Flagellin:allergen fusion proteins as novel vaccines for the treatment of severe type I allergies [Elektronische Ressource] / Stefan Schülke

Schus

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2010
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Flagellin:allergen fusion proteins as novel 
vaccines for the treatment of  
severe type I allergies 
 
 
 
 
 
 
 
D i s s e r t a t i o n 
zur Erlangung des Grades 
ʺDoktor der 
Naturwissenschaftenʺ 
 
  
 
am Fachbereich Biologie 
der Johannes Gutenberg‐Universität 
in Mainz 
 
  
 
  
 
Stefan Schülke 
geb. in Bad Kreuznach 
 
  
 
Mainz, 2010 
 
 
 



Tag der mündlichen Prüfung: 25.05.2011

IIContent

1 Introduction..........................................................................................................1
1.1 Pathomechanism of type I allergic diseases..............................................................1
1.2 Current strategies of allergen specific immunotherapy..............................................4
1.3 The mode of action of SIT is poorly understood but includes several mechanisms of
action.........................................................................................................................6
1.3.1 SIT influences many clinical parameters ...................................................................6
1.3.2 Modulation of immune responses is tightly controlled by highly specialized cells.....6
1.3.3 DC are promising target cells for immunotherapy .....................................................8
1.4 New strategies for the treatment of allergies .............................................................9
1.4.1 Recombinant allergens, hypoallergens and peptides................................................9
1.4.1.1 Recombinant allergens may be used to improve therapeutic efficacy and safety.....9
1.4.1.2 Hypoallergenic variants hold potential to improve SIT.............................................10
1.4.1.3 Peptide based vaccines have a high risk of adverse reactions ...............................12
1.4.2 Bacterial extracts.....................................................................................................12
1.4.2.1 Heat killed Listeria are potent immune modulators .................................................13
1.4.2.2 HKL activate the innate immune system .................................................................14
1.4.3 TLR-ligands.............................................................................................................15
1.4.3.1 TLR-ligands are promising tools to modulate allergic immune responses ..............15
1.4.3.2 TLR5-ligand flagellin is an interesting adjuvant candidate for allergen specific ..........
immunotherapy........................................................................................................16
1.4.3.3 Flagellin based vaccines .........................................................................................17
1.4.3.4 TLR5 is expressed on many different cell types......................................................18
1.4.3.5 Flagellin has potent adjuvant activities ....................................................................19
2. Aim.........................................................................................................................20
2.1 Working hypothesis.................................................................................................20
2.2 Working program.....................................................................................................
3. Material and methods.....................................................................................22
3.1 Cloning, expression and purification of flagellin, allergens and flagellin:allergen
fusion proteins.........................................................................................................22
3.1.1 Cloning of flagellin A, rOva, and rflaA-Ova fusion protein .......................................
3.1.2 Expression and purification of flagellin A, rOva, and rflaA-Ova fusion protein ........24
3.1.3 cDNA-cloning of rflaA:Pru p 3 and rflaA:Ara h 2 fusion proteins .............................26
3.1.4 ion of recombinant allergens and fusion proteins..............28
3.1.5 Determination of protein concentration....................................................................29
3.1.6 Sodium dodecyl sulphate polyacrylamide gel electrophoresis ................................29
3.1.7 Reduction and alkylation .........................................................................................29
3.1.8 Limulus amebocyte lysate test ................................................................................29
3.1.9 Circular dichroism spectroscopy..............................................................................30
3.2 In vitro assays..........................................................................................................31
3.2.1 TLR5-activation assay.............................................................................................31
3.2.2 Mice.........................................................................................................................
3.2.3 In vitro generation of bone marrow derived murine dendritic cells ..........................31
3.2.4 stimulation of bone marrow derived murinecells32
3.2.5 Flow cytometry and intracellular cytokine staining...................................................33
3.2.6 Cytokine ELISAs......................................................................................................34
3.2.7 Blocking of endocytosis...........................................................................................34
3.2.8 Preparation of CD4 T cells ......................................................................................
3.2.9 Neutralisation assay................................................................................................35
3.3 Prophylactic and therapeutic intervention in the Ova-induced intestinal allergy
model.......................................................................................................................36
3.3.1 The model of Ova-induced intestinal allergy............................................................36
3.3.2 Prophylactic vaccination..........................................................................................36
3.3.3 Therapeutic 37
III3.3.4 Proliferation assay...................................................................................................38
3.3.4.1 Preparation of antigen presenting cells ...................................................................
3.3.4.2 Preparation of CD4 T cells from spleens and mesenterial lymph nodes.................39
3.3.4.3 CD4 T cell proliferation assay..................................................................................39
3.3.5 Staining of regulatory T cells ...................................................................................40
3.3.6 Determination of Ova-specific IgG1, IgG2a and IgE titers.......................................40
3.3.7 Quantification of Ova-specific IgG1, IgG2a and IgE levels in mouse sera ..............41
3.3.8 Multiplex analysis of cytokine level in sera ..............................................................41
3.3.9 Determination of cytokine levels in intestinal homogenates ....................................42
3.4 Statistical analysis...................................................................................................42
3.5 Oligonucleotides......................................................................................................43
3.6 Chemicals................................................................................................................44
3.7 Consumables and Equipment .................................................................................46
3.8 Antibodies50
3.9 Buffers.....................................................................................................................51
3.10 Culture media, cell lines, and animals .....................................................................52
4. Results .................................................................................................................54
4.1 Generation and quality assessment of recombinant proteins..................................54
4.1.1 cDNA-cloning and protein expression of rOva and rflaA:Ova54
4.1.2 cDNA-cloning and protein expression of rflaA:Ara h 2 ............................................55
4.1.3 Generation of rflaA, rflaA:Pru p 3, rPru p 3 and rAra h 2.........................................56
4.1.4 Recombinant proteins can be produced with high yield and purity using E. coli .....56
4.1.5 Flagellin fusion proteins aggregate due to intermolecular disulfide bonds ..............58
4.1.6 Recombinantly expressed proteins show a considerable amount of secondary
structure...................................................................................................................59
4.1.7 rflaA and rflaA:Ova are able to bind an