dc.contributor.author
Kuhn, Jens Holger
dc.date.accessioned
2018-06-07T15:48:01Z
dc.date.available
2008-11-21T08:42:30.925Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/1615
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-5817
dc.description
1 TABLE OF CONTENTS 6 2 LIST OF TABLES 11 3 LIST OF FIGURES 12 4 LIST OF
ABBREVIATIONS 17 5 INTRODUCTION 21 5.1 Filoviruses 21 5.1.1 Background 21
5.1.2 Filovirus taxonomy and phylogeny 22 5.1.3 Filovirus epidemiology 26
5.1.4 Ecology of filoviruses 32 5.1.5 Clinical and pathological presentation
of filovirus infections 34 5.1.6 Treatment of filovirus infections 38 5.1.7
Diagnosis of filovirus infections 39 5.1.8 Molecular biology of filoviruses 39
5.1.8.1 Filoviral particles 39 5.1.8.2 Filoviral genomes 41 5.1.8.3 NP gene 42
5.1.8.4 VP35 gene 42 5.1.8.5 VP40 gene 42 5.1.8.6 GP gene 42 5.1.8.7 VP30 gene
43 5.1.8.8 VP24 gene 43 5.1.8.9 L gene 44 5.1.8.10 Filovirus life cycle 45 5.2
Filoviral glycoproteins 47 5.2.1 Ebolaviral secreted glycoprotein 47 5.2.2
Filoviral spike protein 49 5.2.3 Ebolaviral secondary secreted glycoprotein 53
5.3 Filovirus cell entry 54 5.4 Filovirus-vaccine development 57 5.5 Objective
of this dissertation 60 6 MATERIALS AND METHODS 62 6.1 Cells and culture
conditions 62 6.2 Construction of filovirus glycoprotein-encoding genes and
variants 63 6.2.1 General Procedures 63 6.2.2 Construction of Lake Victoria
marburgvirus isolate Musoke GP1-Fc 65 6.2.3 Construction of Lake Victoria
marburgvirus isolate Musoke spike protein GP1,2-C9 69 6.2.4 Construction of
Lake Victoria marburgvirus isolate Musoke GP1-Fc truncation variants 71 6.2.5
Construction of Lake Victoria marburgvirus isolate Musoke spike protein
GP1,2-C9 containing GP1-internal deletions 76 6.2.6 Construction of Lake
Victoria marburgvirus isolate Angola GP1-Fc truncation variants 77 6.2.7
Construction of Lake Victoria marburgvirus isolate Musoke receptor-binding
region mutants 78 6.2.8 Construction of mucin-like domain-deleted Zaire
ebolavirus isolate Mayinga GP1 79 6.2.9 Construction of Zaire ebolavirus
isolate Mayinga GP1-Fc truncation variants 81 6.2.10 Construction of Zaire
ebolavirus isolate Mayinga receptor-binding region mutants 83 6.2.11
Construction of Côte d’Ivoire, Reston, and Sudan ebolavirus receptor-binding
regions 85 6.2.12 Construction of Zaire ebolavirus isolate Mayinga sGP-Fc 86
6.2.13 Construction of Zaire ebolavirus isolate Mayinga ssGP-Fc 87 6.2.14
Construction of ebolaviral Δ-peptides 88 6.2.15 Construction of Sudan
ebolavirus Δ-peptide truncation variants 90 6.2.16 Construction of Sudan
ebolavirus Δ-peptide mutants 92 6.2.17 Construction of the Reston-Sudan
ebolavirus Δ-peptide chimera 94 6.2.18 Construction of the Sudan-Reston
ebolavirus Δ-peptide chimera 96 6.2.19 Construction of plasmids encoding
proteins fused to the Fc region of murine IgG2A 98 6.2.20 Construction and
origin of plasmids encoding control proteins 99 6.3 Evaluation of expression
of filoviral glycoprotein variants and control proteins 100 6.4 Expression of
filoviral glycoprotein variants and control proteins 102 6.5 Cell binding
assays 103 6.6 Cell-binding competition assay 104 6.7 Transduction assay with
pseudotyped gammaretroviruses 105 6.8 Infection assay with recombinant
infectious Zaire ebolavirus 108 6.9 HIV-1 neutralization assay 108 6.10
Cathepsin assay 109 6.11 Immunization and vaccination protocol 110 6.11.1
Animals 110 6.11.2 Preparation of immunogen 110 6.11.3 Immunization protocol
110 6.11.4 Determination of antibody titers 110 6.11.5 Determination of
cytotoxic T-lymphocyte responses 111 6.11.6 Viral challenge 112 7 RESULTS 113
7.1 Filoviruses attach to a common cell-surface receptor 113 7.1.1 Lake
Victoria marburgvirus isolate Musoke GP1 truncation variant 38-188-Fc binds to
filovirus-permissive cells more efficiently than full-length GP1 113 7.1.2
Zaire ebolavirus isolate Mayinga GP1 truncation variant 54-201-Fc binds to
