental groups (Fig. 3.16E). 65 Figure 3.16: IFN+CD8+ Tc1 cells in the spleen and CNS of vaccinated and unvaccinated mice. Top: percentage of CD8+ T cells that produce IFN in the (A) spleen, (B) spinal cord, and (C) brain 10, 15 and 24 d.p.i. Bottom: total IFN+CD8+T cells counts in the (D) spleen, (E) spinal cord, and (F) brain 10, 15 and 24 d.p.i. Analyzed via Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparison tests or via Kruskal-Wallis with Dunn’s multiple comparisons tests. Mean with SEM indicated. n = 3-11 per group. ***p<0.001, **p<0.01, and *p<0.05. 66 In the spleen, the ratio of Tc1 cells to Treg cells is comparable between experimental groups (Fig. 3.17A). In the CNS on EAE day 10, the ratio of Tc1 to Treg cells is highest for the vaccinated group, particularly in the brain, which reflects the increased abundance of Tc1 cells in the brains of vaccinated mice 10 days post EAE induction (Fig. 3.17B-C). However, 15 d.p.i., the Tc1 to Treg ratio is highest for unvaccinated/infected mice in the CNS. Figure 3.17: Ratio of Tc1 to T regulatory cells in the spleen and CNS for vaccinated and unvaccinated mice. Ratio of IFN+CD8+ T cells to FOXP3+CD4+ T cells in the (A) spleen, (B), spinal cord, and (C) brain for vaccinated/infected, unvaccinated/infected and unvaccinated/uninfected mice 10, 15 and 24 d.p.i. Samples with total count of 0 cells for Tc1 or Treg populations were excluded. n = 1-11 per group. Mean with SEM indicated. 67 Production of granzyme B by CD8+ T cells in the spleen and CNS was assessed 10 days post EAE induction. Overall, increased counts of CD8+Granzyme B+ are observed in the spleen and CNS of unvaccinated/infected mice compared to vaccinated mice, significantly so in the spinal cord (Fig. 3.18A). This increased abundance of granzyme B producing CD8+ T cells is not reflected by the percentage of CD8+ T cells that produce granzyme B, which is not statistically significant between experimental groups in any of the organs investigated (Fig. 3.18B). Figure 3.18: CD8+ T cell Granzyme B production in the spleen and CNS of vaccinated and unvaccinated mice 10 days post EAE induction. (A) Total cell counts and (B) percentage of Granzyme B producing CD8+ T cells in the spleen, brain and spinal cord of vaccinated/infected, unvaccinated/infected and unvaccinated/uninfected mice 10 d.p.i. Analyzed via Kruskal-Wallis with Dunn’s multiple comparisons tests. Mean with SEM indicated. n = 6 per group. *p<0.05. 68 3.3.5 Quantification of HV68-specific CD8+ T cells in the spleens and brains of vaccinated and unvaccinated mice before and during EAE The response of CD8+ T cells to HV68 antigens (expressed by ORF6 and ORF61) were measured in a subset of spleen and brain samples by staining with H-2b MHC I tetramers. Previous analyses have revealed that T cell responses to epitopes derived from ORF61 and ORF6 progress with distinct kinetics 141. CD8+ T cells specific for the product of ORF61 (ribonucleotide-reductase large subunit protein) are detected by the p79 tetramer, expand in early latency and continue to circulate at high levels throughout the latent infection period 141. CD8+ T cells specific for the product of ORF6 (single stranded DNA binding protein) are detected by the p56 tetramer and predominate early in infection 141. It has also been established that although levels of p56+CD8+ T cells are similar over the course of infection for WT and ACRTA HV68 infections, there is a significantly higher abundance of p79+CD8+ T cells later in infection in WT compared to ACRTA HV68 infection 141. It is important to note that in this work, tetramer staining was performed only during the latent stage of infection, at least 9-weeks and 5-weeks post ACRTA and WT HV68 infection, respectively. 69 70 Figure 3.19: HV68-specific CD8+ T cells in the spleen and brain of vaccinated and unvaccinated mice. Percent of CD8+ T cells that are (A) p79+CD44+, or (B) p56+CD44+ in the spleens of vaccinated/infected, unvaccinated/infected, and unvaccinated/uninfected mice before EAE induction, and 10, 15 and 24 d.p.i. Total counts of CD8+ (C) p79+CD44+ and (D) p56+CD44+ T cells in the spleens of vaccinated/infected and unvaccinated/infected mice before EAE induction, and 10 and 15 d.p.i. Percent of CD8+ T cells that are CD8+ T cells that are (E) p79+CD44+, or (F) p56+CD44+ in the brains of vaccinated/infected, unvaccinated/infected, and unvaccinated/uninfected mice 10 and 24 d.p.i. Analyzed via Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparison tests or via Kruskal-Wallis with Dunn’s multiple comparisons tests. Mean with SEM indicated. n = 3-11 per group. