The Innate and Adaptive Immune Response to Measles Virus ?· i The Innate and Adaptive Immune Response…

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<p> i </p> <p>The Innate and Adaptive Immune Response </p> <p>to Measles Virus </p> <p> By: </p> <p>Nicole Putnam </p> <p>A thesis submitted to Johns Hopkins University in conformity </p> <p>with the requirements for the degree of Master of Science. </p> <p>Baltimore, Maryland </p> <p>April 2014 </p> <p> Nicole Putnam </p> <p>All Rights Reserved </p> <p> ii </p> <p>Abstract </p> <p>Measles is one of the most important causes of childhood morbidity </p> <p>and mortality worldwide. Although a vaccine is available, the high </p> <p>transmission rate of measles virus requires population of 95% to interrupt its </p> <p>transmission. The World Health Organization and the United Nations </p> <p>Childrens Fund recommend that children that develop measles receive </p> <p>vitamin A supplementation, as a safe, cheap, and efficacious way to reduce </p> <p>the burden of disease. Due to differences between strains and confounding </p> <p>data of measles stocks contaminated with defective interfering RNA </p> <p>particles, the immune response to measles virus infection has not been well </p> <p>defined. Furthermore, the mechanism by which vitamin A protects against </p> <p>severe measles-induced disease is unknown. </p> <p>In this thesis, I investigate the innate and adaptive immune response </p> <p>to measles virus infection. Measles virus strains were purified of defective </p> <p>interfering RNA particles and used for in vitro infections of monocyte derived </p> <p>dendritic cells. Gene expression changes of interferon-stimulated genes and </p> <p>viral stress-induced genes, IFIT1 and Mx1, were upregulated in response to </p> <p>infection with the Edmonston measles virus vaccine strain, as well as the </p> <p>wild-type strains of Bilthoven, IC-B, and C- and V-protein knockout strains, </p> <p>as compared to mock infected cells. Unexpectedly, there were no differences </p> <p>between transcript levels of these genes between C and V protein knockout </p> <p>strains and the respective wild-type infection. Additionally, the absence of </p> <p> iii </p> <p>type I interferon production supports the theory that measles virus induces </p> <p>the transcription of these genes through the viral stress-induced pathway, </p> <p>and not the interferon-stimulated pathway. </p> <p>While a previous study had detected measles virus-specific IL-17-</p> <p>producing T cells in measles virus-infected rhesus macaques, the Th17 </p> <p>response to measles virus has not been characterized. Th17 cell </p> <p>differentiation was inhibited early after measles virus infection in vitro. </p> <p>There was a significant decrease in IL-23A transcript ts and a significant </p> <p>increase in IL-27 transcripts, both of which affect Th17 cell differentiation </p> <p>negatively. However, in a rhesus macaque model of infection, a biphasic Th17 </p> <p>response was observed with peaks at days 18 and 56. </p> <p>The effects of vitamin A supplementation following measles virus </p> <p>infection on the immune response was explored in a rhesus macaque model </p> <p>using supplemented and non-supplemented groups. While some data has yet </p> <p>to be explored, major differences were not observed between the two groups </p> <p>up to three months following infection, in regards to clearance of infectious </p> <p>virus, immune cell composition, or immune cell function. Archived data will </p> <p>elucidate the role of vitamin A in measles virus RNA persistence, and Th1 </p> <p>and T follicular helper cell responses. Data will continue to be analyzed out to </p> <p>six months post infection. A larger cohort will be necessary to elucidate the </p> <p>role of vitamin A in protection against severe disease and death due to </p> <p>measles. </p> <p> iv </p> <p>Acknowledgements </p> <p> First and foremost, I would like thank my advisor, Dr. Diane E. </p> <p>Griffin, for allowing me to do my masters research