Superiority of ear skin for DNA immunization in mouse tumor models [Elektronische Ressource] / presented by Jing Ni

Nijing

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• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Superiority of ear skin for DNA
immunization in mouse tumor models




Dissertation
Submitted to the Faculty of Biosciences
of The Ruprecht-Karls-Universität Heidelberg, Germany
for the Degree of
Doctor of Natural Sciences



Presented by
Jing Ni

thOral examination at September 17 2008

This thesis was written in the Tumor Immunology of German Cancer
Research Centre (DKFZ, Heidelberg, Germany) in the duration period of
October 2005 to July 2008 under the supervision of Prof. Dr. Volker
Schirrmacher.









st1 Supervisor: Prof. Dr. Volker Schirrmacher, Tumor Immunology of German Cancer
Research Centre (Division of Cellular Immunology)
nd2 Supervisor: Prof. Dr. Michael Wink, Institute of Pharmacy and Molecular Biotech-
nology at the University of Heidelberg (Department of Biology)





I herewith declare that I wrote this PhD thesis independently under supervision and used no
other sources and aids than those indicated




…………………… ……….…………………
Date Signature
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"Immunization ranks among the most important health advances of the 20th century. With
the exception of safe drinking water, vaccinology has more effectively reduced mortality
than any other modality."


"Extraordinary advances in biotechnology make DNA vaccines the most promising area of
vaccinology."


James Mark Simmerman
http://findarticles.com/p/articles/mi_qa3958/is_200201/ai_n9053796/pg_1




“Tumor antigens are being rapidly revealed, and can be expressed on cell surface or more
commonly, as peptides in association with the major histocompatibility complex class I (or
II) molecules. DNA vaccines can be designed to activate antibody and /or T-cell responses,
providing focused immune attack on selected antigens.”


“DNA vaccines offer a precise but flexible strategy for delivering antigens to the immune
system, and additional sequences encoding molecules to manipulate outcome can be
included.”

