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Characterization of Alpha-Galactosylceramide As a Mucosal Adjuvant

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Characterization of Alpha-Galactosylceramide As a Mucosal Adjuvant The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 8-2010 CHARACTERIZATION OF ALPHA-GALACTOSYLCERAMIDE AS A MUCOSAL ADJUVANT Amy N. Courtney Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Immunology and Infectious Disease Commons Recommended Citation Courtney, Amy N., "CHARACTERIZATION OF ALPHA-GALACTOSYLCERAMIDE AS A MUCOSAL ADJUVANT" (2010). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 63. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/63 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. CHARACTERIZATION OF ALPHA-GALACTOSYLCERAMIDE AS A MUCOSAL ADJUVANT by Amy N. Courtney, BS Approved: ________________________________________________ Dr. K Jagannadha Sastry, PhD, Supervisory Professor ________________________________________________ Dr. Mary K. Estes, PhD ________________________________________________ Dr. Jeffrey K. Actor, PhD ________________________________________________ Dr. Pramod N. Nehete, PhD ________________________________________________ Dr. Chengming Zhu, PhD ________________________________________________ Dr. Dapeng Zhou, MD, PhD Approved: __________________________________ Dr. George M. Stancel, PhD Dean, The University of Texas Health Science Center at Houston Graduate School of Biomedical Sciences CHARACTERIZATION OF ALPHA-GALACTOSYLCERAMIDE AS A MUCOSAL ADJUVANT A Dissertation Presented to the Faculty of The University of Texas Health Science Center at Houston and The University of Texas M.D. Anderson Cancer Center Graduate School of Biomedical Sciences In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY By Amy N. Courtney, BS Houston, Texas August, 2010 ACKNOWLEDGEMENTS I would first like to thank my family, especially my mom Ellen and my husband Cary for all of their love and support throughout my career as a PhD student. Their support has given me the strength and the patience to complete my requirements, and without them I would not have this degree. I would also like to thank my mom for instilling a love of science in me as a child. I would like to thank my mentor, Dr. K. Jagannadha Sastry for all of his help and guidance over the years. He has supported me and my development into a scientist, and he has pushed me to be the best that I can. He has truly been an exceptional mentor, and I cannot express how much I appreciate his help and support. Additionally, I would like to thank the members of my supervisory committee, Drs. Jeffrey Actor, Pramod Nehete, Dapeng Zhou, Chengming Zhu and Mary Estes for their help and advice as I developed my PhD project. Finally, I would like to thank all of the members, past and present, of the Sastry lab. Especially Dr. Pramod and Bharti Nehete who trained me when I first began, Dr. Hong He who trained me on how to perform flow cytometry, Dr. Danielle Fontenot who was the co-student in the lab and my partner in crime, and Sachin Shenoy and Ameerah Wishahy for all of their help and technical support. I would also like to thank Prakash Thapa from Dr. Dapeng Zhou’s lab for his help and technical support. i CHARACTERIZATION OF ALPHA-GALACTOSYLCERAMIDE AS A MUCOSAL ADJUVANT Publication No. _______ Amy N. Courtney, BS Supervisory Professor: K. Jagannadha Sastry, PhD Adjuvants are essential components of vaccine formulations that enhance adaptive immune responses to antigens, particularly for immunizations targeting the tolerogenic mucosal tissues, which are more biologically relevant for protective immunity against pathogens transmitted by the mucosal routes. Adjuvants possess the inherent capacity to bridge innate and adaptive immune responses through activating innate immune mediators. Here evidence is presented in support of the effectiveness of a synthetic glycolipid, alpha-Galactosylceramide ( α-GalCer), as an adjuvant for mucosal immunization with peptide and protein antigens, by oral and intranasal routes, to prime antigen-specific immune responses in multiple systemic and mucosal compartments. The adjuvant activity of α-GalCer delivered by the intranasal route was manifested in terms of potent activation of NKT cells, an important innate immunity mediator, along with the activation of dendritic cells (DC) which serve as the professional antigen- presenting cells. Data from this investigation provide the first evidence for mucosal delivery as an effective means to harness the adjuvant potential of α-GalCer for priming as well as boosting cellular immune responses to co-administered immunogens. Unlike systemic administration where a single dose of α-GalCer leads to anergy of responding NKT cells and thus hinders delivery of booster immunizations, we demonstrated that administration of multiple doses of α- GalCer by the intranasal route affords repeated activation of NKT cells and the induction of broad systemic and mucosal immunity. This is specifically advantageous, and may be even essential, for vaccination regimens against mucosal pathogens such as the human immunodeficiency virus (HIV) and the human papillomavirus (HPV), where priming of durable protective immunity at the mucosal portals of pathogen entry would be highly desirable. ii TABLE OF CONTENTS Page Acknowledgements......................................................................................................................i Abstract....................................................................................................................................... ii Table of Contents....................................................................................................................... iii List of Figures ........................................................................................................................... vii List of Abbreviations................................................................................................................... ix CHAPTER 1 Background and Significance ............................................................................. 1 1.1 Mucosal Immunity ....................................................................................3 1.1.1 Common mucosal immune system ...............................................4 1.1.2 Tolerance in mucosal immunization ..............................................5 1.1.3 Mucosal routes of immunization....................................................6 1.2 Adjuvants .................................................................................................7 1.2.1 Types of adjuvants........................................................................8 1.2.2 Mucosal adjuvants ......................................................................10 1.3 α-Galactosylceramide ( α-GalCer)...........................................................13 1.3.1 α-GalCer modulates NKT cell activity..........................................16 1.3.2 α-GalCer as a T-cell adjuvant .....................................................22 1.3.3 Pharmokinetics of α-GalCer........................................................23 1.4 NKT Cell Anergy.....................................................................................24 iii 1.4.1 Role of APCs in modulating NKT cell anergy ..............................24 1.4.2 Role of inhibitory markers in inducing NKT cell anergy ...............25 1.5 Significance............................................................................................26 2 Statement of Objectives.....................................................................................28 3 Materials and Methods.......................................................................................30 3.1 Animals ..................................................................................................30 3.2 Cell lines and cell cultures......................................................................30 3.3 Peptides and reagents ...........................................................................31 3.4 Adjuvants ...............................................................................................31 3.5 Immunizations........................................................................................32 3.6 Analysis of antigen-specific cytolytic activity by 51 Cr release assay ........33 3.7 Preparation of target cells expressing HIV env gp160 ............................33 3.8 IFN γ ELISpot assay ...............................................................................34 3.9 Fluorescence labeled antibodies and flow cytometry..............................34 3.10 Intracellular cytokine staining .................................................................35 3.11 OT-1 proliferation assay.........................................................................36 3.12 In vivo
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