in her laboratory. Her </p> <p>guidance and support was invaluable throughout my time here. I would like </p> <p>to thank her for the opportunity to get involved in the dynamic, challenging, </p> <p>and rewarding research that she had entrusted with me. Furthermore, a </p> <p>huge thank you to Dr. Rupak Shivakoti for passing down his knowledge of </p> <p>the basics of how to work with measles virus and acquainting me with Dr. </p> <p>Griffins lab in general. Additional thanks go out to Rupak to teaching me </p> <p>many, many techniques. Although he was available to ask questions while he </p> <p>was here, it was helpful that he encouraged me to jump right in and </p> <p>conduct my experiments independently early on. I would like to also thank </p> <p>Rupak for being responsive to questions much after he had graduated from </p> <p>the laboratory, which was especially helpful. </p> <p> I would like to thank Dr. Wendy Lin, for providing me with her </p> <p>knowledge of the logistics of working with measles virus in rhesus macaques. </p> <p>Her ability to pass down her understandings and techniques was invaluable. </p> <p>Furthermore, I would like to thank Wendy for taking time from her career at </p> <p>Columbia University to come down to Baltimore to meet with us, as well as </p> <p>making herself available to talk about techniques or data analysis. </p> <p>Importantly, this project would not have run as smoothly as it did without </p> <p>the help of Ashley Nelson, the PhD student with whom I shared </p> <p> v </p> <p>responsibility in this project. With Ashleys flexibility to work around my </p> <p>schedule, we were able to make sure the assays for the monkey study could </p> <p>be completed and analyzed in a timely manner so I could complete my thesis </p> <p>work. I would also like to thank Ashley for her support and friendship </p> <p>throughout my time here! </p> <p>The vitamin A/monkey study was largely a success due to the time and </p> <p>effort of Dr. Bob Adams and Dr. Tori Baxter. I would like to thank them </p> <p>immensely for their time and expertise in handling the monkeys, obtaining </p> <p>samples, and for being flexible with their schedules around the holidays, </p> <p>while also granting this project many of their early mornings. </p> <p>I would like to give a huge thanks to my roommate, Dr. Cailin Deal, </p> <p>who was able to provide me her knowledge and skills in so many areas of </p> <p>virology and immunology as a whole. Her expertise in writing in science was </p> <p>crucial to the process of editing my thesis, as well as her general knowledge </p> <p>of techniques and data analysis. Furthermore, I would like to thank Debbie </p> <p>Hauer for her assistance in teaching me techniques and processing samples </p> <p>that were essential for my projects and this thesis. </p> <p>I would like to extend my warmest thanks to the rest of Dr. Griffins </p> <p>laboratory for being so welcoming to me as a masters student, for passing </p> <p>down their expertise and insight when I needed assistance, and for their </p> <p>general support and friendship. Dr. Kim Shulz, Dr. Tori Baxter, Dr. Kirsten </p> <p>Kulscar, Stephen Goldstein and Siva Manivannan, your help was </p> <p> vi </p> <p>instrumental towards my experience as a student in this laboratory. </p> <p>Additionally, I would like to acknowledge Gui Nilaratanakul, Rachy </p> <p>Abraham, and Nina Martin for their presence and livelihood in the lab. </p> <p>Finally, I would like to thank my mother, father, and brother Ryan, as </p> <p>well as my extended family and friends for providing their unwavering </p> <p>support. As a little girl, my parents told me I could do whatever I put my </p> <p>mind to, and when I decided to pursue a science and research their </p> <p>enthusiasm was there to match my own. This thesis is the product of the </p> <p>hard work and support of many people, and I would again like to extend a </p> <p>tremendous thanks to all of the people by my side! </p> <p> vii </p> <p>Table of Contents </p> <p>Abstract. ii </p> <p>Acknowledgements.. iv </p> <p>List of Tables...... ix </p> <p>List of Figures. x </p> <p>Chapter 1: Introduction to measles virus 1 </p> <p> Public health implications... 