Christopher Howard Thesis

Identification of Epitopes on the
Dengue Virus Type 4 Envelope

Glycoprotein Involved in
Neutralisation by Antibodies
A thesis submitted in 2006

for a Doctor of Philosophy degree
by
Christopher Bruce Howard

Bachelor of Science (Honours)
Centre for Molecular Biotechnology

School of Life Sciences
Queensland University of Technology
Australia
KEYWORDS
Dengue virus, envelope protein, neutralisation, monoclonal antibody, epitope,
neutralisation escape mutant, chimeric E protein, site directed mutagenesis, peptide
display, tetravalent vaccine.
2
ABSTRACT
Dengue virus (DENV) is the causative agent of dengue fever (DF), the most prevalent
arthropod-borne viral disease in the world and therefore is considered an emerging
global health threat. The four DENV serotypes (DENV-1, DENV-2, DENV-3 and
DENV-4) that infect humans are distinguished from one another by unique antigenic
determinants (epitopes) on the DENV envelope (E) protein. The E protein is the primary
antigenic site of the DENV and is responsible for inducing neutralising antibody (Ab)
and cell mediated immune response in DENV infected hosts. The DENV E protein also
mediates attachment of virions to host cell receptors and entry of virions into host cells
by membrane fusion.
The study of epitopes on DENV E protein is necessary for understanding viral function
and for the design of unique polyvalent vaccines capable of inducing a neutralising
antibody response against each DENV serotype. Reverse genetics using infectious
cDNA clones has enabled the construction of functional intertypic DENV, where the E
protein of one DENV serotype is put in the genetic background of a different DENV
serotype. In addition, observations from our laboratory indicate that chimeric E
proteins, consisting of E protein structural domains from different DENV serotypes can
fold into functional proteins. This suggests that there is potential to engineer viruses
with intertypic DENV E proteins as potential DENV vaccine candidates, which is the
long term goal of studies within our research group. However, if a chimeric E protein
was to be constructed containing epitopes involved in antibody mediated neutralisation
of each DENV serotype, then knowledge of the location of these epitopes on the E
protein of each DENV serotype would be essential.
Prior to this study, monoclonal antibodies (MAbs) had been used to identify epitopes
involved in antibody mediated neutralisation on the E protein of all DENV serotypes,
except DENV-4. The primary objective of this study was to identify epitopes on the
DENV-4 E protein involved in neutralisation by antibodies. In order to achieve this
objective, a panel of 14 MAbs was generated against DENV-4 in BALB/c mice and
characterised using various serological and functional assays.
3
The identification of DENV-4 specific neutralising MAbs in the panel was essential for
subsequent experiments aimed at determining antigenic domains, structural domains or
specific epitopes (peptides or amino acids) involved in the neutralisation of DENV-4.
The majority of MAbs (11/14) generated against DENV-4 recognised the E protein. The
remaining three MAbs reacted with the non-structural (NS) 1 protein. The majority of
MAbs against the E protein were DENV or Flavivirus group reactive, but four MAbs
were DENV-4 specific. All MAbs against the E protein recognised conformationally
dependent epitopes and were able to capture DENV-4 in an enzyme linked immuno-
adsorbent assay (ELISA).
Eighty percent (9/11) of the anti-E MAbs produced for this study neutralised infection of
cells by DENV-4 in vitro. Three of the neutralising MAbs (F1G2, 18F5 and 13H8) were
DENV-4 specific and also demonstrated the strongest neutralisation activity of the
panel, reducing DENV-4 infectivity by 100-1000 fold. The amount of virus neutralised
by the MAbs was not related to the avidity of the MAbs. The DENV-4 specific MAbs
F1G2, 18F5 and 13H8 were used to identify epitopes involved in neutralisation of
DENV-4.
The MAbs that effectively captured DENV-4 were used in competitive binding assays
(CBAs) to determine spatial relationships between epitopes and therefore define
antigenic domains on the DENV-4 E protein. The CBAs indicated that the epitopes
recognised by the panel of MAbs segregated into two distinct domains (D4E1 and
D4E2) and both contained epitopes involved in neutralisation. CBAs incorporating
human serum from DENV-4 infected patients suggested that the MAbs recognised the
same, or spatially related, epitopes in domain D4E2 as antibodies from humans who had
experienced natural dengue infections, indicating the clinical relevance of such epitopes
for the development of DENV vaccines. The reactivity of the capture MAbs with low
pH treated DENV-4 was also evaluated in an attempt to identify epitopes that might be
more accessible during low pH-mediated virus fusion. Only one of the MAbs (13H8)
recognised an acid resistant epitope.
4
Initial attempts to identify epitopes on the DENV-4 E protein involved in neutralisation
followed the traditional epitope mapping approach of selecting subpopulations of
DENV-4 which escaped neutralisation by MAbs. These attempts were unsuccessful so a
variety of strategies for mapping epitopes were used including DENV-4 variant analysis
and site directed mutagenesis of the DENV-4 E protein, MAb screening of chimeric
DENV-3/4 E proteins and MAb screening of a bacterial peptide display library.
DENV-4 variants including DENV-4 isolates from different geographical locations or

