New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy:
Gespeichert in:
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| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2004
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| Ausgabe: | [Maschinenschr.] |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Erlangen-Nürnberg, Univ., Diss., 2004 |
| Beschreibung: | 108 Bl. Ill., graph. Darst. |
Internformat
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| 245 | 1 | 0 | |a New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy |c von Mohamed Ibrahim Hussein El-Sayed |
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Datensatz im Suchindex
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| adam_text | Titel: New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination
Autor: Sayed, Mohamed Ibrahim Husseiny el-
Jahr: 2004
Contents
CONTENTS
1 INTRODUCTION 1
1.1 Vaccine development 1
1.2 Live attenuated bacteria as antigen delivery systems 2
1.3 Modulation of the immune responses stimulated by live bacterial carriers 3
1.4 Pathogenicity of Salmonella 4
1.4.1 Pathogenicity Islands of Salmonella 5
1.5 The immune response to Salmonella 9
1.5.1 The acquired immune response to S. Typhimurium 9
1.6 Current vaccines against Salmonella 11
1.6.1 Subunit vaccines 12
1.6.2 Live attenuated S. enterica serovar Typhimurium vaccines 12
1.7 S. Typhimurium as carrier for delivery of heterologous antigens 14
1.7.1 Current limits in the development of recombinant Salmonella vaccines 15
1.8 Aims of the work 18
2 MATERIALS AND METHODS 19
2.1 Bacterial strains and culture conditions 19
2.2 Genetic Methods 23
2.2.1 DNA-Standard methods 23
2.2.2 Amplification of DNA by PCR 23
2.3 Protein analysis and Western blotting 25
2.3.1 Western-Blot-Analysis 25
2.4 Cell culture and infection studies 26
2.4.1 Bacterial infection of macrophages and survival assay 26
2.4.2 Bacterial infection of HeLa cells 27
2.4.3 Immunofiuorescence 27
2.5 Construction of expression plasmids 27
2.5.1 Construction of OVA expression cassettes in pWSK29 27
2.5.2 Construction of OVA expression cassettes in pLAT389 28
2.5.3 Construction of GFP expression cassette in pLAT389 28
2.5.4 Construction of P60 and Llo expression cassettes in pWSK29 29
2.5.5 Construction of SseF-OVA-M45 29
- -
Contents
2.5.6 Chromosomal integration 30
2.6 Animal immunization 32
2.6.1 Immunization protocols 32
2.6.2 Determinations of bacterial counts in mouse organs 32
2.6.3 Sample collection for antibody titer analysis 33
2.6.4 Mice, adoptive transfer, and immunization 33
2.6.5 Protection assay 34
2.6.6 Mouse mixed infection studies 34
2.7 Quantification of antibody titers 34
2.8 Antibodies 35
3 RESULTS 37
3.1 Construction of the expression plasmids 37
3.2 Regulated expression of heterologous antigens by a SPI2-derived promoter 39
3.2.1 Analysis of gene expression in vitro by Western blot 39
3.2.2 Synthesis of P60 or Llo proteins by recombinant Salmonella strains 41
3.2.3 Analysis of gene expression in vitro by FACS 42
3.3 Expression of model antigens by recombinant Salmonella inside host cells 44
3.3.1 Analysis of gene expression in RAW macrophages by FACS 44
3.3.2 Analysis of gene expression in RAW macrophages by
immunofiuorescence 48
3.3.3 Intravacuolar expression of heterologous antigens from SPI2 promoters... 50
3.4 Plasmid stability 51
3.4.1 In vitro stability of plasmid in recombinant Salmonella strains 51
3.4.2 In vivo persistence of recombinant strains 53
3.5 Humoral immune responses 54
3.5.1 Generation of antibody response after oral vaccination 54
3.5.2 Induction of mucosal immune response after oral vaccination 55
3.6 Cellular immune responses 57
3.6.1 Ovalbumin model antigen 57
3.6.2 Demonstration of T cells in organs after CFSE-labeling 58
3.6.3 Induction of OVA-specific T cells 59
3.7 Elicitation of protective immunity 62
3.8 Chromosomal integration of expression cassettes for antigens
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Contents
( knock-in approach) 64
3.8.1 Rationale for the integration of expression cassettes into the Salmonella
chromosome 64
3.8.2 Stability of expression after chromosomal integration 66
3.8.3 Attenuation of virulence by knock-in mutagenesis 68
3.8.4 Expression cassettes are functional after insertion into various loci 69
3.9 Analysis of translocation of vaccine antigens by the TTSS of Salmonella 73
3.9.1 Synthesis of a SseF-OVA fusion protein in vitro 73
3.9.2 Translocation of the SseF-OVA-M45 fusion protein by intracellular SPI2 74
3.9.3 Translocation of a SseF-Llo fusion protein by the SPI2-encoded TTSS 75
4 DISCUSSION 78
4.1 Intracellular-activated promoters for vaccination 78
4.2 Requirement for low copy plasmid constructs 79
4.3 A SPI2-deficient strain as vaccine carrier 80
4.4 Modulation of immune responses 81
4.5 Chromosomal integration (Knock-in approach) 83
4.6 Translocation of fusion proteins by the SPI2-encoded TTSS 84
4.7 Conclusion and Outlook 85
5 SUMMARY 87
6 REFERENCES 89
7 ABBREVIATIONS 104
8 CURRICULUM VITAE 107
9 ACKNOWLEDGMENT 108
_
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| spelling | Sayed, Mohamed Ibrahim Husseiny el- 1969- Verfasser (DE-588)129731781 aut New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy von Mohamed Ibrahim Hussein El-Sayed [Maschinenschr.] 2004 108 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Erlangen-Nürnberg, Univ., Diss., 2004 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=013015486&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Sayed, Mohamed Ibrahim Husseiny el- 1969- New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy |
| subject_GND | (DE-588)4113937-9 |
| title | New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy |
| title_auth | New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy |
| title_exact_search | New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy |
| title_full | New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy von Mohamed Ibrahim Hussein El-Sayed |
| title_fullStr | New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy von Mohamed Ibrahim Hussein El-Sayed |
| title_full_unstemmed | New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy von Mohamed Ibrahim Hussein El-Sayed |
| title_short | New approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy |
| title_sort | new approaches to optimize salmonella enterica serovar typhimurium strains as tools for vaccination and immunotherapy |
| topic_facet | Hochschulschrift |
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