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05. Prior to the induction of EAE, the spleens of vaccinated mice display elevated percentages of CD8+ T cells specific to HV68 compared to unvaccinated/infected mice, and this difference is more pronounced for p56+CD8+ T cells compared to p79+CD8+ cells (Fig 3.19A-B). Following EAE induction, percentages of p79+CD8+T cells remain moderately higher in the spleens of vaccinated mice compared to unvaccinated/infected mice. However, the total count of p79+CD44+CD8+ T cell begins higher in the spleens of unvaccinated/infected mice compared to vaccinated mice prior to EAE induction, and this remains true at 10 d.p.i. (Fig 3.19C). By 15 d.p.i., there is an expansion in the abundance of p79+CD44+CD8+ T cells in the spleens of vaccinated mice and a reduction in counts for unvaccinated/infected mice. Counts of splenic p56+CD44+CD8+ T cells begins higher for vaccinated mice than unvaccinated/infected mice prior to EAE induction, however counts are reduced for both groups by 10 and 15 d.p.i. (Fig 3.19C). CD8+ T cells specific to HV68 are also observed in the brains of vaccinated and unvaccinated infected mice 10 and 24 d.p.i. (Fig. 3.19E-F). It should be noted that overall, the p79 tetramer binds with more specifity than the p56 tetrameter, as more non-specific binding from T cells of unvaccinated/uninfected mice is observed for the p56 tetramer in the spleen and brain samples. 71 3.3.6 Vaccinated mice display increased levels of NK cells in the spleen 10 d.p.i compared to unvaccinated mice In both humans and mice, NK cells are activated following gammaherpesvirus infection, although a dominant role of NK in the control of gammaherpesvirus infection has not been found 142. Depletion of NK cells in mice results in an increased severity and relapsing pattern of EAE in C57Bl/6 mice, suggesting a regulatory role of NK cells in EAE 143. NK cells have further been shown to exert a direct cytotoxic effect on autoantigen-specific, encephalitogenic T cells in EAE mice 144. As such, the potential contribution of NK cells to the attenuation of EAE in vaccinated mice was assessed. The stages of murine NK cell development are characterized by differential expression of surface markers 145. For instance, in the immature NK (iNK) stage, NK cells express receptors including NK1.1, while expression of CD49b defines the initial stage of mature NK (mNK) cell development 145. As such, iNK and mNK cell populations were assessed using NK1.1 and CD49b (Fig. 3.20A). The spleens of unvaccinated/infected mice display significantly fewer percentages of both iNK and mNK cells 10-days post EAE induction compared to vaccinated mice (Fig. 3.20B-C). However, unvaccinated/infected mice display a slightly elevated abundance of splenic NK cells 10 d.p.i. compared to both other experimental groups (Fig. 3.20D-E). 72 Figure 3.20: NK cells in the spleens of vaccinated and unvaccinated mice 10 d.p.i. (A) Representative flow cytometric gating strategy for identification of NK cells in the spleen. Percentage of all CD45+ cells that are (B) NK1.1+CD49+ mNK cells and (C) NK1.1+CD49- iNK cells. Total counts of (D) NK1.1+CD49b+ and (E) NK1.1+CD49b- cells. Analyzed via Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparison tests or via Kruskal-Wallis with Dunn’s multiple comparisons tests. Mean with SEM indicated. n = 6 per group. **p<0.01, and *p<0.05. 73 Functional NK cell maturation can further be defined by the differential surface expression of CD27 and CD11b145. Specifically, maturation occurs in a 4-stage process, starting with CD11blowCD27low, and continuing to CD11blowCD27high, then CD11bhighCD27high, and finally CD11bhighCD27low 146. However, 10 days post EAE induction, no significant differences are observed in percentages of functional NK cell subsets between the three experimental groups (Table 3.4). The vast majority of mNK cells are of the CD11bhighCD27low subtype, which are considered the most mature NK cells 145. Table 3.4: Functional NK cell subtypes in the spleen 10 dp.i. Mean and standard deviation of percentages of functional CD11b ±CD27 ± NK cells in the spleens of vaccinated/infected, unvaccinated/infected