chemically mutagenised DENV-4 were screened with neutralising MAbs to identify
neutralisation escape mutant (n.e.m.) viruses. Site directed mutagenesis of the DENV-4
E protein confirmed whether amino acid changes identified in DENV-4 n.e.m.s were
essential for the binding of neutralising MAbs to an epitope.
The MAb screening of DENV-4 variants identified n.e.m.s with amino acid changes at
residues E95, E96, E156, E157, E203, E329 and E402 of the DENV-4 E protein. Site
directed mutagenesis of the DENV-4 E protein identified two epitopes recognised by the
DENV-4 specific neutralising MAbs F1G2 and 18F5 at specific amino acid residues
within domains II and III of the DENV-4 E protein. No specific epitopes were identified
for the MAb 13H8; however this MAb did recognise domain I and II of the DENV-4 E
protein, when screened against DENV-3/4 chimeric DENV E proteins.
The first epitope, which was recognised by the MAb F1G2, contained residue E95 which
was located in domain II of the DENV-4 E protein. The aspartate (Asp) to alanine (Ala)
change at E95 prevented the binding of F1G2 to the DENV-4 E protein. The binding of
F1G2 to the E95 residue was confirmed using the pFlitrX bacterial peptide display
library, which demonstrated binding of F1G2 to a peptide homologous with residues
E99-E104. No peptides recognised by 13H8 and 18F5 were identified by this method.
The MAb F1G2 also bound to the domain III region (E300-E495) of the DENV-4 E
protein when screened against DENV-3/4 chimeric DENV E proteins. This implied that
F1G2 may be recognising a discontinuous epitope consisting of domains II and III.
5
The second epitope, which was recognised by MAb 18F5, contained residue E329 which
was located in domain III of the DENV-4 E protein. The alanine (Ala) to threonine
(Thr) change at E329 prevented the binding of 18F5 to the DENV-4 E protein. MAb
18F5 also bound to the domain III region (E300-E495) of the DENV-4 E protein when
screened against DENV-3/4 chimeric E proteins, thus confirming the E329 epitope.
The potential mechanisms by which the DENV-4 specific MAbs neutralise virus
infection were evaluated by the virus overlay protein binding assay (VOPBA). The
binding of MAb 18F5 to a domain III (E329) epitope of the DENV-4 E protein and the
binding of MAb F1G2 to domain II (E95, E99-E104) and domain III epitopes (chimeric
E protein) of the DENV-4 E protein, prevented the attachment of DENV-4 to a 40 kDa
C6/36 cell protein. In contrast the binding of MAb 13H8 to domains I and II of the
DENV-4 E protein did not prevent attachment of DENV-4 to the same protein.
This was preliminary evidence that the binding of domain III epitopes by the MAbs
F1G2 and 18F5 may be important in preventing virus attachment. The binding of MAb
13H8 to domains I and II, and the ability of this MAb to recognise DENV-4 treated at
low pH, suggested that MAb 13H8 may block epitopes exposed at low pH that are
required for low pH mediated virus fusion to host cell membranes.
Overall, the different methods used in this study identified epitopes involved in the
neutralisation of DENV-4. The distribution of epitopes involved in neutralisation
throughout the DENV-4 E protein were similar to the distribution of epitopes involved
in neutralisation on the DENV-1, 2 and 3 E proteins. This suggested that it might be
possible to elicit neutralising antibodies against multiple DENV serotypes using
chimeric E-proteins derived from two or more DENV serotypes and therefore, facilitate
the design of novel tetravalent DENV vaccines.
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TABLE OF CONTENTS
KEYWORDS___________________________________________________________2
ABSTRACT ___________________________________________________________3
LIST OF FIGURES ____________________________________________________10
LIST OF TABLES _____________________________________________________12
ABBREVIATIONS ____________________________________________________14
AMINO ACID ABBREVIATIONS ________________________________________18
DECLARATION OF ORIGINAL AUTHORSHIP ___________________________19
ACKNOWLEDGEMENTS ______________________________________________20
1 INTRODUCTION _________________________________________________21
1.1 The pathogenesis of DHF/DSS________________________________________ 25
1.2 Antibody dependent enhancement (ADE) ______________________________ 28
1.3 Viral virulence ____________________________________________________ 30
1.4 The dengue virus___________________________________________________ 32
1.5 Structural proteins of flaviviruses_____________________________________ 37
1.5.1 The core protein _________________________________________________________ 37
1.5.2 The membrane proteins (prM and M) ________________________________________ 37
1.5.3 The envelope protein _____________________________________________________ 39
1.6 Structural model of the envelope protein _______________________________ 45
1.6.1 Domain I_______________________________________________________________ 45
1.6.2 Domain II ______________________________________________________________ 45
1.6.3 Domain III _____________________________________________________________ 48
1.6.4 Stem anchor region_______________________________________________________ 49
1.6.5 Dimeric interactions ______________________________________________________ 51
1.7 Dengue virus E protein models _______________________________________ 51
1.7.1 Antigenic model _________________________________________________________ 51
1.7.2 Structural model of DENV E protein dimer ____________________________________ 52
1.7.3 Structural model of the DENV E protein dimer post fusion________________________ 53
1.8 Analysis of functional sites on the flavivirus E protein ____________________ 54
1.9 Dengue vaccine design ______________________________________________ 70
1.10 Objectives ________________________________________________________ 73
2 MATERIALS AND METHODS ______________________________________74
2.1 Cells _____________________________________________________________ 74
2.2 Virus_____________________________________________________________ 75
2.2.1 Preparation of working stocks ______________________________________________ 75
2.2.2 Concentration of DENV-4 by precipitation with polyethylene glycol ________________ 77
2.2.3 Preparation of lysate of DENV-4 infected, and uninfected, cells____________________ 77
2.3 Production of hybridomas and anti-DENV-4 MAbs______________________ 77
2.4 Serological and Functional Assays ____________________________________ 82
2.4.1 Hemagglutination and hemagglutination inhibition ______________________________ 82
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2.4.2 Indirect ELISA _________________________________________________________ 83
2.4.3 Indirect immunofluorescence assay (Indirect IFA) ______________________________ 84
2.4.4 Polyacrylamide gel electrophoresis (PAGE) and immunoblotting of DENV proteins ___ 85
2.4.5 Infectivity and neutralisation_______________________________________________ 86
2.4.6 Capture ELISA _________________________________________________________ 89
2.5 Molecular Biology __________________________________________________ 91
2.5.1 RT-PCR and sequencing __________________________________________________ 91
2.5.2 Site directed mutagenesis _________________________________________________ 95
2.5.3 DNA transfection of BHK cells ____________________________________________ 97
2.6 Peptide Display_____________________________________________________ 98
2.7 Virus Overlay Protein Binding Assay (VOPBA) ________________________ 101
3 RESULTS _______________________________________________________103
3.1 Production and characterisation of monoclonal antibodies________________ 103
3.1.1 Serological assays ______________________________________________________ 105
3.1.2 Functional assays ______________________________________________________ 110
3.1.3 Capture of DENV-4 ____________________________________________________ 111
3.2 Identification of antigenic domains on the DENV-4 envelope protein _______ 118
3.2.1 Competitive capture ELISAs _____________________________________________ 118
3.2.2 Competitive capture ELISAs with human serum ______________________________ 122
3.3 Identification of epitopes on the DENV-4 envelope protein involved in
neutralisation____________________________________________________________ 125
3.3.1 Selection of DENV-4 that escaped neutralisation by MAbs ______________________ 125
3.3.2 Chemical mutagenesis of DENV-4 and selection of neutralisation escape mutant
viruses________________________________________________________________________127
3.3.3 Ability of anti-DENV-4 MAbs to recognise different strains of DENV-4 virus_______ 132
3.3.4 Analysis of MAb binding sites using site directed mutagenesis of the DENV-4 E
protein and chimeric DENV E proteins. ____________________________________________ 136
3.3.5 Bacterial peptide display library ___________________________________________ 139
3.3.6 Virus Overlay Protein Binding Assay (VOPBA) ______________________________ 139
4 DISCUSSION____________________________________________________142
4.1 Production and Characterisation of MAbs against DENV-4_______________ 142
4.2 Strategies for the identification of epitopes on the DENV-4 envelope
protein involved in neutralisation ___________________________________________ 143
4.3 Identification of antigenic domains on the DENV-4 envelope protein
involved in neutralisation __________________________________________________ 145
neutralisation. ___________________________________________________________ 150
4.4.1 Identification of an epitope involved in neutralisation by the DENV-4 specific MAb
F1G2._______________________________________________________________________ 151
4.4.2 Identification of an epitope involved in neutralisation by the DENV-4 specific MAb
18F5.___ ____________________________________________________________________ 163
4.4.3 Domain I and II epitopes recognised by the DENV-4 specific neutralising MAbs 13H8
and 1H10. ___________________________________________________________________ 174
4.4.4 DENV and Flavivirus group-reactive epitopes ________________________________ 175
