Characterization of the molecular and immunological properties of Acanthocheilonema viteae tropomyosin [Elektronische Ressource] / von Michal Janusz Sereda

Michal Janusz Sereda

Sujets

• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2009
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

Characterization of the molecular and immunological properties
of Acanthocheilonema viteae tropomyosin.


Dissertation
zur Erlangerung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Michał Janusz Sereda (M.Sc. Biology)
geboren am 04.06.76 in Warschau, Polen




Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Christoph Markschies
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Dr. Christian Limberg

Gutachter/innen: 1. Prof. Dr Richard Lucius
2. Prof. Dr Norbert Brattig
3. PD Dr Susanne Hartmann

Tag der mündlichen Prüfung: 14.02.08 Table of contents
I. Summary

II. Zusammenfassung

11 1. Introduction………………………………………………………………………………..
1.1. Filarial infections......................................................................................................... 11
1.2. Filarial proxi models for research………………………………………………………... 13
1.3. The A. viteae proxi model for O. volvulus……………………………………………… 13
1.4. Immunological aspects of Helminth infections……………………………………….. 15
1.5. Tropomyosin................................................................................................................ 16
1.6. Structure and function of tropomyosin…………………………………………………. 17
1.7. Conserved structure of tropomyosin......................................................................... 18
1.8. Tropomyosin isoforms in nematodes........................................................................ 19
1.9. Allergenicity of tropomyosin and cross-reactivity among different species……. 19
1.10. Tropomyosin as a vaccine candidate…………………………………………………… 20
1.11. Aims and objectives of this study………………………………………………………. 22

23 2. Results……………………………………………………………………………………….
2.1. Molecular cloning of A. viteae and O. volvulus tropomyosins……………………... 23
2.2. Alignment of tropomyosin sequences of A. viteae and O. volvulus with other
invertebrates and vertebrates reveals a great level of similarity…………………... 23
2.3. Purification of filarial tropomyosins.......................................................................... 25
2.3.1. Recombinant tropomyosin of A. viteae and O. volvulus…………………………….. 25

2.3.2. Purification of tropomyosin by electroelution from SDS- PAGE gel……………….. 26

2.3.3. Purification of native A. viteae tropomyosin by affinity chromatography with
monoclonal antibody…………………………………………………………………….. 27
2.3.4. Both eAvTropo and rAvTropo retain the secondary structure of an -helical
27 protein…………………………………………………………………………………......
2.4. Transfected COS7 cells express A. viteae tropomyosin after 48 h………………... 28
2.5. Use of tropomyosin based vaccine against A. viteae challenge infections……… 29
2.5.1. Recombinant protein vaccine efficacy depends on adjuvant used……………….... 30 2.5.2. Vaccination with worm derived A. vitae tropomyosin………………………………... 31
2.5.3. Single peptide vaccination....................................................................................... 32
2.5.4. cDNA based vaccines effects.................................................................................. 33
2.5.5. Effect of vaccination on parasite length and development………………………….. 35
2.6. Analysis of antibody responses of immunized jirds and mice……………………... 35
2.6.1. rAvtropo vaccination elicits IgM and IgG that are not restimulated in the course of
infection…………………………………………………………………………………… 35
2.6.2. IgG subclasses........................................................................................................ 37
2.6.3. Immunization with A. viteae tropomyosin induces IgE responses…………………. 38
2.6.4. The role of specific anti-tropomyosin antibodies in protecton against L3
challenge…………………………………………………………………………………. 39
2.7. Analysis of antibody responses of immunized jirds and mice……………………... 40
2.7.1. Screening of peptide libraries revealed 13 IgG epitopes……………………………. 40
2.7.2. A. viteae tropomyosin has 11 IgE epitopes…………………………………………… 42
2.7.3. Key amino acid residues within A. viteae tropomyosin IgE epitopes are important
for antibody binding……………………………………………………………………… 45
2.8. Allergenicity of tropomyosin...................................................................................... 50
2.8.1. A. viteae tropomyosin is a potent IgE inducer………………………………………... 50
2.8.2. Nematode tropomyosins are strongly cross-reactive……………………………… 52
2.8.3. Determination of the cross-reactivity degree between tropomyosins by
competition assay……………………………………………………………………….. 54
2.9. Raising monoclonal antibodies against A. viteae tropomyosin……………………. 55
2.9.1. NR1 but not R21 and N11 mAbs is specific to invertebrate tropomyosins………... 55
2.9.2. Epitopes of NR1 and R21 mAb are located in C-terminal part of A. viteae
tropomyosin………………………………………………………………………………. 57
2.9.3. N11 is presumably targeted against posttranslational modification on A. viteae
tropomyosin……………………………………. 58
2.9.4. Tropomyosin is a surface component of A. viteae L3……………………………….. 59
2.9.5. Tropomyosin specific mAb help to kill microfilariae in vitro by ADCC……………… 60
2.10. T cell responses to tropomyosin………………………………………………………… 64
2.10.1. Both, the whole molecule and synthetic peptides based on A. viteae tropomyosin
are capable to stimulate T cell proliferation…………………………………………… 64
2.11. A. viteae tropomyosin influences expansion of various cell populations after
immunization………………………………………………………………………………… 66
2.11.1. Splenocytes from animals immunized with A. viteae tropomyosin produce IL-4
66 and IL-10 but no INF-
+ +
2.11.2. Immunization with A. vitae tropomyosin expand cells Gr1 /CD11b in spleen……. 67 + +
2.11.3. Gr1 /CD11b subpopulation of splenocytes is responsible for production of IL-4
and IL-10………………………………………………………………………………….. 70
+ +
2.11.4. The subpopulation of Gr1 /CD11b cells raises in peritoneal exudate after
immunization with rAvTropo……………………………………………………………. 72
2.11.5. The constitution of PECs after immunization with A. viteae tropomyosin…………. 74