and unvaccinated/uninfected mice 10 d.p.i. n = 6 per group. In healthy mice, NK cells make up a small fraction (1.1±0.14%) of total immune cells in the CNS compartments, and generally reside in the boundaries such as the meninges and choroid plexus 147. During EAE, NK cells are mobilized from the periphery and accumulate in the inflamed CNS 148,149. Overall, no significant differences in percentages or counts of iNK or mNK cells are observed in the brains of vaccinated or unvaccinated EAE mice (Fig. 3.21A-E). Naïve-EAE mice demonstrate mildly elevated percentages of both iNK and mNK cells compared to infected mice, however this is not reflected by the total abundance of NK cells. ACRTA γHV68 Vaccinated and WT γHV68 infected Mock vaccinated and WT γHV68 infected Mock vaccinated and mock infected %CD11bloCD27lo 6.87 ± 1.2 8.59 ± 3.2 6.23 ± 2.3 %CD11bloCD27hi 3.46 ± 0.67 4.04 ± 1.6 2.62 ± 1.3 %CD11bhiCD27lo 82.5 ± 3.7 80.67 ± 4.0 86.7 ± 5.5 %CD11bhiCD27hi 7.14 ± 2.6 6.71 ± 0.85 5.13 ± 2.6 74 Figure 3.21: NK cells in the brains of vaccinated and unvaccinated mice 10 d.p.i. (A) Representative flow cytometric gating strategy for identification of NK cells in the brain. Percentage of all CD45hi+ cells that are (B) NK1.1+CD49+ mNK cells and (C) NK1.1+CD49- iNK cells. Total counts of (D) NK1.1+CD49b+ and (E) NK1.1+CD49b- cells. Analyzed via Kruskal-Wallis with Dunn’s multiple comparisons tests. Mean with SEM indicated. n = 6 per group. 75 CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS 4.1 Summary and significance of findings The growing body of evidence implicating EBV as a causal factor in MS has sparked interest in the potential of EBV-targeting therapies to treat the disease. One such approach is by preventing or controlling primary EBV infecting using a prophylactic vaccine. Assessing the potential of EBV vaccination to prevent MS is challenging due to the human-tropic nature of the virus, and the lack of an approved EBV vaccine. However, these challenges can be overcome using animal models of EBV and MS such as ACRTA and WT HV68, and EAE. The results of this work demonstrate that non-sterilizing vaccination against HV68 significantly attenuates viral enhancement of EAE. Vaccinated mice display reduced clinical EAE scores and a significant delay in time before EAE onset compared to unvaccinated mice. In the spinal cord, early in the course of EAE, vaccinated mice display significantly lower amounts of infiltrating CD45hi+ immune cells, and reduced abundances of CD3+ T cells, Th1 and Th17 cells compared to unvaccinated/infected mice. Vaccinated mice also have significantly higher proportions of NK cells, which are believed to play a regulatory role in EAE. Interestingly, in the brain, vaccinated mice tend to display higher amounts of Tc1 cells compared to unvaccinated mice. Viral DNA is detectable via PCR in the spleens of vaccinated mice 10-12 weeks post ACRTA HV68 vaccination and 6-9 weeks post WT HV68 infection. It is known that ACRTA HV68 is no longer detectable in the spleens of mice 2-weeks post infection 130, and as such, it is unlikely that the viral DNA detected in the spleens of vaccinated mice via PCR 10-12 weeks post vaccination is from ACRTA HV68. Therefore, it is probable that at the dose, route of administration and timeline used, ACRTA HV68 is a non-sterilizing vaccine. Importantly, qPCR analysis revealed that the viral load of the latent infection in vaccinated mice is significantly 76 reduced compared to unvaccinated mice. The copy numbers of HV68 DNA detected via qPCR from splenocyte samples in this work are low compared to previously published work. However, the statistically significant difference between the HV68-infected and naïve mice as well as the comparable loads of the PTGER2 gene between groups supports the validity of the results. Furthermore, dilutions of 2x10-3 copies of HV68 DNA were detectable from an ORF50 gene fragment sequence for the purpose of the standard curve analysis, highlighting the sensitivity of the reaction. As hypothesized, vaccinated mice with reduced HV68 viral loads presented with a milder course of EAE compared to the unvaccinated/HV68-infected mice that displayed HV68-driven EAE enhancement 136. Interestingly, vaccinated mice also consistently presented with improved EAE outcomes compared to naïve-EAE mice. Possibly the most striking difference in the immunological outcomes