4.5 Proposed neutralisation mechanisms used by DENV-4 specific MAbs. ______ 176
4.6 Epitopes involved in the neutralisation of DENV ________________________ 177
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5 Conclusion ______________________________________________________180
6 APPENDIX A: SOLUTIONS _______________________________________182
6.1 Solutions used for serological assays__________________________________ 182
6.1.1 25 x PBS (Phosphate buffered saline) pH 7.4 _________________________________ 182
6.1.2 1 x PBS pH 7.4_________________________________________________________ 182
6.1.3 Borate saline pH 9.0 _____________________________________________________ 182
6.1.4 3M Hydrochloric acid (HCl) ______________________________________________ 183
6.1.5 Crystal violet-formalin stain solution ________________________________________ 183
6.2 Solutions used for PAGE and western blotting _________________________ 183
6.2.1 Resolving buffer (1.5M Tris pH 8.8) ________________________________________ 183
6.2.2 Stacking buffer (1.0M Tris pH 6.8)._________________________________________ 183
6.2.3 10% ammonium persulfate________________________________________________ 183
6.2.4 2 x PAGE sample buffer _________________________________________________ 184
6.2.5 10% SDS solution ______________________________________________________ 184
6.2.6 5 x PAGE Running Buffer ________________________________________________ 184
6.2.7 CAPS transfer buffer ____________________________________________________ 184
6.2.8 10 x Tris-buffered saline (TBS) ____________________________________________ 185
6.2.9 1 x TBS ______________________________________________________________ 185
6.3 Recipes for Polyacrylamide Gels_____________________________________ 185
6.3.1 10% resolving polyacrylamide gel __________________________________________ 185
6.3.2 5% stacking polyacrylamide gel____________________________________________ 186
6.4 Molecular Biology_________________________________________________ 186
6.4.1 DEPC-treated water _____________________________________________________ 186
6.4.2 50 x Tris acetate EDTA (TAE) buffer _______________________________________ 186
6.4.3 1 x TAE buffer _________________________________________________________ 186
6.4.4 6 x DNA loading dye ____________________________________________________ 187
6.4.5 3M Sodium acetate pH 5.2 ________________________________________________ 187
6.4.6 Luria broth (LB) medium _________________________________________________ 187
6.4.7 Luria broth (LB) agar ____________________________________________________ 187
7 APPENDIX B: DATA _____________________________________________188
7.1 Affect of 6M urea treatment on MAb adsorption to ELISA plates _________ 188
7.2 Competitive binding assay results____________________________________ 189
7.3 Amino acid changes in DENV-4 n.e.m.s E protein sequences _____________ 196
7.3.1 Wildtype DENV-4:______________________________________________________ 196
7.3.2 DENV-4 5FU induced n.e.m.s _____________________________________________ 196
7.3.3 DENV-4 natural n.e.m.s __________________________________________________ 196
REFERENCES ______________________________________________________197
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LIST OF FIGURES
Figure 1.1 The worldwide distribution of dengue and the vector Aedes aegypti (Gubler, 1998).
_________________________________________________________________________________ 22
Figure 1.2 The structure of the immature and mature flavivirus virion (Heinz et al., 1994). _________ 33
Figure 1.3 Structure of (A) mature and (B) immature DENV-2 particles as determined by cryoelectron
microscopy and image reconstruction (Kuhn et al., 2002; Zhang et al., 2003a). ___________________ 34
Figure 1.4 The organisation of E protein dimer sets (circled) on the surface of a mature DENV-2 particle
(A) (Kuhn et al., 2002). _______________________________________________________________ 41
Figure 1.5 A ribbon diagram of the three dimensional structural model of the TBEV envelope protein
dimer (Rey et al., 1995). ______________________________________________________________ 44
Figure 1.6 Schematic diagram of the TBEV E protein monomer including the functional determinants of
the stem-anchor region (Allison et al., 1999).______________________________________________ 50
Figure 2.1. Diagram of the pFliTrx bacterial peptide display library. __________________________ 99
Figure 3.1 Western blot analysis of selected anti-DENV-4 MAbs using lysate of (A) DENV-4 infected
C6/36 cells and (B) uninfected C6/36 cells. ______________________________________________ 106
Figure 3.2 Reaction of MAbs with western blots of PEG-concentrated DENV-4 (-) or with the same virus
preparation treated with 2ME (+). _____________________________________________________ 107
Figure 3.3 Differences between the reactivity of anti-E MAbs and anti-NS1 MAbs with DENV-4 infected
C6/36 cells in IFAs, represented by the MAbs 13H8 (anti-E) and F12A3 (anti-NS1). ______________ 109
Figure 3.4 The capture of DENV-4 at different dilutions by MAb F1G2 coated to an ELISA plate at
1ug/ml. __________________________________________________________________________ 113
Figure 3.5 Effects of different concentrations of urea on the capture of DENV-4 by DENV-4 specific
neutralising MAbs 13H8, 1H10, 18F5 and F1G2. _________________________________________ 116
Figure 3.6. The ability of MAbs to inhibit binding of the HRP labelled 6B6C1 detection MAb to DENV-4
in a capture ELISA.. ________________________________________________________________ 121
Figure 3.7 The potential affects of 5FU treatment on the genetic diversity of a DENV-4 population and
selection of DENV-4 n.e.m.s, demonstrated by a chromatogram of the nucleotide sequence of (A) DENV-4
NM (no 5FU treatment, A at nucleotide 284), (B) DENV-4 W10 (10uM 5FU treatment, A/C at nucleotide
284] and (C) DENV-4 n.e.m. _________________________________________________________ 129
Figure 3.8. Reactivity of MAbs 13H8, F2D1 and F1G2 with C6/36 cells infected with wildtype DENV-4
(NM) or DENV-4 n.e.m. _____________________________________________________________ 131
Figure 3.9. The reactivity of MAb F1G2 with BHK cells transfected with (A) pVAX DENV-4-C-prM-E/
E95 (Asp) and (B) pVAX DENV-4-C-prM-E/ E95 (Asp-Ala).. ________________________________ 138
10
Figure 3.10. The amino acid sequences of (A) the seven peptides recognised by the MAb F1G2 in the
bacterial peptide display library and the (B) alignments of these peptides with the DENV-4 H241 E
protein sequence between residues E91 and E112. _________________________________________ 140
Figure 3.11. The binding of DENV-4 to a 40kDa protein from uninfected C6/36 cell lysate (+C) in the
VOPBA. __________________________________________________________________________ 141
Figure 4.1. Antigenic model of the DENV-4 E protein derived from competitive binding assays._____ 146
Figure 4.2 Location of the epitope involved in neutralisation by the DENV-4 specific MAb F1G2 at
amino acid residue E95 on an (A) overhead and (B) side view of the DENV-4 E protein in its pre-fusion
conformation (model derived from DENV-2 E protein model; 2.75A resolution; pdb file:1OAN; Modis et
al., 2003). _________________________________________________________________________ 154
Figure 4.3 The alignment of amino acid residues of the E protein of different DENV-4 isolates and the
prototype strains for each DENV serotype, associated with the E95 residue, involved in DENV-4
neutralisation by the MAb F1G2._______________________________________________________ 155
Figure 4.4 The amino acid substitutions occurring at residue E95 of the E protein in different DENV-4
and the affects on the surface exposure of the residue, and potential reactivity with the MAb F1G2. __ 158
Figure 4.5 (A) The potential interdimeric epitope for the MAb F1G2, formed by interactions between
domain II and domain III residues located on opposite subunits of the DENV-4 E protein dimer. _____ 160
Figure 4.6 Phylogenetic analysis of the E protein of DENV-4 used in IFA with MAb 18F5._________ 164
Figure 4.7 The location of the epitope recognised by the DENV-4 specific MAb 18F5 at residue E329,
coloured pink, on the (A) overhead and (B) side views of the DENV-4 E protein (pdb file 1OAN). ____ 165
Figure 4.8 The alignment of amino acid residues of the E protein of different DENV-4 isolates and the
prototype strains for each DENV serotype, associated with the E329 residue, involved in DENV-4
neutralisation by the MAb 18F5. _______________________________________________________ 166
Figure 4.9 Relative position of DENV and Flavivirus neutralisation epitopes on domain III of the DENV-
4 E protein structural model. __________________________________________________________ 169
Figure 4.10 The relative position of functional epitopes identified in DENV or flaviviruses on the Domain
III portion of the DENV-4 E protein structural model. ______________________________________ 172
Figure 4.11 Location of amino acid residues involved in the neutralisation of DENV on the structural
model of the DENV-4 E protein. _______________________________________________________ 179
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LIST OF TABLES
Table 1.1 Flavivirus proteins _________________________________________________________ 36
Table 1.2 Epitopes on the flavivirus envelope protein located using competitive binding experiments with
monoclonal antibodies. _______________________________________________________________ 55
Table 1.3 Epitopes in the flavivirus envelope protein identified using linear peptides, fusion proteins and
phage display. ______________________________________________________________________ 58
Table 1.4 Epitopes on the flavivirus envelope protein involved in neutralisation identified using
monoclonal antibodies. _______________________________________________________________ 62
Table 1.5. Functional determinants defined by comparing the phenotype of viral variants __________ 64
Table 1.6. Functional determinants identified by infectious clones_____________________________ 68
Table 2.1 Dengue viruses used in this study _______________________________________________ 76
