Immunomonitoring technologies for the evaluation of modified vaccinia virus Ankara expressing HIV-1 nef as a vaccine against AIDS [Elektronische Ressource] / Sarah Kutscher

Sarah Kutscher

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 142
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der LudwigMaximiliansUniversität München



Immunomonitoring technologies for the evaluation of
Modified Vaccinia Virus Ankara expressing HIV1 nef as
a vaccine against AIDS



Sarah Kutscher

aus

HeilbronnNeckargartach

2010


Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 von Frau Professor Ulrike Protzer betreut und von Herrn Professor Martin Biel
vor der Fakultät für Chemie und Pharmazie vertreten.






Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfsmittel erarbeitet.




München, am 10.02.2010






………………………………………….
(Unterschrift des Autors)










Dissertation eingereicht am: 11.02.2010

1. Gutachter Frau Professor Ulrike Protzer

2. Gutachter Herr Professor Martin Biel

Mündliche Prüfung am: 05.07.2010
I TABLE OF CONTENS

I TABLE OF CONTENS................................................................................... 3
II ABBREVIATION LIST ................................................................................. 6
1 ZUSAMMENFASSUNG.............................................................................. 8
2 SUMMARY.................................................................................................10
3 INTRODUCTION ......................................................................................12
3.1 HUMAN IMMUNODEFICIENCY VIRUS (HIV)......................................................................... 12
3.1.1 Epidemiological profile ................................................................................................... 12
3.1.2 Virological profile ........................................................................................................... 13
3.1.3 Clinical profile................................................................................................................. 17
3.1.4 Therapy options ............................................................................................................... 18
3.2 VACCINATION ...................................................................................................................... 21
3.2.1 Prophylactic HIV vaccination ......................................................................................... 21
3.2.2 Therapeutic HIV vaccination........................................................................................... 23
3.3 MODIFIED VACCINIA VIRUS ANKARA (MVA) AND MVANEF........................................... 24
3.3.1 MVA................................................................................................................................. 24
3.3.2 Nef.................................................................................................................................... 25
3.3.3 MVA'HIV'1LAI'nef (MVA'nef) ......................... .............................................................. 26
3.4 IMMUNONMONITORING TECHNOLOGIES .............................................................................. 27
3.4.1 Flow cytometry ................................................................................................................ 27
3.4.2 T'cell immunomonitoring ................................................................................................ 28
4 RESULTS....................................................................................................31
4.1 DEVELOPMENT OF FLOWCYTOMETRY BASED ASSAYS FOR THE CHARACTERIZATION OF T
CELL IMMUNE RESPONSES.................................................................................................... 31
4.1.1 Establishment of 9 – color Intracellular cytokine staining (ICS).................................... 31
4.1.2 Establishment of a Carboxyfluorescein succinimidyl ester (CFSE) 'based proliferation
assay ................................................................................................................................ 35
4.1.3 Data processing and storage........................................................................................... 36
4.1.4 Characterization of the phenotype in ICS and MHC class I tetramer staining............... 38
4.1.5 Establishment of an analysis system based on the evaluation of of IFN'γ+ MIP'1β+ T'
cells.................................................................................................................................. 40 I TABLE OF CONTENS

4.1.6 Comparison of the IFNγ+ MIP1β+ data analysis system in comparison to the ELISPOT
assay ................................................................................................................................ 44
4.1.7 Influence of the variation in cell number input in the ICS assay..................................... 47
4.2 REEVALUATION OF A THERAPEUTIC VACCINATION TRIAL USING MVANEF IN HIV1
INFECTED INDIVIDUALS........................................................................................................ 48
4.2.1 MVA'nef vaccination increases the magnitude of the total Nef'specific CD4 T'cell
response........................................................................................................................... 48
4.2.2 MVA'nef vaccination induces Nef'specific CD4 and CD 8 T cells able to proliferate .... 50
4.2.3 MVA'nef vaccination increases the grade of functionality of CD4 T cells...................... 51
4.2.4 Detailed analysis of the CD4 T'cell immune response throughout the trial in a single
subject V04 ...................................................................................................................... 53
4.2.5 Functional characteristics of the proliferating Nef'specific CD4 and CD8 T cells........ 55
4.2.6 Correlation between CD4 T'cell responses and CD8 T' cell proliferative activity ......... 56
4.2.7 Vaccine induced polyfunctional CD4 T cells produce high quantities of cytokines........ 57
4.2.8 Comparison of the CD4 T'cell immune response in MVA'nef vaccinated subjects to CD4
T'cell responses in persistent but controlled viral infections regarding functionality.... 60
5 DISCUSSION..............................................................................................62
5.1 ESTABLISHMENT OF FLOWCYTOMETRY BASED ASSAYS FOR THE CHARACTERIZATION OF T
CELL IMMUNE RESPONSES.................................................................................................... 63
5.2 REEVALUATION OF A THERAPEUTIC VACCINATION TRIAL USING MVANEF IN HIV1
INFECTED INDIVIDUALS........................................................................................................ 66
6 CONCLUSIONS.........................................................................................71
7 MATERIALS AND METHODS...............................................................73
7.1 PATIENTS AND SAMPLES ...................................................................................................... 73
7.1.1 Mva'nef vaccination trial ................................................................................................ 73
7.1.2 IFN'γ+ MIP'1β+ data evaluation and ELISPOT comparis on........................................ 74
7.1.3 Evaluation of CMV and EBV specific immune responses in healthy volunteers............. 74
7.2 MATERIALS .......................................................................................................................... 75
7.2.1 Reagents and solutions .................................................................................................... 75
7.2.2 Peptides ........................................................................................................................... 76
7.2.3 Antibodies ........................................................................................................................ 78
7.2.4 Consumables.................................................................................................................... 79
7.2.5 Laboratory equipment ..................................................................................................... 79
7.2.6 Software........................................................................................................................... 80
7.3 METHODS ............................................................................................................................. 81
4 I TABLE OF CONTENS

7.3.1 Purification of Peripheral Blood Mononuclear Cells (PBMC) using Ficoll density
gradient....................