Characterization of a major neutralizing epitope on the yellow fever virus envelope protein using human recombinant monoclonal antibody fragments generated by phage display [Elektronische Ressource] / vorgelegt von Stephane Daffis

Stephane Xxx

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• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2006
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Aus dem Medizinischen Zentrum für Hygiene und Infektionsbiologie
der Philipps-Universität Marburg
Institut für Virologie
Geschäftsführender Direktor: Prof. Dr. Hans-Dieter Klenk
Characterization of a major neutralizing
epitope on the yellow fever virus
envelope protein using human
recombinant monoclonal antibody
fragments generated by phage display
Inaugural-Dissertation
zur Erlangung des Doktorgrades der Humanbiologie
(Dr. rer. physiol.)
dem Fachbereich Medizin
der Philipps-Universität Marburg
vorgelegt von
Stephane Daffis
aus Montgivray, Frankreich.
Marburg, Deutschland
May 2006Angenommen vom Fachbereich Medizin
der Philipps-Universität Marburg am: 17. Mai 2006
Gedruckt mit Genehmigung des Fachbereichs
Dekan: Herr Prof. Dr. Bernhard Maisch
Referent: Herr PD. Dr. Jan ter Meulen
Korreferent: Herr Prof. Dr Tim Plant To my wife, Fanny
To my parentsINDEX
SUMMARY 1
I-INTRODUCTION 4
I-1 . Yellow fever (YF). 4
I-1-1. Disease. 4
I-1-2. Epidemiology. 4
I-1-3. Resurgence of yellow fever as a major public health problem. 5
I-1-4. Transmission cycles. 5
I-2. Yellow fever virus (YFV). 6
I-2-1. Taxonomy of flaviviruses. 6
I-2-2. Classification of the yellow fever virus. 6
I-2-3. Structure of the virion. 7
I-2-4. Pathogenesis. 9
I-2-5. The Flavivirus life cycle. 10
I-2-6. Cellular receptor(s) for Flaviviruses. 10
I-2-7. Flavivirus fusion with host cell membranes. 11
I-3. The YFV envelope protein (the E protein). 12
I-3-1. Molecular Structure. 13
I-3-2. Antigenic structure. 13
I-4 The Yellow Fever vaccine. 17
I-4-1. The French Neurotropic Vaccine (FNV). 17
I-4-2. The 17D vaccine. 18
I-5 Immune Response to YFV. 20
I-5-1. Innate immune response. 20
I-5-2. Adaptive immune response. 21
I-6. Adverse effects following YF 17D vaccination. 22
I-6-1. The YELlow fever-Associated Neurotropic Disease (YEL-AND). 22
I-6-2. The YELlow fever-Associated Viscerotropic Disease (YEL-AVD). 23
I-7. Antibodies. 23
I-7-1. Structure of antibodies. 24
I-7-2. Germline organization of the genetic loci of antibodies. 26
I-7-3. Somatic recombination, affinity maturation and isotype switching in
antibody diversity. 26
I-7-4. Antiviral antibodies and their mechanism of action. 28
I-7-5. Monoclonal antibodies and biological applications. 28
I-8. Phage Display technology. 29
I-8-1. Principle. 29
I-8-2. Filamentous phages. 30INDEX
I-8-3. Phage display system. 30
I-8-4. Phage-displayed scFv libraries. 32
I-8-5. Selection of antibody libraries: “Biopanning”. 34
I-8-6. Human viruses neutralized by recombinant antibody fragments. 36
I-9. Aim of the work 37
II-MATERIAL 39
II-1. Plasticware. 39
II-2. Chemicals. 39
II-3. Enzymes. 40
II-4. Antibodies 40
II-5. Radioactive amino acids. 40
II-6. Commercial Kits. 40
II-7. Vectors. 41
II-8. Viruses. 41
II-9. Bacteria and phages. 42
II-10. Eukaryotic Cells. 42
II-11. PCR primers. 42
II-11-1. Degenerated primers specific for the VH and VL genes. 42
II-11-2. pHEN3-specific primers. 44
II-11-3. YFV E, NS1 and prM proteins-specific primers. 44
II-12. Buffers and solutions. 44
II-12-1. Virus purification. 45
II-12-2. Phage purification. 45
II-12-3. DNA electrophoresis. 45
II-12-4. ELISA. 45
II-12-5. SDS PAGE and Western Blot. 45
II-12-6. Dot Blot buffers. 46
II-12-7. Protein purification 46
II-12-8. pH shift buffers. 47
II-13. Media for bacterial culture. 47
II-14. Media for cell culture. 48
III-METHODS 50
III-1. Schematic representation of the construction of the phage libraries
expressing recombinant antibody fragments (scFvs). 50
III-2. Isolation of lymphocytes from two African donors who recovered from
yellow fever. 52INDEX
III-3. First strand cDNA synthesis. 52
III-4. PCR amplification of the Variable Kappa Light chain (VLκ), Variable Lambda
Light chain (VLλ) and Variable Heavy chain (VH) genes of antibodies. 53
III-4-1. PCR conditions and protocol. 53
III-4-2. Combination of degenerated primers used to amplify the Variable
Kappa Light chains (VLκ). 54
III-4-3. Combination of degenerated primers used to amplify the Variable
Lambda Light chains (VLλ). 55
III-4-4. Combination of degenerated primers used to amplify the Variable
Heavy chains (VH). 56
III-5. Cloning of VLκ and VLλ PCR products into the pHEN3 phagemid to generate
VLκ and VLλ bacterial sub-libraries. 56
