INVESTIGATION AND CHARACTERIZATION OF THE ENHANCED HUMORAL RESPONSE FOLLOWING IMMUNIZATION WITH THE LETHAL AND EDEMA TOXINS OF BACILLUS ANTHRACIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Karen E. Brenneman, B. S. ***** The Ohio State University 2007 Dissertation Committee: Approved by: Dr. Darrell R. Galloway, Adviser Dr. Brian Ahmer ____________________________ Dr. Paula W. Bryant Adviser Dr. Richard F. Mortensen Graduate Program in Microbiology ABSTRACT Bacillus anthracis is the causative agent of the disease anthrax. Although the organism produces multiple virulence factors during an infection, a large portion of the disease pathology can be attributed the effects of two A-B toxins: lethal and edema toxin. These toxins selectively target innate immune effector cells, preventing containment and clearance of the developing infection. Additionally, lethal and edema toxin have been shown to impair the priming and proliferation of adaptive immune cells. Despite these immunosuppressive effects, lethal toxin has been shown to stimulate a strong immune response the context of an immunization. Co-immunization with the two protein components of lethal toxin (lethal factor and protective antigen) increases antibody response to each component. In this work, the functional activities of lethal toxin were investigated in order to identify the toxin actions responsible for the enhanced immune response. The effects of each step of the cellular intoxication process were examined, as the toxin proteins specifically target immunological cells and shuttle through different antigen processing compartments during the course of intoxication. The catalytic activity of lethal toxin was also examined, since the downstream consequences of toxicity on a developing immune response have not been characterized. To study this, single amino acid changes were ii made in each of the lethal toxin components in order to remove specific functional activities of the toxin complex. The immune response generated by toxin combinations lacking a single functional activity were then compared to the response stimulated by wild type lethal toxin. The work from this project revealed a complicated relationship between lethal toxin and the immune system. The enhanced antibody response depended on the catalytic activity of lethal toxin and was observed only when wild type lethal toxin was present in an immunization. Although the inactive forms of lethal toxin did not produce an enhanced immune response, altering the functional abilities of the toxin complex illuminated a fundamental immunological difference between the two components of lethal toxin. A strong antibody response to lethal factor depended on the production of IFN- and the cytosolic localization of this antigen. In contrast, the antibody response to protective antigen required endosomal processing and the concurrent production of IL-4. These two immune responses antagonized each other, resulting in a competition between the antibody responses against the lethal toxin components that was observed under all circumstances tested. This project also determined that the combination of edema toxin components (protective antigen and edema factor) enhanced antibody production, and subsequently investigated the functional mechanism by which this response was provoked. Although edema toxin forms a protein complex analogous to lethal toxin, edema toxin interacted iii with the immune system in a manner quite different from lethal toxin. The combination of the edema toxin components was able to act as an adjuvant on the immune system. An enhanced immune response to edema toxin did not depend on either the catalytic activity of the toxin or the cellular intoxication process. Competition between the antibody responses against the edema toxin components was observed to a lesser degree than for the lethal toxin components. A robust immune response to edema factor was accompanied by the production of both IL-4 and IFN-, and thus competition between the antibody responses to the edema toxin components was observed only when edema factor was localized to the cytosol and separated from protective antigen. Co-localization of edema factor and protective antigen in the endosome resulted in an enhanced immune response to both proteins. iv Dedicated to my husband Rob, who still isn’t sure what antibodies do, but who has always supported and encouraged me v ACKNOWLEDGMENTS This document would not be possible without the help of numerous individuals. First, I would like to thank my adviser, Dr. Darrell Galloway, for his commitment to me and to this project. My committee also deserves recognition for their flexibility in allowing this project to be completed under