filovirus-permissive cells more efficiently than mucin-like domain-deleted GP1
119 7.1.3 Lake Victoria marburgvirus isolate Angola and Musoke GP1 truncation
variants bind to filovirus-permissive cells with comparable efficiency 125
7.1.4 Both Lake Victoria marburgvirus isolate Musoke GP1 truncation variant
38-188-Fc and Zaire ebolavirus isolate Mayinga GP1 truncation variant
54-201-Fc specifically inhibit entry of gammaretroviruses pseudotyped with
functional spike proteins of either filovirus 128 7.1.5 Both Lake Victoria
marburgvirus isolate Musoke and isolate Angola 38-188 Fc inhibit entry of
gammaretrovirus particles pseudotyped with Lake Victoria marburgvirus isolate
Musoke GP1,2 more efficiently than other GP1 truncation variants 132 7.1.6
Lake Victoria marburgvirus isolate Musoke 38-188-Fc and Zaire ebolavirus
isolate Mayinga 54-201-Fc inhibit the replication of infectious Zaire
ebolavirus 134 7.1.7 All filoviruses use a common cell-entry factor 135 7.2
Identification of ebolaviral Δ-peptides as potent filovirus cell-entry
modulators 142 7.2.1 Zaire ebolavirus Δ-peptide-Fc, but not secreted
glycoprotein or secondary secreted glycoprotein, binds to filovirus-permissive
cells 142 7.2.2 Zaire ebolavirus Δ-peptide-Fc, but not secreted glycoprotein
or secondary secreted glycoprotein, inhibits entry of gammaretroviruses
pseudotyped with filoviral spike protein 147 7.2.3 Côte d’Ivoire, Sudan, and
Zaire, and to much lesser extent Reston, ebolaviral Δ-peptide Fc fusion
proteins inhibit filoviral GP1,2- mediated entry in a dose-dependent manner
150 7.2.4 Côte d’Ivoire, Sudan, and Zaire, but not Reston, ebolaviral Δ-
peptide Fc fusion proteins inhibit replication of infectious Zaire ebolavirus
157 7.2.5 Ebolaviral Δ-peptides inhibit filoviral GP1,2-mediated entry
specifically 159 7.2.6 Mutational analysis of Sudan ebolavirus Δ-peptide 163
7.2.7 Sudan ebolavirus Δ-Fc and Lake Victoria marburgvirus isolate Musoke
38-188-Fc may compete for the same cell-surface binding factor 170 7.2.8
Ebolaviral Δ-peptides do not inhibit cathepsin B activity 172 7.3 Filoviral
Fc-conjugated receptor-binding regions are strongly immunogenic filovirus
candidate vaccines 174 7.3.1 C57/BL6 mice inoculated with Lake Victoria
marburgvirus isolate Musoke 38-188-Fc or Zaire ebolavirus isolate Mayinga
54-201-Fc develop strong humoral and cytotoxic T-lymphocyte immune responses
174 7.3.2 Sera from C57/BL6 mice inoculated with Lake Victoria marburgvirus
38-188-Fc or Zaire ebolavirus isolate Mayinga 54-201-Fc neutralize infectious
Zaire ebolavirus in vitro 178 7.3.3 C57/BL6 mice inoculated with Lake Victoria
isolate Musoke 38-188-Fc or Zaire ebolavirus isolate Mayinga 54-201-Fc are
partially and fully protected against challenge with infectious Zaire
ebolavirus, respectively 180 8 DISCUSSION 181 9 SUMMARY 212 10 REFERENCES 216
11 PUBLICATIONS AND PRESENTATIONS 242 12 DECLARATION 252 13 CURRICULUM VITAE
253
dc.description.abstract
The GP1,2 spike proteins of filoviruses (marburgviruses and ebolaviruses)
mediate viral cell-surface attachment, membrane fusion, and entry into cells
expressing the unknown filovirus receptor(s). Here, it is shown that a 151
amino-acid fragment of the Lake Victoria marburgvirus GP1 subunit (residues
38-188), fused to the Fc region of human IgG1, bound filovirus-permissive cell
lines more efficiently than full-length GP1. An analogous 148 amino-acid
fragment of the Zaire ebolavirus GP1 subunit (residues 54-201) similarly bound
the same cell lines more efficiently than a series of longer GP1-truncation
variants. Neither the marburgvirus GP1 fragment, nor that of ebolavirus, bound
to filovirus-resistant lymphocyte cell lines thought not to express the
filovirus receptor. Both Lake Victoria marburgvirus 38-188-Fc and Zaire
ebolavirus 54-201-Fc specifically inhibited the replication of infectious
Zaire ebolavirus, as well as transduction of filovirus-permissive cells by
gammaretroviruses pseudotyped with either the Lake Victoria marburgvirus or