2 </p> <p> Measles virus pathogenesis. 2 </p> <p> Prevention of measles virus infection... 3 </p> <p> Measles virus virology.. 6 </p> <p> Measles virus infection. 7 </p> <p> Defective replication of measles virus genome 8 </p> <p> Innate immune response to viral infection.. 9 </p> <p> TLRs.. 9 </p> <p>Cytoplasmic PRRs 10 </p> <p>Type I interferon.. 11 </p> <p>Interferon-inducible antiviral proteins... 12 </p> <p>Innate immune response to measles virus infection 12 </p> <p>Role of dendritic cells.. 14 </p> <p> Adaptive immune response to measles virus infection... 14 </p> <p>Antibody response 15 </p> <p>T lymphocyte response 16 </p> <p>Effector CD4+ T lymphocytes 16 </p> <p>Immunosuppression following measles virus infection.. 18 </p> <p>Figures 20 </p> <p>Chapter 2: Comparision of in vitro immune responses to wild-type </p> <p>measles virus with C and V protein-knock out strains; wild-type and </p> <p>vaccine strains of measles virus 24 </p> <p> Introduction.. 25 </p> <p>Measles virus immune evasion. 25 </p> <p> Block of type I interferon production.. 25 </p> <p> Block of type I interferon signaling. 27 </p> <p> Interferon-stimulated genes (ISGs) and virus stress-induced </p> <p>genes (VSIGs)... 28 </p> <p> Role of dendritic cells in measles virus infection.. 29 </p> <p> Defective interfering (DI) particles.. 29 </p> <p>Th17 response to viral infection... 30 </p> <p>Th17 response to measles virus infection.. 30 </p> <p>Materials and methods... 31 </p> <p>Results 38 </p> <p>Discussion.. 46 </p> <p>Tables. 53 </p> <p>Figures... 54 </p> <p> viii </p> <p>Chapter 3: Effects of vitamin A supplementation on the immune </p> <p>response and the Th17 response to measles virus infection in rhesus </p> <p>macaques. 63 </p> <p> Introduction.. 64 </p> <p>Vitamin A and measles infection. 64 </p> <p>Role of vitamin A in CD4+ T cell differentiation.. 66 </p> <p>Vitamin A supplementation.. 67 </p> <p>Vitamin A: Potential roles in improving measles outcome.... 69 </p> <p> Repair of lung epithelium.. 69 </p> <p> Effect on lymphopenia and T cell-mediated viral clearance.. 69 </p> <p>Inhibition of viral replication 70 </p> <p>Enhanced antibody response. 71 </p> <p>Th17 response to measles infection. 71 </p> <p> Materials and methods... 72 </p> <p>Results 81 </p> <p>Discussion.. 91 </p> <p>Tables. 98 </p> <p>Figures. 100 </p> <p>Chapter 4: Discussion of the immune responses to measles virus </p> <p>infection in vitro and in vivo 120 </p> <p> The innate immune response to measles virus infection. 121 </p> <p>Type I interferon 121 </p> <p> The role of measles virus and its C and V proteins on interferon- </p> <p>stimulated genes (ISGs) and virus stress-induced genes </p> <p>(VSIGs). 122 </p> <p> The early adaptive immune response to measles virus infection... 122 </p> <p> Th17 regulatory cytokine expression to measles virus infection 123 </p> <p> The Th17 response to measles virus infection 124 </p> <p> The measles virus antibody response 124 </p> <p> The role of vitamin A on the immune response to measles virus </p> <p>infection 125 </p> <p>References.... 127 </p> <p>Curriculum vitae....... 144 </p> <p> ix </p> <p>List of Tables </p> <p>Chapter 2: </p> <p>Table 1: PCR primers, targets, and cycling conditions for P gene sequencing </p> <p>and detection of measles virus standard and defective genomes. 53 </p> <p>Chapter 3: </p> <p>Table 1: PCR primers, targets, and cycling conditions for detection of the </p> <p>measles virus N gene...... 98 </p> <p>Table 2: Measles virus shedding in respiratory secretions... 98 </p> <p>Table 3: IL-17A ELISAs....... 99 </p> <p> x </p> <p>List of Figures </p> <p>Chapter 1: </p> <p>Figure 1: Measles virus infection and pathogenesis.. 20 </p> <p>Figure 2: 2012 immunization coverage rates with measles-containing </p> <p>vaccines in infants... 21 </p> <p>Figure 3: Measles virus structure, RNA genome, and replication.. 