Table 2.2 Reference MAbs used in this study______________________________________________ 81
Table 2.3 Oligonucleotide primers used for PCR and DNA sequencing _________________________ 93
Table 2.4 Thermal cycling conditions for PCR ____________________________________________ 94
Table 2.5 Oligonucleotide primers used for site directed mutagenesis __________________________ 96
Table 3.1 Characteristics of anti-DENV-4 MAbs and reference MAbs 4G2, 6B6C1 and 1H10 used in this
study ____________________________________________________________________________ 104
Table 3.2 The ability of anti-DENV-4 MAbs to combine with PEG-concentrated DENV-4 in antibody 112
Table 3.3 Capture of DENV-4 by MAbs before and after exposure of the virus to pH 6.0 for 15 minutes.
________________________________________________________________________________ 115
Table 3.4 The effect of 6M urea on the capture of DENV-4 by anti-DENV-4 MAbs _______________ 117
Table 3.5. Inhibition of capture of DENV-4 by MAbs when virus was pre-incubated with the homologous
or heterologous MAbs_______________________________________________________________ 120
Table 3.6. The ability of human serum containing anti-dengue or anti-flavivirus antibodies to inhibit
capture of DENV-4 by anti-DENV-4 MAbs ______________________________________________ 123
Table 3.7. Genotypic and phenotypic properties of DENV-4 derived by treatment with 10µM 5FU and
BHK cell passage __________________________________________________________________ 128
Table 3.8. Reaction of anti-DENV-4 MAbs with C6/36 cells infected with DENV-4 isolated from different
geographical regions _______________________________________________________________ 133
Table 3.9. Variation in the amino acid sequences of the E proteins of the DENV-4 strains used in the IFA
in Table 3.8. ______________________________________________________________________ 134
12
Table 3.10. Comparison of the genotypic and phenotypic properties of DENV-4 strains recognised by
MAb 18F5 with those not recognised by this MAb. _________________________________________ 135
Table 3.11. Indirect IFA using selected MAbs as primary antibody and BHK cells transfected with
plasmids containing wildtype, chimeric and mutagenised DENV-4 E genes. _____________________ 137
Table 4.1 Characteristics of amino acids at residue E95 in different DENV-4 ____________________ 157
Table 4.2 Neutralisation epitope clusters in Domain III of the Flavivirus E protein. ______________ 168
Table 4.3 Virulence determinants in Domain III of the Flavivirus E protein _____________________ 173
13
ABBREVIATIONS
Abbreviation Definition
Å Angstrom
aa Amino acid
Ab Antibody
Abs Absorbance
ADE Antibody dependent enhancement
AGRF Australian Genome Research Facility
AMV Avian myeloblastosis virus
AR Analytical reagent
BHK Baby hamster kidney
C Core
cDNA Complementary DNA
CAPS 3-(Cyclohexylamino)-1-propanesulfonic acid
CBAs Competitive Binding Assays
CDC Centre for Disease Control
CHO Chinese hamster ovary
CMC Carboxymethylcellulose
CTLs Cytotoxic T lymphocytes
CV Crystal violet
cryoEM Cryoelectron microscopy
DENV Dengue virus
DENV-1 Dengue virus type 1
DEPC Diethylpyrocarbonate
DF Dengue fever
DHF Dengue haemorrhagic fever
DMSO Dimethyl sulphoxide
DNA Deoxyribose-nucleic-acid
DSS Dengue shock syndrome
E Envelope
ER Endoplasmic reticulum
ELISA Enzyme linked immuno-adsorbent assay
FCS Fetal calf serum
FFU Foci forming units
FFWI Fusion from within
14
FFWO Fusion from without
FITC Fluorescein Isothiocyanate
FLAVI Flavivirus
FPK FliTrx panning kit
FRhL Fetal rhesus lung
5FU 5-Fluorouracil
GAGs Glycosaminoglycans
HA Hemagglutination
HAT Hypoxanthine aminopterin thymidine
HCl Hydrochloric Acid
HCV Hepatitis C virus
HI Hemagglutination inhibition
HRP Horse radish peroxidase
HS Heparan sulphate
HT Hypoxanthine thymidine
IFA Immunofluorescence assay
IFN-γ Interferon gamma
Ig Immunoglobulin
IL-2 Interleukin 2
i.p. Intraperitoneal
i.v. Intravenous
JEV Japanese encephalitis virus
Kb Kilobase
kDa Kilodalton
KUNV Kunjin virus
LB Luria broth
LBA Luria broth agar
LCMV Lymphocytic choriomeningitis virus
LGTV Langat virus
LIV Louping ill virus
2ME 2-mercaptoethanol
M Membrane
MAbs Monoclonal antibodies
MES 2-(N-morpholino) ethanesulfonic acid
MHC Major histocompatibility complex
MVEV Murray valley encephalitis virus
Mw Molecular weight
n Number of values
N Neutralisation
15
NCR Non coding regions
n.d. Not determined
n.e.m. Neutralisation escape mutant
NI Neutralisation index
NMR Nuclear magnetic resonance
NP-40 Nonidet P-40
NS Non-structural
NS1 Non-structural 1
ORF Open reading frame
p Probability value (Student T test)
PAGE Polyacrylamide gel electrophoresis
PBMCs Peripheral blood mononuclear cells
PBS Phosphate buffered saline
PBST Phosphate buffered saline with Tween-20
PCR Polymerase chain reaction
PDB Protein data base
PDK Primary dog kidney
PEG Polyethylene glycol
PFHM Protein-free hybridoma medium
PFU Plaque forming units
prM Pre-membrane
PRNT Plaque reduction neutralisation test
PS-EK Porcine-equine kidney
QUT Queensland University of Technology
RGD Arg-Gly-Asp
RNA Ribose-nucleic-acid
RSPs Recombinant subviral particles
RT Reverse transcriptase
s.d. Standard deviation
SDM Site directed mutagenesis
SDS Sodium dodecyl sulphate
sE Soluble envelope protein
SLEV Saint Louis encephalitis virus
SRID Single radial immuno-diffusion
SMB Suckling mouse brain
TAE Tris-acetate/EDTA
TBEV Tick-borne encephalitis virus
TBS Tris-buffered saline
TBST Tris-buffered saline with Tween-20
16
TCS Tissue culture supernatant
TCID Tissue culture infectious dose
TEMED N,N,N’,N’-tetramethylethylenediamine
TMB 3', 5, 5'-TetraMethylBenzidine
TNF-α Tumour necrosis factor alpha
3D Three dimensional
VOPBA Virus overlay protein binding assay
WHO World Health Organisation
WNV West Nile virus
WRAIR Walter Reed Army Institute of Research
UQ University of Queensland
YARU Yale Arbovirus Reference Centre
YFV Yellow fever virus
17
AMINO ACID ABBREVIATIONS
Amino Acid 3 Letter Code 1 Letter Code
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic Acid Asp D
Cysteine Cys C
Glutamine Gln Q
Glutamic Acid Glu E
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
18
DECLARATION OF ORIGINAL AUTHORSHIP
The work contained in this thesis has not been previously submitted for a degree or
diploma at any other higher education institution. To the best of my knowledge and
belief, the thesis contains no material previously published or written by another person
except where due reference is made.
Signature: __________________
Date:___________________
19
ACKNOWLEDGEMENTS
The completion of this manuscript would not have been possible without the assistance
and encouragement of family, friends and work colleagues. In particular, I would like to
acknowledge my supervisor Dr John Aaskov for his mentoring, support and
encouragement throughout my research, and his commitment to improving my science.
I would also like to thank members of the dynamic QUT Arbovirology team for their
continued support and friendship. In particular, I would like to thank Kym Lowry, Steve
Liew, Scott Craig, Deema Al Sheikly and Dave Hammond for making working in a
laboratory interesting and most importantly, a lot of fun. I also would like to extend my
gratitude to work colleagues at CBio Ltd, for their patience and understanding regarding
my thesis commitments. Finally and most importantly I would like to thank my wife,
Belinda, and my family for their continued support, which greatly assisted in the
completion of this manuscript.
20
1 INTRODUCTION
Dengue is the most prevalent arthropod-borne viral disease of humans with more than
2.5 billion people at risk of infection in tropical and subtropical regions of Africa, Asia,
Australia and the Americas (Gubler, 1998; Gubler, 2002) (Figure 1.1). There are four
serologically related but antigenically distinct dengue virus (DENV) serotypes (DENV-
1, DENV-2, DENV-3 and DENV-4). The principal vectors of DENV are mosquitoes of
the Aedes genus, predominately Aedes aegypti. The dramatic rise in the number of
dengue cases over the last decade has been due to demographic changes, which favour
human contact with Aedes aegypti mosquitoes, as well as the ecological changes that
facilitate vector breeding (Gubler, 1998). The failure of vector control programs,
increased air travel providing the means for rapid movement of viremic humans,
dissemination of multiple dengue serotypes and establishment of hyperendemic cycles
of dengue transmission have also contributed to the increased incidence of this disease
(Gubler and Meltzer, 1999; Monath, 1994).
Humans are the only hosts that develop symptoms following an infection with DENV.
Lower primates are permissive to DENV infection but the duration and magnitude of
viremia is low (Halstead et al., 1973a). Infection of humans with DENV can be
asymptomatic or progress to undifferentiated fever, classical dengue fever (DF), or to
the severe disease forms; dengue haemorrhagic fever (DHF) and dengue shock
syndrome (DSS). At the beginning of the 21st century an estimated 100 million cases of
DF and more than 250,000 cases of DHF/DSS occur annually (Gubler, 1998; Gubler,
2002). More than 100 countries have endemic dengue, and DHF has been documented
in more than 60 of these countries (Gubler, 1998) (Figure 1.1).
21
This figure is not available online.
Please consult the hardcopy thesis
available from the QUT Library
Figure 1.1 The worldwide distribution of dengue and the vector Aedes aegypti (Gubler,
1998). The grey shading represents areas infested with Aedes aegypti, whereas the
darker shading represents areas infested with Aedes aegypti and with dengue epidemic
activity.
22
Classical DF is an acute, incapacitating disease characterised by high fever, severe
headache, retro-orbital pain, muscle and bone or joint pains, nausea, vomiting, rash,
leukopenia and lymphadenopathy (Siler et al., 1926; Simmons et al., 1931; Sabin, 1952).
A saddleback fever is sometimes observed in dengue patients as well as haemorrhagic
complications such as epistasis, gingival and gastrointestinal bleeding, hematuria and
menorrhagia (Halstead et al., 1969).
DHF is characterised by a high fever, haemorrhage, increased vascular permeability,