the Zaire ebolavirus GP1,2 spike protein. Similarly, GP1-Fc fusion fragments
of Côte d’Ivoire ebolavirus, Reston ebolavirus, and Sudan ebolavirus,
corresponding to Zaire ebolavirus GP1 residues 54-201, inhibited
gammaretroviruses pseudotyped with Lake Victoria marburgvirus GP1,2. These
studies identified the receptor-binding regions (RBRs) of marburgviruses and
ebolaviruses, and demonstrated that all filoviruses utilize at least one
common receptor. In addition to the GP1,2 spike glycoprotein, ebolaviruses,
but not marburgviruses, express two secreted glycoproteins, sGP and ssGP, from
the GP gene by cotranscriptional editing. All three proteins have identical
N-termini that include residues 54-201. However, it is shown that neither sGP-
Fc nor ssGP-Fc binds to filovirus-permissive cells. Both proteins were unable
to inhibit transduction of such cells by gammaretroviruses pseudotyped with
the Lake Victoria marburgvirus GP1,2 spike protein, indicating that they do
not bind to the filovirus receptor. Instead, it is shown that Fc-conjugated
Δ-peptide, which is a short C-terminal cleavage product of sGP bearing no
sequence similarity to the filoviral RBRs, inhibited pseudotyped
gammaretroviruses and infectious Zaire ebolavirus specifically and in a dose-
dependent manner. Δ-Fc derived from Côte d’Ivoire, Sudan, and Zaire ebolavirus
sGP inhibited Lake Victoria marburgvirus GP1,2 spike protein-mediated entry
and replication of infectious Zaire ebolavirus comparably or better than Zaire
ebolavirus 54-201-Fc. Interestingly, Δ-Fc derived from sGP of Reston
ebolavirus, thought to be the only filovirus apathogenic for humans, had
little or no effect. These data suggest that Δ-peptides modulate filovirus
cell entry and may be important virulence factors. Last, the immunogenic
properties of filoviral RBR-Fcs were evaluated in a lethal ebolavirus mouse
model. C57/BL6 mice were immunized on days 0, 21, and 35 with Zaire ebolavirus
54-201-Fc + RIBI adjuvant or Lake Victoria marburgvirus 38-188-Fc + RIBI
adjuvant and challenged with mouse-adapted Zaire ebolavirus on day 62. All
mice immunized with Zaire ebolavirus 54-201-Fc survived otherwise lethal
challenge without showing any signs of disease. Half of the mice immunized
with Lake Victoria marburgvirus 38-188-Fc also survived otherwise lethal Zaire
ebolavirus infection. Sera collected from mice immunized with Zaire ebolavirus
54-201-Fc or Lake Victoria marburgvirus 38-188-Fc neutralized Zaire ebolavirus
infection of Vero E6 cells, and strong cytotoxic T-lymphocyte responses were
detected in mice immunized with either RBR-Fc. This is the first report of a
cross-protective filovirus candidate vaccine.
de
dc.description.abstract
Das Oberflächenprotein der Filoviren (Marburg- und Ebolaviren), GP1,2, ist
verantwortlich für die Oberflächenadsorption an, Membranfusion mit, und
Penetration von Zellen, welche den noch unbekannten Filovirusrezeptor
exprimieren. Hier wird gezeigt, dass ein aus 151 Aminosäuren bestehendes und
mit der Fc-Region von humanem IgG1 fusioniertes Lake Victoria marburgvirus
GP1-Fragment (Aminosäuren 38-188; 38-188-Fc) mit höherer Affinität an
filovirusempfängliche Zellen bindet als unverändertes GP1. Das analoge Zaire
ebolavirus GP1-Fragment, bestehend aus 148 Aminosäuren (Aminosäuren 54-201)
und der Fc-Region von humanem IgG1 (54-201-Fc), band an die gleichen Zellen
mit ebenfalls höherer Affinität als längere Fragmente. Weder das Marburgvirus,
noch das Ebolavirus GP1-Fragment vermochte es, an filovirusresistente
Lymphozyten zu binden, von welchen vermutet wird, dass sie den
Filovirusrezeptor nicht exprimieren. Sowohl Lake Victoria marburgvirus
38-188-Fc, als auch Zaire ebolavirus 54-201-Fc, hemmten die Replikation von
infektiösem Zaire ebolavirus und die Transduktion filovirusempfänglicher
Zellen durch Gammaretroviren, die mit Lake Victoria marburgvirus oder Zaire
ebolavirus GP1,2-Oberflächenproteinen pseudotypisiert waren. Analoge Fc-
