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Effect of Mycobacterium Vaccae NCTC 11659 (Standard Or Recombinant) on Cytokine Production by Human and Murine Cells

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Effect of Mycobacterium Vaccae NCTC 11659 (Standard Or Recombinant) on Cytokine Production by Human and Murine Cells Effect of Mycobacterium vaccae NCTC 11659 (standard or recombinant) on cytokine production by human and murine cells Elaine Anne Bayley A thesis submitted in partial fulfillment of the degree of Doctor of Philosophy of the University of London Department of Bacteriology Windeyer Institute of Medical Sciences University College London March 1999 1 ProQuest Number: 10609099 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10609099 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 Abstract Killed M. vaccae is in clinical trials as an immunotherapeutic agent and adjuvant, but understanding of its mode of action is incomplete. To test its potential as a recombinant carrier organism and the nature of the responses evoked, recombinant strains were generated that expressed p27 of SIV. In mice these primed for specific production of IFNy in the presence of p27, and induced serum IgG2a and, at higher doses, IgGl responses to M. vaccae sonicate. Flow cytometry for intracellular cytokines after a single subcutaneous injection of 109 M. vaccae revealed accumulation of IFNy-secreting CD8+ cells in lymph nodes. This finding, together with data generated simultaneously by another research group, implied an unusual adjuvant effect, not shared by other killed mycobacteria. In order to investigate this adjuvanticity the effects of killed M. vaccae on cytokine release from the human THP-1 monocyte line were investigated. M. vaccae , BCG and soluble bacterial preparations were all able to induce IL-12, IL-10 and TNFa production in vitro to varying extents. Dose of mycobacterium and the addition of IFNy influenced the balance of cytokines. Attempts to define active components in the IL-12 induction system were not successful, but it was noted that M. vaccae differed strikingly from the other killed mycobacteria in that its induction of IL-12 was greatly enhanced by exposure to lysozyme. The induction of IL-12 proved sufficiently reproducible to be used as a potency assay for material manufactured for clinical use. In conclusion, M. vaccae has been shown to be a potent Thl inducer at appropriate doses, with the added ability to induce expansion of the CD8+ IFNy+ population, perhaps via IL-12 release following exposure to lysozyme in vivo. CD8+ cells are strongly implicated in the clinical situations where M. vaccae has shown benefit. 2 Acknowledgements I should like to thank my supervisor, Professor Graham Rook, for his invaluable guidance throughout my work and studies. I should also like to thank the many collaborators with whom I have worked during my career, especially those who gave assistance with my PhD: Dr J. Thole and Miss S. Ely (molecular biology) and Dr C.C. Wang (mouse work). I especially thank my husband, James, for his encouragement throughout the years and my Mother for helping with Francesca. 3 Table of Contents Abstract ..................................................................................................................................2 Acknowledgements .............................................................................................................. 3 Table of Contents ................................................................................................................. 4 List of Tables ........................................................................................................................ 9 List of Illustrations ............................................................................................................. 10 Abbreviations ......................................................................................................................13 Chapter 1 Introduction ........................................................................................................ 15 1.1 Lymphoid system organisation ....................................................................15 1.2 Innate and adaptive immunity ......................................................................16 1.3 Polarised type 1 or type 2 responses and disease ........................................17 1.3.1 The possibility of a ‘ switch’ in predominant immune responses during chronic disease ............................................................................................19 1.4 Factors polarising T cell responses .............................................................20 1.5 Cytokine stimulation of type 1 responses: ..................................................20 1.5.1 Interleukin-12 ...............................................................................................20 1.5.2 Interferon y...................................................................................................25 1.5.3 Tumour necrosis factor a ............................................................................ 27 1.5.4 Regulators of Thl responses: IL-4, IL-13 and IL-10 .................................28 1.6 Multiple antigen processing pathways in immunity ..................................29 1.6.1 Dendritic cells and T cell stimulation .........................................................31 1.6.2 Possible roles for all presentation pathways in mycobacterial infections32 1.7 Vaccines ....................................................................................................... 33 1.7.1 Attenuated vaccines .....................................................................................33 1.7.2 Designer vaccines ........................................................................................34 1.7.3 Considerations for vaccine design .............................................................. 37 1.8 History of vaccination with BCG and M. vaccae ......................................40 1.8.1 The selection of M. vaccae for investigation as an immunotherapeutic agent and vaccine ........................................................................................43 1.9 Recent uses of M. vaccae immunotherapy ................................................ 45 1.9.1 Pilot studies in tuberculosis ........................................................................ 45 1.9.2 GCP trials in tuberculosis and/or HIV ........................................................46 1.9.3 Use in other conditions ............................................................................... 46 1.10 Aims of the project ......................................................................................48 4 Chapter 2 Materials and Methods ...................................................................................... 49 2.1 Materials and methods used in molecular biology....................................49 2.1.1 Cutting vectors and inserts with restriction enzymes ............................... 49 2.1.2 Wizard DNA clean up system ....................................................................49 2.1.3 Calf intestine alkaline phosphatase treatment of cut vector DNA ............50 2.1.4 Amplification by PCR of SIVpl7/27..........................................................50 2.1.5 Separation of DNA on agarose gels ............................................................52 2.1.6 QLAEX DNA gel extraction protocol ........................................................ 52 2.1.7 Ligation using T4 DNA ligase ....................................................................53 2.1.8 Culture of E. coli K12 DH5a, transformation and freezing ..................... 54 2.1.9 Plasmid isolation protocols .........................................................................55 2.1.10 Culture and electroporation of M. vaccae .................................................. 56 2.1.11 DNA sequencing using T7 polymerase ......................................................57 2.1.12 SDS-PAGE and immunoblotting ................................................................60 2.1.13 Production of autoclaved recombinant M. vaccae vaccine ....................... 62 2.2 Materials and methods used in analysis of murine responses to recombinant M. vaccae .............................................................................. 62 2.2.1 Antigen preparation for spleen cell stimulation and antibody detection. 62 2.2.2 Culture of spleen cells and collection of supernatants for cytokine measurement ............................................................................................... 63 2.2.3 Capture ELISA for the detection of murine IFNy in supernatants ...........64 2.2.4 Capture ELISA for the detection of murine interleukin 2 and interleukin 4 in supernatants ........................................................................................ 65 2.2.5 ELISA to detect specific immunoglobulins in the mouse sera ................. 65 2.3 Materials and methods used in flow cytometric analysis of murine responses to standard M. vaccae ................................................................ 66 2.3.1 Vaccination of mice ...................................................................................
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