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

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Aus dem Medizinischen Zentrum für Hygiene und Infektionsbiologieder Philipps-Universität MarburgInstitut für VirologieGeschäftsführender Direktor: Prof. Dr. Hans-Dieter KlenkCharacterization of a major neutralizingepitope on the yellow fever virusenvelope protein using humanrecombinant monoclonal antibodyfragments generated by phage displayInaugural-Dissertationzur Erlangung des Doktorgrades der Humanbiologie(Dr. rer. physiol.)dem Fachbereich Medizinder Philipps-Universität Marburgvorgelegt vonStephane Daffisaus Montgivray, Frankreich.Marburg, DeutschlandMay 2006Angenommen vom Fachbereich Medizinder Philipps-Universität Marburg am: 17. Mai 2006Gedruckt mit Genehmigung des FachbereichsDekan: Herr Prof. Dr. Bernhard MaischReferent: Herr PD. Dr. Jan ter MeulenKorreferent: Herr Prof. Dr Tim Plant To my wife, Fanny To my parentsINDEXSUMMARY 1I-INTRODUCTION 4I-1 . Yellow fever (YF). 4I-1-1. Disease. 4I-1-2. Epidemiology. 4I-1-3. Resurgence of yellow fever as a major public health problem. 5I-1-4. Transmission cycles. 5I-2. Yellow fever virus (YFV). 6I-2-1. Taxonomy of flaviviruses. 6I-2-2. Classification of the yellow fever virus. 6I-2-3. Structure of the virion. 7I-2-4. Pathogenesis. 9I-2-5. The Flavivirus life cycle. 10I-2-6. Cellular receptor(s) for Flaviviruses. 10I-2-7. Flavivirus fusion with host cell membranes. 11I-3. The YFV envelope protein (the E protein).

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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 residues on the E protein associated with
resistance to scFv-7A neutralization. 93
IV-10. Location of amino acid substitutions associated with resistance to scFv-7A
neutralization in the E protein homodimeric crystal structure of YFV. 93
IV-11. Mutations in the E protein of the YFV Senegal 90 strain. 95
IV-11. Importance of the mutations E-71 and E-155 in terms of neutralization
escape using human polyclonal sera from YF patients and 17D vaccinees. 96INDEX
V-DISCUSSION 99
V-1 Quality of antibody libraries and results of selection. 99
V-1-1. Construction of two human immune antibody phage display libraries and
their panning against purified YFV 17D-204-WHO virions. 99
V-1-2. Isolation of closely-related scFvs specific for the YFV E protein. 100
V-2.Nature of the epitopes. 101
V-2-1. The epitopes are conformation-dependent and pH-sensitive. 101
V-2-2. The scFv-7A epitope is formed by amino acid residues from domain I (E-
153, E-154 and E-155) and from domain II (E-71) in the E protein. 101
V-3. The scFv-7A epitope. 102
V-3-1. E-71 is extremely conserved among all sequenced YF strains and critical
for neutralization. 102
V-3-2. Aa residues E-153, E-154 and E-155 contribute to a lesser extent in the
scFv-7A epitope. 102
V-4. Three isolated scFvs exhibit a broad and potent neutralizing activity in vitro.
105
V-5. Homology of the scFv-7A with previously described YFV neutralizing epitopes
of monoclonal mouse antibodies. 107
V-6. Neutralization of scFv-7A escape mutants generated from a 17D 204-WHO
vaccine lot with sera from 17D-immunized travelers. 107
V-7. Neutralization of the YFV Senegal 90 strain. 108
V-8. Potential mechanism of the scFv neutralization. 109
V-9. Potentiel use of these scFvs as a therapeutical tool to treat YF. 111
V-10. Implications for the design of a cDNA-based YF vaccine. 112
VI-LITERATURE. 115
VII-ANNEXES 132
VII-1. Figures & Tables. 132
VII-2. Abbreviations. 134
VII-3. Publications, presentations and posters. 136
VII-4. Curriculum Vitae. 138
VII-5. Aknowledgments. 142SUMMARY
SUMMARY
Yellow fever virus (YFV) is a mosquito-transmitted, enveloped, positive stranded
RNA virus belonging to the genus flavivirus, which causes hemorrhagic fever in
humans in Africa and South America. The YFV is responsible for 200 000 clinical
infections per year including 40 000 deaths. Despite the presence of a highly
effective YF vaccine called 17D vaccine, this disease is now strongly re-emerging
and has to be considered as a public health problem. The present live attenuated
17D vaccine has two major drawbacks: 1) the ancient production method by
inoculating viable embryonated eggs which limits the vaccine production capacity
and, therefore, impairs attempts to control the disease and may contribute to
vaccine supply shortage. 2) this vaccine is a non clonal vaccine which is constituted
of heterogenous virion sub-populations. Furthermore, recent reports of several
cases of viscerotropic and neurotropic disease associated with 17D vaccination have
raised the obvious question of vaccine safety. Taken together, these data show that
it appears essential to design a new clonal vaccine which could be based on
infectious cDNA clone and produced in animal cell culture. For this purpose, the
knowledge of YFV neutralizing epitopes is essential. Because YFV immunity is
mainly antibody-mediated, we wanted to isolate human neutralizing antibodies
specific for YFV and use them as a tool to characterize the neutralizing epitopes of
YFV. The phage display technology provides one of the most convenient systems to
isolate such neutralizing recombinant antibody fragments. We generated YF
patient-derived antibody phage libraries which were screened against purified
virions of the YFV-204-WHO vaccine strain. This step led to the isolation of several
single-chain antibody fragments (scFv) which recognized conformational and pH
sensitive epitopes in the envelope E protein. Three genetically closely-related and
competing scFvs were found to be able to neutralize in vitro the 17D vaccine strain
and five wild-type African strains of YFV. To map their epitopes, neutralization
escape variants of the YFV-17D-204-WHO were generated using one high-affinity
scFv (scFv-7A). Amino acids (aa) E-153, E-154 and E-155 in domain I and aa E-71
in domain II of the E protein were shown to be the critical components of one
complex neutralizing epitope. These aa do not form a contiguous epitope on the
monomeric E protein, but are in close vicinity in the dimeric form the E protein is
predicted to adopt, based on the crystal structures of related flaviviruses. The
neutralizing epitope is thus predicted to be formed by contribution of aa from
domain I and II of opposing E monomers. The nature of this epitope was supported
by the analysis of one wild-type YFV strain (Senegal 90) which is naturally resistant
to neutralization by scFv-7A. Microneutralization assays using sera from YFV-
1