III-5-1. Digestion of the VLκ and VLλ PCR products (reactions 1b to 17b
obtained from PCR 1b) and the phagemid pHEN3. 56
III-5-2. Dephosphorylation of the pHEN3 vector. 57
III-5-3. Ligation. 57
III-5-4. Transformation of electrocompetent TG1 E.coli cells by electroporation
and plating of VLκ and VLλ sub-libraries. 58
III-5-5. Estimation of the library size. 58
III-5-6. DNA preparation of VLκ and VLλ sub-libraries. 59
III-6 Cloning of VH PCR products into VLκ-pHEN3 and VLλ-pHEN3 phagemids to
generate the VLκ-VH and VLλ-VH final bacterial libraries. 59
III-7. Colony PCR. 60
III-8. Rescue of recombinant phages displaying scFV fragments from the final
bacterial libraries. 61
III-8-1. Preparation of a helper phage working stock. 61
III-8-2. Rescue of recombinant phages from constructed scFv libraries. 62
III-9. Preparation and purification of YFV 17D-204-WHO particles. 63
III-9-1. Preparation of a YFV 17D-204-WHO master stock. 63
III-9-2. Titer determination by plaque assay. 63
III-9-3. Purification of YFV 17D-204-WHO particles. 64
III-9-4. Determination of purified YFV 17D-204-WHO particles antigenicity in
ELISA. 64
III-10. Biopanning. 65
III-10-1. Selection step. 65
III-10-2. Rescue of phages isolated from the first round of selection. 65
III-10-3. Polyclonal phage ELISA. 66
III-10-4. Monoclonal phage ELISA. 66INDEX
III-10-5. BstNI fingerprintings. 66
III-11. Expression and purification of scFvs in E.coli TG1 cells. 67
III-11-1. Cloning in the prokaryotic expression plasmid pAB1. 67
III-11-2. Expression of scFvs in E.coli cells. 67
III-11-3. ScFv purification by Immobilized Metal ion Affinity Chromatography
(IMAC). 68
III-11-4. Determination of scFvs by Coomassie staining and Western Blot
analysis. 68
III-11-5. scFv ELISA. 69
III-12. Dot Blot analysis. 70
III-13. Radioimmunoprecipitation assay (RIPA). 70
III-13-1. Production of radiolabeled soluble viral proteins from radiolabeled YF
17D-204-WHO virions. 70
III-13-2. RIPA. 71
III-14. pH sensitivity experiments. 71
III-15. Competition ELISA using biotinylated antibodies. 71
III-15-1. Biotinylation. 71
III-15-2. Competition ELISA. 72
III-16. Plaque reduction neutralization test (PRNT). 72
III-17. Generation of escape mutants. 72
III-18. Microneutralization assay. 73
III-18-1. Determination of the 50% Tissue Culture Infectious Dose (TCID ). 7350
III-18-2. Microneutralization assay. 73
III-19. Sequencing analysis. 74
III-19-1. Sequencing analysis of scFvs displayed by YFV-17D-204-WHO specific
monoclonal phages 74
III-19-2. Sequencing analysis of prM and E proteins of all YFV strains (17D-204
WHO, wild-type strains and escape mutants). 74
III-20. Molecular Modelling. 75
IV-RESULTS 77
IV-1. Generation of two phage libraries displaying recombinant antibody
fragments (scFvs) from two recovered yellow fever patients. 77
IV-1-1. Amplification of the Variable Kappa light chains (VLκ), the Variable
Lambda light chains (VLλ) and the Variable Heavy chains (VH) genes by PCR.77
IV-1-2. Construction of bacterial scFv libraries and rescue of recombinant
phages displaying scFvs on their surface. 78INDEX
IV-2. Isolation of monoclonal phages with a specific affinity for YFV 17D-204-
WHO particles. 79
IV-2-1. Purification of YFV 17D-204-WHO particles. 79
IV-2-2. Enrichment of specific phage binders to the YFV antigen through
biopanning. 80
IV-2-3. Screening of monoclonal phages from round 3 and round 4 of the
selection step. 81
IV-2-4. Genetic diversity of scFvs displayed by YFV 17D-204-WHO-specific
binders. 81
IV-3. Expression and purification of six different scFvs (7A, 5A, R3(27), 1A, 2A
and R3(9)) with a specific affinity for YFV 17D-204-WHO virions. 82
IV-3-1. Expression and purification of scFv-7A, 5A, R3(27), 1A, 2A and R3(9) as
soluble molecules. 82
IV-3-2. Reactivity of soluble scFv-7A, 5A, R3(27), 1A, 2A and R3(9) with the
YFV 17D-204-WHO antigen. 83
IV-3-3. Sequencing analysis of scFvs-7A, 5A, R3(27), 1A, 2A and R3(9). 84
IV-4. Identification of the YFV proteins recognized by the scFvs-7A, 5A, R3(27),
1A, 2A and R3(9) 85
IV-4-1. Western Blot analysis. 85
IV-4-2. Dot Blot analysis. 85
IV-4-3. Radioimmunoprecipitation assay (RIPA). 86
IV-5. Competition ELISA. 87
IV-6. pH sensitivity of the epitopes. 88
IV-7. Neutralization assays. 89
IV-7-1. Plaque Reduction Neutralization Test (PRNT) using the YFV 17D 204-
WHO (vaccine strain) and YFV wild-type strain Asibi. 89
IV-7-2. Plaque Reduction Neutralization Test (PRNT) using 5 wild-type YFV
strains representing three of the five known African genotypes. 90
IV-8. Generation of YFV 17D-204 WHO variants exhibiting resistance to scFv-7A
neutralization. 92
IV-9. Identification of amino acid