21 </p> <p>Figure 4: Defective interfering particle formation from the measles virus </p> <p>genome... 22 </p> <p>Figure 5: Biopsies of the measles virus rash show CD4+ and CD8+ </p> <p>lymphocyte infiltration... 22 </p> <p>Figure 6: Measles virus RNA over the course of infection... 23 </p> <p>Figure 7: Potential mechanisms leading to measles virus-induced immune </p> <p>suppression... 23 </p> <p>Chapter 2: </p> <p>Figure 1: Signaling pathways leading to virus stress-inducible gene (VSIG) </p> <p>induction.... 54 </p> <p>Figure 2: Generation of wild-type measles virus defective for the C or V </p> <p>protein.... 55 </p> <p>Figure 3: Primer binding sites for sequencing.... 55 </p> <p>Figure 4: Sequencing confirms correct viral sequences. .. 56 </p> <p>Figure 5: Gel of measles virus stocks. .. 57 </p> <p>Figure 6: Measles virus standard and DI genome PCR products of measles </p> <p>virus stocks used in in vitro experiments. 58 </p> <p>Figure 7: mRNAs for interferon-stimulated genes, IFIT1 and Mx1, are </p> <p>upregulated in moDCs by the Edmonston measles virus vaccine strain and </p> <p>Bilt Wt strain at MOIs of 0.4 and 4.0.. 59 </p> <p>Figure 8: mRNAs for interferon-stimulated genes, IFIT1 and Mx1, are </p> <p>comparably upregulated moDCs in response to Wt measles virus, and its </p> <p>respective C- and V-protein KO strains at MOIs of 0.01 and 0.1. 59 </p> <p>Figure 9: Positive regulators of Th17 cell differentiation, IL-1, IL-23A and </p> <p>IL-6, mRNA expression levels from moDCs in response to Edmonston and </p> <p>Bilthoven measles virus infection at MOIs of 0.4 and 4.0.. 60 </p> <p>Figure 10: Positive regulators of Th17 cell differentiation, IL-1 and IL-6, </p> <p>mRNA expression levels from moDCs in response to wild-type measles virus </p> <p>and its respective C- and V-protein knock out strains at MOIs of 0.01 and </p> <p>0.1.... 61 </p> <p>Figure 11: Negative regulators of Th17 cell differentiation, IL-27 and IL-10, </p> <p>mRNA expression levels from moDCs in response to Edmonston and </p> <p>Bilthoven measles virus infection at MOIs of 0.4 and 4.0.. 61 </p> <p> xi </p> <p>Figure 12: Negative regulators of Th17 cell differentiation, IL-27 and IL-10, </p> <p>mRNA expression levels from moDCs in response to wild-type measles virus </p> <p>and its respective C- and V-protein knock out strains at MOIs of 0.01 and </p> <p>0.1.... 62 </p> <p>Chapter 3: </p> <p>Figure 1: Vitamin A (retinol) status and usage is impaired during </p> <p>infection 100 </p> <p>Figure 2: Plasma retinol levels averaged between two rhesus macaques after </p> <p>measles infection... 100 </p> <p>Figure 3: Time course of measles virus clearance 101 </p> <p>Figure 4: Viremia is present by day 7, and is cleared in all animals by day </p> <p>18. .... 101 </p> <p>Figure 5: Change in total body weight over course of measles virus </p> <p>infection... 102 </p> <p>Figure 6: Maculopapular rash was very robust on monkey 50Y on day 10 </p> <p>post-infection.. 102 </p> <p>Figure 7: Rash histology of skin biopsies... 103 </p> <p>Figure 8: Histology of lymph node biopsies 104 </p> <p>Figure 9: Vitamin A levels begin to drop at day 21 in the non-supplemented </p> <p>group of monkeys (17Y, 31Y, 46Y) but remain stable in vitamin A-</p> <p>supplemented monkeys (14Y, 24Y, 50Y). 105 </p> <p>Figure 10: Comprehensive blood counts and differential leukocyte counts </p> <p>following rash..... 106 </p> <p>Figure 11: Frequency of CD4+ and CD8+ cells within the CD14-CD20- live </p> <p>cell population, and CD4:CD8 cell ratio... 107 </p> <p>Figure 12: Measles virus H, N, and F protein-specific IFN- secreting T cells </p> <p>peak at 21 days post-infection.... 108 </p> <p>Figure 13: Intracellular staining for IL-17A..... 109 </p> <p>Figure 14: Intracellular staining for IL-21 110 </p> <p>Figure 15: Frequency of IL-17+ cells as a percentage of total CD4+ cells </p> <p>peaked at day 18... 111 </p> <p>Figure 16: Frequency of IL-21+ cells as a percentage of total CD4+ T cells </p> <p>showed peaks...</p>

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