Fusionskonstrukte, welche die zum 54-201-Fragment analogen Sequenzen der
GP1-Proteine von Côte d’Ivoire, Reston, oder Sudan ebolavirus enthielten,
hemmten ebenfalls die Zelltransduktion durch Lake Victoria marburgvirus GP1,2
pseudotypisierte Gammaretroviren. Diese Experimente identifizierten die
Rezeptorbinderegionen (RBR) der Marburg- und Ebolaviren und zeigen, dass all
Filoviren mindestens einen gemeinsamen Rezeptor benutzen. Mittels
kotranskriptionalen Editings des GP-Gens exprimieren Ebolaviren, nicht aber
Marburgviren, neben dem GP1,2-Oberflächenprotein zwei sezernierte
Glykoproteine (sGP, ssGP). Alle drei Proteine besitzen identische N-Termini,
welche die Aminosäuren 54-201 beinhalten. Hier wird gezeigt, dass weder sGP-
Fc, noch ssGP-Fc, an filovirusempfängliche Zellen binden konnte. Keines der
beiden Proteine konnte die Zelltransduktion dieser Zellen mit Lake Victoria
marburgvirus GP1,2 pseudotypisierten Gammaretroviren hemmen, was vermuten
lässt, dass sie nicht an den Filovirus-Rezeptor banden. Stattdessen wird
gezeigt, dass Fc-konjugiertes Δ-Peptid – ein kurzes, C-terminales Spaltprodukt
von sGP ohne Sequenzähnlichkeit zu filoviralen Rezeptorbinderegionen (Δ-Fc) –
sowohl die Zelltransduktion mit pseudotypisierten Gammaretroviren hemmte, als
auch die Replikation von infektiösem Zaire ebolavirus. Côte d’Ivoire, Sudan
und Zaire ebolavirus Δ-Fc hemmten den durch Lake Victoria marburgvirus
GP1,2-vermittelten Zelleintritt und die Replikation von infektiösem Zaire
ebolavirus vergleichbar zu oder besser als Zaire ebolavirus 54-201-Fc.
Interessanterweise hatte Δ-Fc des Reston ebolavirus, welches als einziges der
Filoviren als humanapathogen gilt, geringen oder keinen Effekt. Diese
Ergebnisse lassen vermuten, dass Δ-Peptide an der Zellpenetration durch
Filoviren beteiligt sind und möglicherweise wichtige Virulenzfaktoren
darstellen. Zuletzt wurden die immunogenen Eigenschaften der filoviralen Fc-
konjugierten Rezeptorbinderegionen in einem letalen Mausmodell untersucht.
C57/BL6-Mäuse wurden nach 0, 21 und 35 Tagen mit Zaire ebolavirus 54-201-Fc +
RIBI-Adjuvans oder Lake Victoria marburgvirus 38-188-Fc + RIBI-Adjuvast
immunisiert und nach 62 Tagen mit mausadaptiertem Zaire ebolavirus infiziert.
Alle mit Zaire ebolavirus 54-201-Fc-immunisierten Mäuse überlebten die
ansonsten tödliche Infektion ohne sichtliche Krankheitssymptome. Die Hälfte
der mit Lake Victoria marburgvirus 38-188-Fc-immunisierten Mäuse überlebten
ebenfalls die ansonsten tödliche Infektion mit Zaire ebolavirus. Sera, die mit
Zaire ebolavirus 54-201-Fc oder Lake Victoria marburgvirus 38-188-Fc
immunisierten Mäusen entnommen wurden, hemmten die Infektion von Vero E6
Zellen mit Zaire ebolavirus. Darüber hinaus konnte eine starke zytotoxische
T-Zellantwort in immunisierten Mäusen nachgewiesen werden. Hiermit wird
erstmalig ein Filovirusimpfstoffkandidat beschrieben, der sowohl partiell vor
Marburg-, als auch vollständig vor Ebolaviren schützt.
de
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject
hemorrhagic fever
dc.subject.ddc
500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie
dc.title
Filoviruses attach to a common cell-surface molecule via distinct and strongly
immunogenic receptor-binding regions, and modulate cell entry through
Δ-peptides
dc.contributor.contact
jenshkuhn@comcast.net
dc.contributor.firstReferee
Assoc. Prof. Michael R. Farzan, PhD
dc.contributor.furtherReferee
Prof. Dr. rer. nat. Volker Haucke
dc.date.accepted
2008-11-12
dc.identifier.urn
urn:nbn:de:kobv:188-fudissthesis000000006165-0
dc.title.translated
Filoviren binden an ein gemeinsames Zelloberflächenmolekül mittels
abgetrennter und stark immunogener Rezeptorbinderegionen und modulieren den
Zelleintritt durch Δ-Peptide
de
refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000006165
refubium.mycore.derivateId
FUDISS_derivate_000000004688
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access