Jason Rice et al.
Nat Rev Cancer 2008, 8: 108-20
Table of Contents
Table of Contents
IAcknowledgements
IIIContributions
IVAbstract
Zusammenfassung V
VIList of abbreviations
11 Introduction
11.1 Vaccine
1.1.1 The development of vaccine 1
1.1.2 Vaccine and immune responses 2
1.1.3 DNA vaccine 4
1.1.4 Improvement of DNA vaccine 6
1.1.4.1 Delivery methods 6
1.1.4.2 Adjuvants 7
1.1.5 Functions of dendritic cells in DNA vaccine 9
1.1.6 DNA immunization to ear pinna 9
1.2 Cancer therapy 11
1.2.1 Traditional cancer therapy 11
1.2.2 Cancer immunotherapy 12
1.3 Newcastle Disease Virus (NDV) 14
1.3.1 Application of NDV for cancer therapy 14
1.3.2 Functional study of NDV molecules 15
172 Aims of this thesis
183 Materials and Methods
3.1 Equipment 18
3.2 Molecular biological methods 20
3.2.1 Buffers and solutions 20
3.2.2 Preparation of DNA from bacteria 20
3.2.3 Cloning of DNA vectors 20
3.2.3.1 Preparation of DNA fragments by enzyme-cutting 20
Table of Contents
3.2.3.2 Preparation of DNA fragments by PCR 21
3.2.3.3 Extraction of DNA fragments from the gel 22
3.2.3.4 Commercial and ready-to-use plasmids 23
3.2.3.5 Cloning strategies 24
3.2.4 Determination of nucleic acid concentration 25
3.3 Cell biological methods 26
3.3.1 Buffers and solutions 26
3.3.2 Cell culture methods 27
3.3.2.1 Culture of cells 27
3.3.2.2 Freezing and thawing of cells 28
3.3.2.3 Determination of cell number and viability 28
3.3.3 Preparation of human PBMC 29
3.3.4 Generation of dendritic cells from murine bone marrow 29
3.3.5 Transfection of mammalian cells with jetPEI 30
3.3.6ammalian cells with lipofectamine 2000 30
3.3.7 Transfection of dendritic cells with Amaxa machine 31
3.3.8 Stable transfection of mammalian cells with jetPEI and polyMag 31
3.3.9 In vitro promoter activity 31
3.3.10 In vitro luciferase assay 32
3.3.10.1 Firefly luciferase assay 32
3.3.10.2 Firefly/Renilla dual luciferase assay 32
3.3.11 X-gal staining 32
3.3.12 FDG staining 33
3.4 Immunobiological methods 35
3.4.1 Buffers and solutions 35
3.4.2 ELISA 35
3.4.2.1 β-gal ELISA 35
3.4.2.2 Mouse IFN- γ and IL-4 ELISA 36
3.4.2.3 Human IFN- α ELISA 36
3.4.2.4 Mouse IFN- α ELISA 36
373.4.2.5 TGF- β ELISA
3.4.2.6 IL-10 ELISA 37
513.4.3 Cr release assay 37
3.4.4 Flow cytometry 38
3.4.5 Hemadsorption assay 41
Table of Contents
3.4.6 Immunohistochemistry 41
3.4.7 Preparation of cell lysates 41
3.5 In vivo experiments 43
3.5.1 Buffers and solutions 43
3.5.2 DNA immunization and electroporation 43
3.5.3 Tumor inoculation 44
3.5.4 Preparation of mouse serum 44
3.5.5 In vivo imaging of luciferase expression 44
3.5.6 Preparation of single cell suspension from murine organs 45
3.5.6.1 Spleen 45
3.5.6.2 Lymph node 45
3.5.6.3 Peripheral blood 45
3.5.6.4 Bone marrow 46
3.5.6.5 Tumor 46
3.5.6.6 Ear 46
3.5.7 Staining of metastases 46
3.6 Statistical methods 47
4 Results 48
4.1 Superiority of ear pinna to flank skin for antigen expression and
induction of immune responses by DNA immunization 48
4.1.1 Comparison of antigen expression in ear pinna and flank skin 48
4.1.2 Humoral responses by ear pinna or flank skin DNA immunization 49
4.1.3 Cellular responses by ear pinna or flank skin DNA immunization 50
4.1.3.1 Cytotoxicity 50
514.1.3.2 IFN- γ and IL-4 secretion
4.2 Adjuvant effect of Newcastle disease virus HN gene for ear pinna DNA
vaccination with beta-galactosidase as a surrogate tumor antigen 52
4.2.1 in vitro activity of the HN molecule 52
4.2.1.1 Construction of a plasmid encoding the HN gene 52
4.2.1.2 Cell binding activity of HN 53
4.2.1.3 IFN- α induction activity of HN 53
4.2.2 in vivo activity by HN DNA injection 54
4.2.2.1 Serum IFN- α induction by NDV administration 54
4.2.2.2 Serum IFN- α induction by HN DNA ie immunization 54
4.2.2.3 Prophylactic anti-tumor effect 55
Table of Contents
4.2.3 Adjuvant effect of HN in prophylactic mouse lymphoma models 55
4.2.3.1 Construction of plasmids encoding HN and lacZ genes 56
4.2.3.2 Adjuvant effect of HN in the Eb-lacZ tumor model 57
4.2.3.3 Adjuvant effect of HN in the ESb-lacZ tumor model 57
4.3 Adjuvant effect of HN gene for ear pinna DNA vaccination with tumor
associated antigen EpCAM 62
4.3.1 Construction of plasmids encoding HN and EpCAM genes 62
4.3.2 Adjuvant effect of HN in prophylactic mammary carcinoma models 63
4.3.3 Adjuvant effect of HN in a prophylactic colon carcinoma models 66
4.3.3.1 Generation of CT26EP with stable human EpCAM expression 66
4.3.3.2 MHC I expression on the cell surface with IFN- α treatment 66
4.3.3.3 Improvement of prophylactic anti-tumor effect by HN 67
4.3.4 Adjuvant effect of HN in therapeutic mouse tumor models 68
4.3.4.1 DNA treatment started from day 4 after tumor cell inoculation 68
4.3.4.1.1 Therapeutic anti-tumor effect 69
4.3.4.1.2 Serum antibody level in tumor-bearing mice 70
4.3.4.1.3 Lung metastases 72
4.3.4.2 DNA treatment started from day 7 after tumor cell inoculation 72
4.3.5 Influence of humoral and cellular immune responses by HN 76
4.3.5.1 Influence of humoral immune responses 76
4.3.5.1 Influence of cellular immune responses 76
4.3.6 Adoptive transfer of antigen specific splenocytes 78
4.3.6.1 Stable transfection of firefly luciferase in DA3/DE 79
4.3.6.2 Adoptive transfer of antigen specific splenocytes 83
4.3.7 Adjuvant effect of HN in immuno-deficient mice 85
4.3.8 Adjuvant effect of HN in tumor infiltrated lymphocytes 87
4.4 Improvement of ear pinna DNA vaccination by electroporation 90
4.4.1 Parameters for electroporation 90
4.4.2 Optimization of DNA injection volume to ear pinna and flank skin 91
4.4.3 Optimization of DNA electroporation voltage 91
4.4.4 Improvement of long-term antigen expression 94
4.4.5 Improvement of humoral immune responses 94
4.4.6 Improvement of cellular immune 96
4.4.6.1 Cytotoxicity 96
4.4.6.2 IFN- γ and IL-4 secretion 97
4.4.7 Improvement of prophylactic anti-tumor effect 97
Table of Contents
4.4.8 Improvement of therapeutic anti-tumor effect 98
4.4.9 Down-regulation of suppressive factors 100
4.5 Crucial function of dendritic cells in ear pinna DNA immunization 101
4.5.1 Distribution of dendritic cells in ear pinna and flank skin 101
4.5.2 Generation of a short murine CD11c promoter 102
4.5.2.1 Verification of the functional region of murine CD11c promoter 102
4.5.2.2 in vitro specific activity of the CD11cS and CD11cL promoters 104
4.5.2.3 in vivo activity of the CD11cS and CD11cL promoters in mice 105
4.5.3 Verification of the specific activity of the CD11cS promoter in vivo 106
4.5.3.1 in vivo activity in mouse muscle tissue 106
4.5.3.2 in vivo activity in dendritic cell-depleted mice 106
4.5.4 Comparison of CMV and CD11c