hepatomegaly and moderate to marked thrombocytopenia (Nimmannitya et al., 1969).
The increase in vascular permeability leads to plasma leakage, hemoconcentration,
haemorrhage, low pulse pressure, hypotension and other signs of shock. DHF grades 1
and 2 are differentiated from classical dengue fever by the presence of
thrombocytopenia, hepatomegaly and hemoconcentration. DHF grades 3 and 4 are
classified as DSS, a more severe disease manifestation differentiated from DHF by
circulatory failure due to a rapid and weak pulse, narrow pulse pressure or hypotension
with cold clammy skin and restlessness (Nimmannitya et al., 1969).
DF is rarely fatal (<1%). However, the mortality rates for DHF/DSS vary from 1 to
10% for hospitalised patients and up to 30% if untreated (Gubler, 1998).
The disease that is now considered to be dengue has been recognised since at least the
end of the 18th century in French West India (1635), Batavia (1779), Cairo (1779) and
in Philadelphia (1780) (Carey, 1971). There are doubts as to whether the disease
described in Batavia (Jakarta), Indonesia, and in Cairo, Egypt, in 1779 was dengue or
whether it was what is now recognised as Chikungunya (Carey, 1971). The clinical
features of Chikungunya virus infection resemble contemporary dengue, but the pains
are restricted more to the joints, the febrile period is shorter and not biphasic, and many
patients experience persistent, residual joint pains following the acute episode (Carey,
1971).
23
The first documented epidemic of an illness that is clinically compatible with present-
day ‘dengue fever’ corresponds to the Bilious Remitting Fever or Break-Bone fever
described by Benjamin Rush in Philadelphia in 1780 (Rush, 1789).
Dengue epidemics were common during the 18th and 19th centuries in North America,
the Caribbean, Asia and Australia. In 1897, the first cases of dengue haemorrhagic fever
(DHF) were reported in Charters Towers, Queensland (Hare, 1898). From the closing
years of the nineteenth century, knowledge of dengue was rapidly accumulated in direct
proportion to the progress of medical and public health science. In 1906, Bancroft
demonstrated that the Aedes aegypti mosquitoes could transmit dengue virus to humans
(Bancroft, 1906). In 1907, Ashburn and Craig demonstrated that dengue was caused by
a filterable virus found in blood from human dengue cases (Ashburn and Craig, 1907).
Cleland et al., (1919) also studied the distribution of dengue virus in blood, and found
that the virus remained in circulation for up to 99 hours after onset of disease and
retained infectivity even after storage for 7 days. Siler et al., (1926) confirmed that
Aedes aegypti was the vector of dengue, and Simmons et al., (1931) demonstrated the
efficiency of Aedes albopictus and Aedes polynesiensis as vectors of DENV.
Dengue epidemics were common on the Japanese mainland between 1942 and 1945, as
a result of World War II. Soldiers travelling between Japan and dengue prevalent areas
were continually introducing the virus to Japan and the spread of disease was attributed
to the ideal breeding conditions created for Aedes aegypti (Hotta, 1952). These
epidemics stimulated Japanese scientists to study dengue virus and its transmission and
this resulted in the first successful propagation of DENV in the brains of suckling mice
(Hotta, 1952). American scientists also isolated DENV from the blood of human
volunteers and recognised two distinct DENV serotypes using cross-protection studies in
humans (Sabin, 1952).
24
In 1954-56 the first major epidemics of DHF occurred in the Philippines and in Thailand
(Quintos et al., 1954; Hammon, 1969). During the period 1956-1995 over 3.5 million
cases of DHF/DSS, with over 58,000 resultant deaths, were reported to the World Health
Organisation (WHO), with the number of cases reported in most countries during 1981-
95 equalling or exceeding those reported in the previous 25 years (Halstead, 1999).
1.1 The pathogenesis of DHF/DSS

