IGA MEDIATED DEFENSES AGAINST HIV-1 by ALISON LAING WRIGHT Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Yung T. Huang, Ph.D. Department of Pathology CASE WESTERN RESERVE UNIVERSITY May, 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________Alison Laing Wright candidate for the ______________________degreePh.D *. Dr. Steven Emancipator (signed)_______________________________________________ (chair of the committee) Dr. Yung Huang ________________________________________________ Dr. Michael Lamm ________________________________________________ Dr. Thomas Blanchard ________________________________________________ Dr. David McDonald ________________________________________________ Dr. Clive Hamlin ________________________________________________ (date) _______________________ January 16th 2008 *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents Title Page ...................................................................................................................................................... 1 Committee Signatures ................................................................................................................................ 2 Table of Contents ....................................................................................................................................... 3 List of Tables ............................................................................................................................................... 6 List of Figures ............................................................................................................................................. 7 Acknowledgments ...................................................................................................................................... 8 Abbreviations .............................................................................................................................................. 9 Abstract ...................................................................................................................................................... 12 Chapter I Introduction ............................................................................................................................ 14 Origin and Classification of Human Immunodeficiency Virus ......................................... 14 HIV Infection Levels ................................................................................................................. 16 HIV Genome and Structure .................................................................................................... 17 HIV Life Cycle ............................................................................................................................ 18 HIV Co-Receptor Usage .......................................................................................................... 20 Mucosal Surface .......................................................................................................................... 21 HIV Entry at the Mucosal Surface ......................................................................................... 22 Early HIV Infection ................................................................................................................... 23 HIV Infection ............................................................................................................................. 26 Mucosal Immunological Responses ....................................................................................... 28 Immunoglobulin A Pathway .................................................................................................... 30 Polymeric Immunoglobulin Expression ................................................................................ 32 Immunoglobulin A Functions ................................................................................................. 32 IgA Neutralization of Viruses .................................................................................................. 34 Antibodies against HIV............................................................................................................. 35 Chapter II Intracellular Neutralization ................................................................................................. 39 Introduction ....................................................................................................................................... 39 Intracellular Neutralization ....................................................................................................... 39 Heterogeneity of HIV ............................................................................................................... 41 Epithelial Infection .................................................................................................................... 41 Intrabodies ................................................................................................................................... 42 Gag and RT ................................................................................................................................. 43 Materials and Methods ..................................................................................................................... 46 Cell Culture .................................................................................................................................. 46 Polarized Cell Preparation ........................................................................................................ 47 Virus .............................................................................................................................................. 48 Monoclonal Antibodies ............................................................................................................. 48 Transcytosis of Ab ..................................................................................................................... 50 Analysis of Ig content by ELISA ............................................................................................ 51 Traditional HIV Neutralization ............................................................................................... 52 Intracellular HIV Neutralization ............................................................................................. 53 HeLa Blue Cell Assay ................................................................................................................ 54 HIV p24 Assay ........................................................................................................................... 55 Preparation of Conditioned Media ......................................................................................... 56 3 Intracellular Colocalization of IgA and HIV Proteins ........................................................ 57 Data Analysis ............................................................................................................................... 58 Intracellular Neutralization Results ................................................................................................ 59 Monoclonal Antibodies ............................................................................................................. 59 Transcytosis of RT and Gag IgA ............................................................................................ 59 Intracellular HIV Neutralization ............................................................................................. 60 Concentration Dependence of Neutralization ..................................................................... 62 Confirmation that Neutralization is Intraepithelial .............................................................. 65 Effect on p24 Synthesis ............................................................................................................ 66 No Intracellular Neutralization Synergy ................................................................................ 67 Intracellular Colocalization of Viral Antigen and Antibody .............................................. 69 Intracellular Neutralization Discussion ......................................................................................... 71 Chapter III Excretion of HIV-1 by Immunoglobulin A .................................................................. 77 Introduction ....................................................................................................................................... 77 Excretion of HIV ....................................................................................................................... 77 Materials and Methods ..................................................................................................................... 80 Cell Culture .................................................................................................................................. 80 Virus .............................................................................................................................................. 81 Monoclonal Antibodies ............................................................................................................