76 3. Discussion…………………………………………………………………………………
3.1. Tropomyosin of A. viteae as a vaccine…………………………………………………. 76
3.2. Allergenicity of A. viteae tropomyosin………………………………………………….. 84
3.3. mAb against A. viteae tropomyosin, their epitopes and specificity……………….. 90
3.4. Cellular alteration and cytokine profile induced by A. viteae tropomyosin……… 92
Outlook………………………………………………………………………………………... 96 3.5


97 4. Methods…………………………………………………………………………………......
4.1. Parasitological methods…………………………………………………………………… 97
4.1.1. Laboratory animals………………………………………………………………………. 97
4.1.2. Maintenance of the life cycle of Acanthocheilonema viteae………………………… 97
4.1.3. Immunisation experiments……………………………………………………………… 97
+ +
4.1.4. Antigen injection in Gr1 /CD11b experiments……………………………………….. 98
4.1.5. Quantification of microfilarial load in blood of jirds…………………………………… 98
4.1.6. Isolation of adult A. viteae from M. unguiculatus……………….. 98
4.1.7. Isolation of L3 stages from the vector Ornithodoros moubata……………………… 99
4.2. Cell culture methods……………………………………………………………………..... 99
4.2.1. Maintenance of mammalian cells……………………………………………………… 99
4.2.2. Preparation of stocks……………………………………………………………………. 99
4.2.3. Transfection of COS7 and HeLa cells…………………………………………………. 100
4.2.4. Raising the monoclonal antibody………………………………………………………. 100
4.2.5. Subcloning………………………………………………………………………………... 101
4.2.6. Rat Basophil Leukemia (RBL) cell mediator release assay………………………… 101
4.2.7. T cells proliferation assay………………………………………………………………. 101
4.3. Immunochemical and immunological methods……………………………. 102
4.3.1. Bleeding of animals for production of sera…………………………………………… 102
4.3.2. Western blot……………………………………………………………………………… 102
4.3.3. Detection of specific antibody in sera of M. unguiculatus and BALB/c mice……… 103
4.3.4. Immunostaining of A. viteae larvae……………………………………………………. 103
4.3.5. Creation of the synthetic peptides libraries…………………………………………... 103 4.3.6. Screening of synthetic peptide libraries……………………………………………….. 104
4.3.7. ELISA………………………………………………………………... 104
4.3.8. IgE ELISA……………………………………………………………………… 105
4.3.9. Cytokine ELISA………………………………………………………………………….. 105
4.3.10. Flow cytometry (FACS)…………………………………………………………………. 105
4.3.11. Depletion of IgG antibodies from sera of immunized mice and jirds……………….. 106
4.3.12. Deglycosylation of proteins on ELISA plate…………………………………………... 106
4.4. Protein analytical methods………………………………………………………………... 107
4.4.1. Preparation of soluble protein extracts………………………………………………... 107
4.4.2. Determination of protein concentration………………………………………………... 107
4.4.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)……….. 107
4.4.4. Maleylation of rAvTropomyosin………………………………………………………… 108
4.4.5. Preparation of gel-eluted antigen……………………………………………. 108
4.4.6. Maleyation of recombinant A. vitae trop