DHF/DSS is a significant cause of morbidity and mortality in children in most Southeast
Asian countries, where all serotypes are endemic (Halstead, 1980). The pathogenesis of
DHF/DSS and the principal sites of DENV replication in humans remain to be
elucidated. Post-mortem studies of fatal DHF/DSS cases revealed diffuse petechial
haemorrhages in most organs, effusions in serous cavities as well as retroperitoneal
oedema (Bhamarapravati et al., 1967). Microscopy suggested that no injury was
sustained to the endothelial cells or to blood vessels but hepatomegaly was evident with
mild to moderate necrosis and apoptotic Councilman bodies common (Bhamarapravati
et al., 1967). Studies of tissues from patients with DHF revealed viral antigens in the
liver, lymph nodes, spleen and bone marrow (Kurane et al., 1994; Rosen et al., 1999).
Several host cells are permissive to DENV infection, particularly mononuclear

phagocytes (Halstead and O’Rourke, 1977; Daughaday et al., 1981; Halstead, 1989;
Kurane et al., 1990). Monocytes and macrophages have been recognised as the primary
targets of DENV replication in humans, and also are important targets for antibody
enhanced infection (Halstead and O’Rourke, 1977). DENV has been identified in
Kupffer cells, primary endothelial cells and in monocytes from the liver of dengue
patients and viral replication has been detected in cultures of human hepatoma cells,
endothelial cells, T cells, dendritic cells, epithelial cells and fibroblasts (Kurane et al.,
1992; Marianneau et al., 1997; Mentor and Kurane, 1997; Avirutnan et al., 1998;
Bonner and O’Sullivan, 1998; Marianneau et al., 1999; Huang et al., 2000; Wu et al.,
2000; Ho et al., 2001; Marovich et al., 2001; Thepparit and Smith, 2004; Thepparit et
al., 2004).
25
The mechanisms by which DENV enter host cells are not completely defined. The
initial step in a DENV infection is the attachment of virus to the surface of the host cell
via cell receptors. It has been proposed that this attachment occurs in 2 stages. First
there may be a generic binding mechanism, in which the attachment of DENV to the
host cell and its concentration at these sites is dependent on the presence of ancillary
molecules such as highly sulphated polysaccharide chains or glycosaminoglycans
(GAGs), particularly heparan sulphate (HS) (Chen et al., 1996a; Chen et al., 1997; Hung
et al., 1999).
GAGs are found on a wide variety of cells and are capable of binding viruses from many
virus families, including flaviviruses. The binding of DENV to heparin and to a highly
sulphated HS has been observed in Vero and Chinese hamster ovary (CHO) cell lines
(Chen et al., 1997). It also has been observed that the binding of the DENV E protein to
host cells requires a highly sulphated and highly charged oligosaccharide (Marks et al.,
2001). The disruption of heparan sulphate molecules with heparinases also has been
shown to inhibit the binding of DENV to host cells (Chen et al., 1997).
The second stage in attachment of DENV to host cells may be the recognition of a
second virus type-specific cell receptor or virus binding protein that facilitates entry of
virus into the cell. It has been suggested that DENV utilise GAG molecules for initial
tethering to the host cell and that the GAG interaction increases the concentration of the
virus ligand in the vicinity of other receptors or receptor complexes involved in entry of
viruses into the host cell.
DENV have been observed to bind to proteins ranging in size from 40-100 kDa, from
different cell lines including C6/36, Vero, Chinese hamster ovary (CHO), K-562, HL60,
HEPG2, bone marrow and neuroblastoma cells in in vitro assays (Rothwell et al., 1996;
Ramos-Castaneda et al., 1997; Salas-Benito and del Angel, 1997; Bielefeldt-Ohmann,
1998; Munoz et al., 1998; Jindadamrongwech et al., 2004; Thepparit and Smith, 2004).
26
The GRP78 binding protein was identified as a liver cell expressed receptor element for
DENV-2 using mass spectrometry fingerprinting (Jindadamrongwech et al., 2004). The
CD-14 and DC-SIGN receptors also have been identified as receptors for DENV
attachment to human dendritic cells and to monocytes (Chen et al., 1999; Navarro-
Sanchez et al., 2003; Tassaneetrithep et al., 2003).
Following attachment to the cell surface, the flaviviruses appear to be internalised to an