unusual circumstances. Thanks go to Dr. Les Baillie and CAPT Al Mateczun for providing a temporary home with the NMRC/BDRD group. Without the help of CDR Gail Chapman, LT Matthew Weiner, Stephanie Gray, Tatiana Pervaia and Gordon Heissler, it would not have been possible to undertake such comprehensive animal studies. I am grateful to the scientists of the BDRD Vaccine group – Drs. Arya Akmal, Mark Albrecht, Christian Darko, Olga Pomerantseva, John Charles Rodenberry and Ramjay Vatsan – for all their insights and suggestions. Special thanks go to CDR Joe Morris, who taught me everything I needed to know about protein purification and endotoxin. Special thanks also go to Dr. Stan Goldman, who has been invaluable in challenging me and providing focus to my usually scattered ideas. I am extremely grateful to Dr. Bart Legutki for teaching me the importance of planning ahead, and to Matthew Bell for teaching me the importance of having all the controls. Both of them have been more than fellow graduate students over the past six years and I am privileged to have had their support, encouragement and friendship. vi VITA 10 September 1978 ……………………………Born – Raleigh, North Carolina, USA 2000 …………………………………………..B. S. Biochemistry and Molecular Biology, Pennsylvania State University 2000 – 2005 …………………………………...Graduate Teaching and Research Associate, The Ohio State University 2005 – present …………………………………Research Associate, Naval Medical Research Center, Biological Defense Research Directorate PUBLICATIONS Hermanson, G., V. Whitlow, S. Parker, K. Tonsky, D. Rusalov, P. Lalor, M. Komai, R. Mere, M. Bell, K. Brenneman, A. Mateczun, T. Evans, D. Kaslow, D. Galloway and P. Hobart. A cationic lipid-formulated plasmid DNA vaccine confers sustained antibody-mediated protection against aerosolized anthrax spores. PNAS. 2004. 101(37):13601-13606. FIELDS OF STUDY Major Field: Microbiology Minor Field: Immunology and Molecular Biology vii TABLE OF CONTENTS Page Abstract ……………………………………………………………………………..….ii Dedication ………………………………………………………………………...........v Acknowledgments ………………………………………………………………..…...vi Vita ……………………………………………………………………………….......vii List of Tables ……………………………………………………………………….....xi List of Figures …………………………………………………………………….…..xii List of Abbreviations ………………………………………………………………....xv Chapters 1. Introduction ………………………………………………………….…......1 1.1 Bacillus anthracis .………………………….…………………………..1 1.1.1 Microbiology …………………………………………………….....1 1.1.2 Virulence factors and the regulation of virulence ………………….4 1.2 The bipartite anthrax toxins ……...………………………………...…..8 1.2.1 Protective antigen ………………………………………………….9 1.2.2 Lethal factor ………………………………………………………14 1.2.3 Edema factor ……………………………………………………...20 1.3 Anthrax: disease, prevention and treatment ………………………….23 1.3.1 Pathogenesis and disease ……....…………………………………23 1.3.2 Anthrax vaccination ………………………………………………28 1.4 Statement of the problem ……………………………………………..31 viii 2. Materials and Methods ………………………………………………………..33 2.1 Toxin mutagenesis and plasmid construction ………………………..33 2.2 Protein purification ……………………………………………………38 2.3 Characterization of protein activity ………………………………….46 2.4 Animal manipulations and immunological analyses …………………56 3. The humoral immune response to lethal toxin is altered by cellular intoxication as well as MEK proteolysis ……………………………………...68 3.1 Introduction …………………………………………………………...68 3.2 Results ………………………………………………………………...72 3.2.1 Characterization of lethal toxin mutants ……………………...72 3.2.2 Immunization with non-proteolytic mutants of lethal toxin ….78 3.2.3 Immunization with lethal toxin mutants deficient in cellular intoxication …………………………………………...82 3.3 Discussion …………………………………………………………….85 4. Edema toxin is an adjuvant whose activity does not depend on either cAMP production or cellular intoxication …………………………………………..115 4.1 Introduction ………………………………………………………….115 4.2 Results ……………………………………………………………….118 4.2.1 Characterization of edema toxin mutants …………………...118 4.2.2 Immunization with edema toxin mutants lacking adenylyl cyclase activity ………………………………………………123 4.2.3 Immunization with edema toxin mutants deficient in cellular intoxication …………………………………………126 4.3 Discussion …………………………………………………………...131 ix 5. The antibody titers to lethal and edema factor are increased by co-administration of bacterial products with inactive toxin …………………………………….154 5.1 Introduction ………………………………………………………….154 5.2 Results ……………………………………………………………….157 5.2.1 Immunogenicity of inactive lethal and edema toxin components prepared without endotoxin removal …………......................157 5.2.2 Immunization with truncated lethal toxin in the presence of purified lipopolysaccharide and monophosphoryl lipid A ....159 5.3 Discussion