Immunogenicity of antigen displaying virus like particles and their use as a potential vaccine against prion diseases [Elektronische Ressource] / vorgelegt von Patricia Bach

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

Immunogenicity of antigen-displaying
virus-like particles and their use as a potential
vaccine against prion diseases


VLVLPP




Patricia Bach

Immunogenicity of antigen-displaying
virus-like particles and their use as a potential
vaccine against prion diseases



Dissertation zur Erlangung
des naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg



vorgelegt von
Patricia Bach
aus Offenbach am Main



Würzburg, 2007Eingereicht am: 24.07.2007



Mitglieder der Prüfungskommission:


Vorsitzender: Prof. Dr. M. J. Müller

Gutachter: Prof. Dr. M. A. Klein

Gutachter: Prof. Dr. T. Raabe












Tag des Promotionskolloquiums: 12.12.2007


Doktorurkunde ausgehändigt am:




Für meine Eltern























„Zwei Dinge sind zu unserer Arbeit nötig: Unermüdliche Ausdauer und die
Bereitschaft, etwas, in das man viel Zeit und Arbeit gesteckt hat, wieder
wegzuwerfen.“

Albert Einstein
deutscher Physiker und Nobelpreisträger
Table of contents
1 INTRODUCTION..................................................................................... 1
1.1 The immune system protects hosts against infection with pathogens ................... 1
1.1.1 Innate and adaptive immunity...................................................................................... 1
1.1.2 The induction of antibody responses........................................................................... 2
1.1.3 Germinal Centers......................................................................................................... 3
1.2 Interferons....................................................................................................................... 5
1.2.1 The Impact of type I IFN stimulation on immune responses ....................................... 6
1.3 Vesicular stomatitis virus (VSV)................................................................................... 8
1.4 Prion biology .................................................................................................................. 9
1.4.1 Prion diseases ............................................................................................................. 9
1.4.2 The prion protein........................................................................................................ 11
1.4.3 The prion hypothesis.................................................................................................. 13
1.5 Active immunization against the prion protein, possible or impossible? ............. 14
1.5.1 Passive immunotherapy of prion diseases ................................................................ 14
1.5.2 Tolerance and autoimmunity ..................................................................................... 15
1.5.3 PrP-specific tolerance vs. immunity........................................................................... 18
1.5.4 Strategies to overcome B cell self-tolerance ............................................................. 19
1.5.5 Virus-like particles (VLP) ........................................................................................... 20
2 AIMS OF THIS THESIS ........................................................................ 22
3 MATERIAL AND METHODS ................................................................ 24
3.1 Materials........................................................................................................................ 24
3.1.1 Mice............................................................................................................................ 24
3.1.2 Equipment.................................................................................................................. 25
3.1.3 Consumables............................................................................................................. 27
3.1.4 Chemicals 30
3.1.5 Buffers and Media...................................................................................................... 33
3.1.6 Prepared Buffers, Media and other reagents ............................................................ 34
3.1.7 Bacteria, Viruses and Prion Inoculum........................................................................ 38
I3.1.8 Antibodies .................................................................................................................. 38
3.1.9 Plasmids and vectors................................................................................................. 40
3.1.10 Software..................................................................................................................... 41
3.2 Methods......................................................................................................................... 43
3.2.1 Molecular biology....................................................................................................... 43
3.2.1.1 Transformation of competent bacteria .............................................................. 43
3.2.1.2 Plasmid preparation .......................................................................................... 43
3.2.2 Cell culture ................................................................................................................. 44
3.2.2.1 Cells and media ................................................................................................ 44
3.2.2.2 Freezing and thawing of cultured cells 45
3.2.2.3 Manual counting of cells.................................................................................... 45
3.2.3 Protein biochemistry .................................................................................................. 46
3.2.3.1 SDS-polyacrylamide-gelelectrophoresis ........................................................... 46
3.2.3.2 Western blot analysis ........................................................................................ 46
3.2.3.3 In vitro PrP-neutralizing assay .......................................................................... 47
3.2.3.4 Expression and purification of recombinant mouse PrP amino acid 121-231 .. 48
3.2.4 Electron microscopy................................................................................................... 49
3.2.4.1 Immungold-labelling .......................................................................................... 49
3.2.5 Virological methods.................................................................................................... 50
3.2.5.1 Cell transfection and production of VLP............................................................ 50
3.2.5.2 Reverse Transcriptase (RT) activity assay ....................................................... 51
3.2.5.3 Viruses .............................................................................................................. 51
3.2.5.4 VSV neutralization assay .................................................................................. 52
3.2.6 Mouse experiments.................................................................................................... 53
3.2.6.1 Mouse anesthesia ............................................................................................. 53
3.2.6.2 Immunization and infection of mice................................................................... 53
3.2.6.3 Blood sampling for serum analysis 54
3.2.6.4 Isolation of splenocytes..................................................................................... 54
+3.2.6.5 Depletion of CD4 T cells .................................................................................. 54
3.2.7 Immunohistology........................................................................................................ 55
3.2.8 ELISA analysis........................................................................................................... 55
3.2.8.1 Analysis of VSV-specific serum binding............................................................ 55
3.2.8.2 Charaterization and Quantification of VLP or VSV ........................................... 56
3.2.8.3 Analysis of PrP-specific serum binding............................................................. 57
3.2.9 Flow cytometric analysis............................................................................................ 57
C3.2.9.1 Flow cytometric determination of PrP -specific serum binding ........................ 57
3.2.9.2 Detection of germinal center B cells by FACS analysis.................................... 57
3.2.9.3 Purification of PrP-specific memory B cells ...................................................... 58
II3.2.9.4 Competition assay............................................................................................. 58
3.2.9.5 Counting of absolute cell numbers by FACS .................................................... 59
4 RESULTS.............................................................