endosome by receptor mediated endocytosis or in some cases to fuse directly with the
cell membrane (Gollins and Porterfield, 1986; Hase et al., 1989). The reduced pH of the
endosome facilitates fusion of the viral membrane with the endosomal membrane and
the release of the nucleocapsid (Gollins and Porterfield, 1986).
Infection with any DENV serotype provides life long immunity to that serotype, but
prior immunity to a second or third serotype seems to be a risk factor for DHF/DSS.
Individuals with pre-existing antibody against one dengue virus serotype are predisposed
to the more severe DHF/DSS when infected subsequently by a different dengue virus
serotype. In Southeast Asia, more than 95% of children aged one year or above develop
DHF/DSS following sequential dengue infections with different serotypes (Burke et al.,
1988; Halstead, 1988; Thein et al., 1997; Nguyen et al., 2004).
A primary infection with DENV-1, DENV-3 or DENV-4 followed by a secondary

infection with DENV-2 increased the risk of DHF/DSS (Sangkawibha et al., 1984;
Guzman et al., 1990; Thein et al., 1997). A small number of cases of DHF also occur
among children under one year of age following primary DENV infections. These are
attributed to pre-existing anti-dengue virus antibodies transferred across the placenta
from dengue immune mothers to the neonate (Halstead et al., 1969; Marchette et al.,
1979; Kliks et al., 1988).
27
1.2 Antibody dependent enhancement (ADE)
It has been proposed that non-neutralising or sub-neutralising levels of cross-reactive
anti-DENV antibodies, derived from a primary dengue infection or by placental transfer,
bind heterotypic virus and form non-neutralised infectious immune complexes. These
complexes infect Fc receptor-bearing cells such as mononuclear phagocytes via a trypsin
resistant Fc receptor, which interacts with the Fc portion of antibodies bound to virus.
This is believed to augment cell surface binding and internalisation of virions leading to
increased viral multiplication and viral load compared to infection via the trypsin
sensitive, proteinaceous viral receptor (Daughaday et al., 1981; Gollins and Porterfield,
1984). This phenomenon is called antibody dependent enhancement (ADE).
The first in vitro example of ADE of a flavivirus infection was observed when testing
neutralisation of Murray Valley encephalitis virus (MVEV) with antisera from
vertebrates (Hawkes, 1964). It was shown that the infectivity of MVEV on chicken
embryo monolayers was neutralised if the virus was combined with low dilutions of
chicken antisera to MVEV. However, at higher dilutions of antisera infectivity was
enhanced i.e. there were more plaques on monolayers infected with MVEV and high
dilutions of antibody, than on monolayers infected with MVEV alone (Hawkes, 1964).
Fractionation of the anti-MVE antisera revealed that enhancement was associated with
IgG antibodies (Hawkes and Lafferty, 1967).
Enhancement of DENV infection in vivo has been observed using rhesus monkeys
(Halstead et al., 1973b; Halstead, 1979). Monkeys infected with DENV-1, DENV-3 or
DENV-4 and subsequently infected with DENV-2 developed higher levels of viremia
than non-immune monkeys given the same dose of DENV-2 (Halstead et al., 1973b).
Monkeys inoculated intravenously (i.v.) with human cord blood containing anti-DENV
antibodies and subsequently infected with DENV-2 also developed higher levels of
viremia than non-immune monkeys given the same dose of DENV-2 (Halstead, 1979).
28
Halstead and O’Rourke (1977) later demonstrated that dengue immune serum from
humans or monkeys could enhance infection of non-immune human and monkey
peripheral blood mononuclear cells (PBMCs) by DENV-2. The dengue immune serum
only enhanced infection of non-immune PBMCs, when the anti-viral antibodies were
diluted beyond the neutralising endpoint (Halstead and O’Rourke, 1977). In vitro
enhancement of dengue virus infection also can occur with antibodies against other
flaviviruses. For example, individuals immunised with the 17D Yellow Fever virus
(YFV) vaccine were more likely to become infected with attenuated strains of DENV
(Bancroft et al., 1984).
ADE of DENV infection was dependent on the Fc portion of IgG antibodies interacting
with Fc receptors of host cells. IgG purified from dengue immune serum enhanced
infection of Fc receptor bearing cells, whereas Fab fragments derived from the IgG
could not enhance infection (Daughaday et al., 1981). The importance of Fc receptors in
ADE was demonstrated using polyclonal and monoclonal antibodies to block Fc
receptors. The incubation of antibodies with monocytes (Fc receptor-bearing cells)
before the addition of virus-antibody (Ab) complexes inhibited enhancement of DENV
infection (Daughaday et al., 1981). Similarly, a monoclonal antibody directed against
Fc receptors, inhibited the enhanced infection of Fc receptor bearing cells by West Nile
virus (WNV) (Peiris et al., 1981).
It is not known how enhanced viral replication in Fc-receptor bearing cells in vivo might
lead to DHF/DSS. It has been proposed that ADE increases the number of dengue-
infected cells and that the lysis or clearance of these cells leads to the release of
vasoactive mediators and procoagulants that increase vascular permeability and cause
DHF/DSS (Kurane and Ennis, 1997; Lei et al., 2001).
29
Activation of the complement system is common in DHF/DSS patients as indicated by
decreased serum levels of the complement proteins C3 and C5, and increased levels of
C3a and C5a (Bokisch et al., 1973). The immune complexes formed between anti-
DENV antibodies and circulating virus in secondary DENV infections may promote
DHF/DSS by activating the complement pathway and cleaving the complement proteins
C3 and C5 into C3a and C5a. These powerful mediators may stimulate the secretion of
histamines and increase vascular permeability (Bokisch et al., 1973). These immune
complexes also can bind to the surface of platelets (Boonpucknavig et al., 1979). It is
proposed that the destruction of opsonised platelets by complement or cell lysis may
explain the observed thrombocytopenia in DHF/DSS cases (Boonpucknavig et al.,
1979). Anderson et al., (1997) also demonstrated that antibody enhanced infection of
macrophages stimulated the secretion of an unidentified molecule that activated vascular
endothelial cells in vitro.
T lymphocytes also have been linked to the pathogenesis of DHF/DSS. It was proposed
that the enhanced infection of Fc receptor-bearing cells by DENV-Ab infectious immune
complexes in a secondary infection activated DENV cross reactive CD4+ and CD8+
memory cytotoxic T cells. The activation of T cells initiated the release of cytokines
such as interferon gamma (IFN-γ), interleukin 2 (IL-2) and tumour necrosis factor alpha
(TNF-α) and other mediators, which lysed DENV infected cells and increased vascular
permeability leading to signs of DHF/DSS (Kurane et al., 1989; Chaturvedi et al., 2000;
Lei et al., 2001).
1.3 Viral virulence