between vaccinated and both groups of unvaccinated mice was the increase in the CD8+:CD4+ T cell ratio for the vaccinated mice in the CNS, predominantly 10 d.p.i. Although the intuitive function of CD8+ T cells in MS and EAE is that of pathogenesis, several studies have also demonstrated that myelin-specific CD8+ T cells can attenuate both actively induced and adoptive transfer-mediated EAE 150,151. Transfer of MOG-induced CD8+ T cells into mice with EAE seems to predominantly ameliorate the chronic phase of disease as opposed to disease onset 152. Furthermore, one study demonstrated that the disease-ameliorating function of CD8+ T cells requires the production of IFN, as protective function was lost when IFN-deficient MOG-induced CD8+ T cells were transferred before EAE induction, but enhanced when IFN-production was stimulated in MOG-induced cells 151. The role of IFN in both MS and EAE has been extensively studied, and is the subject of an intriguing paradox 152. While several studies attribute this cytokine to a predominantly pathogenic and proinflammatory role in disease, 77 accumulating evidence suggests that IFN may play a protective role, and induce regulatory activity to modulate the effector T cell response 152. The complex role of IFN is thought to be disease-stage and cell type specific. In the context of CD8+ T cells, several mechanisms have been proposed for disease amelioration via IFN production including alteration of indoleamine 2, 3-dioxygenase (IDO) production, increasing DC regulatory capacity, modulation of MHC class I expression on APCs and conversion of effector CD4+ T cells into Tregs 151. In addition to the increased CD8+:CD4+ ratio in the CNS 10 and 24 d.p.i. in vaccinated mice, IFN-producing CD8+ T cells infiltrate earlier and at a greater abundance in the brains of the vaccinated mice compared to unvaccinated mice. Given the possible IFN-dependent protective function of CD8+ T cells in EAE, it is possible that the increased levels of Tc1 cells in the CNS of vaccinated mice are contributing to the ameliorated clinical outcomes. Although previous data highlights a role for protective Tc1 cells during the chronic, but not acute, phase of EAE, Tc1 cells from vaccinated mice may be able to exert protective functions earlier because of previous stimulation as a result of the ACRTA HV68 vaccination, and subsequent WT HV68 infection. Aside from IFN-production, CD8+ T cells exerting a regulatory function can be distinguished by several immune markers, including CD8+CD25+, CD8+CD28+, CD8+CD122+, CD8+CD75s+ and CD cells150. CD8+CD25+ could not be detected in these experiments, however, future experiments should investigate the other markers of regulatory CD8+ T cells. Beyond EAE, an immune regulatory role for autoreactive CNS-specific CD8+ T cells has also been shown in MS, wherein suppressive activity of CD8+ T cells is greatly diminished during relapses and recovered as patients enter remission 153. Intriguingly, the elevated CD8+:CD4+ ratio is observed for vaccinated mice at 10 and 24 d.p.i., but not 15 d.pi. This distinction may potentially be explained by the clinical presentation of 78 vaccinated mice at each of the EAE time points assessed. Days 10 and 24 post EAE induction represent time points when clinical scores of vaccinated mice are increasing, however, 15 d.p.i., the vaccinated mice appear to be experiencing a mild clinical remission. Beyond decreasing clinical scores, relapsing remitting (RR)-EAE is also associated with decreased lesion burden, myelin loss and axonal damage compared to chronic (CH)-EAE in C57Bl/6 mice 154. Future experiments should assess these outcomes to determine whether vaccinated mice are undergoing true RR-EAE during an extended clinical course of EAE. Vaccinated mice display increased levels of NK cells in the spleen 10 d.p.i. compared to unvaccinated/infected mice, and similar levels to naïve mice. Given the ability of NK cells to act in a regulatory manner during EAE, the increase in NK cells in vaccinated mice may be contributing to the ameliorated EAE outcomes of vaccinated mice. However, future experiments should continue assessing functional differences in NK cells between vaccinated and naïve mice to establish any differences in regulatory capacity. 4.2 Limitations and future directions This work demonstrates the potential for vaccination against EBV to prevent or ameliorate MS and highlights the urgency and necessity for the development of an EBV vaccine. While this work shows promise in a murine model, translation to human EBV and MS presents several important considerations, including timing of vaccine delivery to prevent waning immunity, and the feasibility of clinical trials to establish a protective role of an EBV vaccine in MS development. These considerations can be difficult to assess in an animal model given the human-specific tropism of EBV. An attractive solution is the use of humanized mice to allow for direct testing of EBV vaccines. The ability of an EBV vaccine to attenuate EAE in humanized mice may be evaluated in the future using our group’s humanized mouse model for EBV and MS 155. 79 Furthermore, in the C57Bl/6 mouse HV68/EAE model, different timelines, administration routes and doses of vaccination should be assessed. In this work, vaccination and infection were only administrated intraperitoneally at 104 PFU, which resulted in non-sterilizing immunity. Future experiments should investigate vaccination plans that may yield better immunity, such as lengthening the period of time between vaccination and challenge, using a booster dose of the vaccine, or performing the WT HV68 challenge intranasally at a lower dose. When mice were assessed up to 24 days post EAE induction, a mild relapsing-remitting phenotype arose for the vaccinated mice, but not for the unvaccinated mice. However, without the evaluation of markers such as myelin loss and axonal degeneration, it is difficult to determine whether vaccinated mice underwent a true period of remission. As such, future experiments should assess the extent of myelin damage through histological analyses, as well as measuring biomarkers of axonal degeneration such as neurofilament light chain. Furthermore, assessing vaccinated mice along a clinical course longer than 24 days warrants further investigation. While infiltration of HV68-specific CD8+ T cells was assessed in the brain, this cell population was not evaluated in the spinal cord and should be a focus of future research. Additionally, studies demonstrating a protective role for CD8+ T cells in EAE have done so specifically for MOG-specific CD8+ T cells. However, in this work, the specificity of CD8+ towards MOG was not evaluated. Therefore, future investigations should aim to quantify the MOG-specificity of CD8+ T cells in the CNS of both vaccinated and unvaccinated mice during EAE and explore the protective capacity of these cells using markers indicative of regulatory CD8+ T cells. When using a live virus such as ACRTA HV68 as a vaccine, it is important to consider that aside from protecting mice from WT HV68 challenge, the acute infection itself plays a role 80 in the immune environment of vaccinated mice. For example, the acute ACRTA HV68 infection may stimulate CD8+ T cells that eventually play a potential role in the attenuation of EAE. As such, future investigation should work to distinguish the role of the acute ACRTA infection itself from the role of the virus as a vaccine. For example, a low dose infection of LCMV does not establish viral latency but does induce a Th1 and memory CD8+ T cell response and should be used in the future as a control for the acute infection caused by ACRTA HV68 prior to WT HV68 challenge. The increasing incidence of MS coupled with the lack of a cure for this debilitating disease underscores the vital need for the development of preventative interventions. While the murine model of HV68 and EAE cannot capture the clinical heterogeneities and immunological complexities of MS and EBV, this research highlights the potential of an EBV vaccine to prevent MS development, which may ultimately lay the groundwork for the use of vaccination to prevent other EBV-related diseases. This work underscores the urgent need for the development of an effective EBV vaccine. 81 REFERENCES 1. Walton, C. et al. Rising prevalence of multiple sclerosis worldwide: Insights from the Atlas of MS, third edition. Mult Scler 26, 1816–1821 (2020). 2. 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Thesis/Dissertation
eng
Microbiology and Immunology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Horwitz, Marc S.
Vaccination against gammaherpesvirus attenuates viral enhanced disease in a murine model of multiple sclerosis
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