Despite the evidence that there is a significantly increased risk of severe dengue disease
following a secondary infection with a DENV of a different serotype to that causing the
initial infection, there have been cases of DHF/DSS in individuals with no detectable
anti-DENV antibodies (Scott et al., 1976; Rosen, 1977; Thein et al., 1997). It has been
suggested that these DHF/DSS cases are caused by more virulent strains of virus (Rosen,
1977).
30
Epidemiological studies have supported the virus virulence theory. The introduction of
an Asian genotype of DENV-2 into the Americas, where all serotypes were co-
circulating, coincided with the first cases of DHF (Rico-Hesse, 1990; Rico-Hesse et al.,
1997). Similar observations were made with the introduction of DENV-3 into the
Americas (Gubler and Clark, 1995). In contrast, the introduction of an American
genotype of DENV-2 into a community with previous immunity to DENV-1 did not
result in any DHF/DSS case (Watts et al., 1999). The failure of the American genotype
DENV-2 to cause DHF/DSS may have been associated with increased cross-protection
and greater neutralisation of that virus type by pre-existing antibody in the DENV-1
immune population, whereas Asian genotype DENV-2 strains were not neutralised
(Kochel et al., 2002)
There also have been structural differences identified between DENV strains that
correlate with pathogenesis (Leitmeyer et al., 1999). The analysis of the whole genome
sequence of DENV-2 causing either DF (American genotype) or DHF (Southeastern
Asian genotype) revealed that the primary determinants of DHF occur at amino acid 390
of the envelope (E) protein, the downstream loop (nucleotides 68-80) of the 5’non
coding region (NCR) and the upstream 300 nucleotides of the 3’NCR (Leitmeyer et al.,
1999). Cologna and Rico-Hesse (2003) evaluated the affects of mutating residue 390 of
the E protein and or replacing the 5’ and 3’ NCR on the infectivity of DENV-2 in human
primary cell cultures. These changes were responsible for changes in DENV-2
replication of human cells and virulence (Cologna and Rico-Hesse, 2003).
It also has been reported that high DENV viremia was associated with increased disease
severity, with the peak viral titre in a DSS patient, 100-1000 fold higher than the virus
titre of dengue fever patients (Vaughn et al., 2000). Recent studies using assays that
measure the growth and infectivity of DENV-2 in mosquito (C6/36) cells and dendritic
cells, suggested that DENV-2 (Southeastern Asian genotype), which causes DHF, can
out compete DENV-2 (American genotype) (Cologna et al., 2005). These authors
proposed that the DENV-2 Southeastern Asian genotype will continue to cause DHF
epidemics by displacing other viruses.
31
1.4 The dengue virus
The dengue viruses (DENV) are members of the genus Flavivirus within the family
Flaviviridae. There are at least 70 species of flaviviruses and the most important human
pathogens are dengue virus (DENV), yellow fever virus (YFV) and Japanese
encephalitis virus (JEV). On the basis of cross-neutralisation tests using polyclonal
hyperimmune antisera, the flaviviruses have been divided into antigenic complexes, the
dengue complex (DENV-1, DENV-2, DENV-3 and DENV-4) is one of those (Calisher
et al., 1989). Phylogenetic analysis of DENV based on the amino acid sequence of the
envelope proteins (E) has produced relationships similar to those identified by the cross-
neutralisation studies (Monath and Heinz, 1996).
Flaviviruses are spherical particles approximately 40-60 nm in diameter consisting of a

30 nm isometric nucleocapsid core surrounded by a lipid envelope approximately 10 nm
thick (Henchal and Putnak, 1991). The nucleocapsid is composed of core (C) proteins
and houses the viral genomic RNA. The envelope (E) and pre-Membrane (prM)/
membrane (M) structural proteins, are embedded in the lipid envelope by C-terminal
hydrophobic anchors. Immature intracellular virions contain the precursor form of the
membrane protein called the pre-membrane (prM) protein, whereas mature virions
contain the membrane protein (M) (Heinz et al., 1994) (Figure 1.2).
Cryoelectron microscopy (cryoEM) and image reconstruction techniques indicated that

mature and immature DENV-2 particles have significant structural differences. The
mature DENV-2 particles are icosahedral in symmetry and have a smooth surface with a
diameter of approximately 500 angstrom (Å) (Kuhn et al., 2002) (Figure 1.3). In
contrast the immature form is 15% larger in diameter and is covered with 60 three
pronged spikes called trimers that jut from the virus surface (Zhang et al., 2003a)
(Figure 1.3)
32
Figure 1.2 The structure of the immature and mature flavivirus virion (Heinz et al.,
1994). The membrane proteins prM, M and E are embedded in the lipid envelope by C-
terminal anchors. The core (C) protein forms the nucleocapsid, which is icosahedral in
symmetry. The immature (intracellular) virion is characterised by the presence of the
pre-membrane (prM) protein which is a molecular chaperone for the envelope (E)
protein. The prM protein is cleaved during virus maturation leaving the membrane (M)
protein in the mature virion form.
33
A.

B.

Figure 1.3 Structure of (A) mature and (B) immature DENV-2 particles as determined
by cryoelectron microscopy and image reconstruction (Kuhn et al., 2002; Zhang et al.,
2003a). The colors in (A) represent the three domains of the E protein monomer. Red
is domain I, yellow is domain II and blue is domain III. The circled region in (B)
represents the trimeric spike of the immature virion.
34
The flavivirus genome is a positive sense single-stranded RNA molecule of
approximately 11 Kb, which has a type I cap at its 5’ end (m7GpppAmp) and lacks a
poly A tail (Wengler and Wengler, 1981; Chambers et al., 1990). A single uninterrupted
open reading frame (ORF) representing 95% of the genome (approximately 10 Kb) is
translated into a single polyprotein precursor of approximately 3388 amino acids. Signal
and stop transfer sequences direct the translocation of the polyprotein back and forth
across the membrane of the endoplasmic reticulum (ER) where it is co- or post-
translationally cleaved into ten individual proteins (Chang, 1997; Lindenbach and Rice,
2003) (Table 1.1). Three of the proteins (C, prM/M, E) are structural proteins, whereas
the other seven proteins are non-structural (NS).
A host signalase located in the lumen of the endoplasmic reticulum cleaves at the C-
prM, prM-E, E-NS1 and NS4a-NS4b junctions, while the cleavage of the NS1-NS2a
junction is performed by an unknown enzyme, most probably a host proteinase (Falgout
et al., 1989; Nowak et al., 1989; Lin et al., 1993; Stocks and Lobigs, 1995). The virus
encoded proteinase is a complex of the NS2b and NS3 proteins and cleaves the NS2a-
NS2b, NS2b-NS3, NS3-NS4a and NS4b-NS5 junctions at specific consensus sequences
composed of basic amino acid residues (Falgout et al., 1991; Cahour et al., 1992).
Internal proteolysis of the C, NS2a, NS3 and NS4a proteins is also catalysed by the viral
proteinase (Nowak et al., 1989; Arias et al., 1993; Lin et al., 1993; Teo and Wright,
1997).
Flanking the ORF of the flavivirus genome are 5’ and 3’ non-coding regions (NCR)
which are approximately 100 and 100-600 nucleotides in length, respectively (Rice,
1990; Zeng et al., 1998). The terminal nucleotide sequences of both NCRs form highly
conserved secondary structures such as stem loops and pseudoknots, which are cis acting
elements of the RNA genome that facilitate RNA replication by binding to host cell
proteins and the viral replicase complex (NS1, NS2a, NS3, NS4a, NS5). Modifications
to these secondary structures result in virulence changes, which are a focus for vaccine
development (Proutski et al., 1997; Mandl et al., 1998; Whitehead et al., 2003).
35
Table 1.1 Flavivirus proteins
Protein Molecular Weight Number of Function

(kDa) amino acids
Core 13-16 113 Core protein
Pre-Membrane 19-23 166 Chaperone protein for E;

precursor to M
Membrane 8-8.5 75 Component of viral

envelope
Envelope 51-60 495 Major envelope protein
NS1 44-49 350-410 Putative RNA replication

cofactor
NS2a 16-21 150-210 Putative RNA replication

cofactor. Coordinates the
shift between RNA
packaging and RNA
replication
NS2b 12-15 130 Cofactor for serine protease

in viral protease complex.
NS3 67-76 615 Viral protease; RNA

replication cofactor;
NTPase and putative
helicase
NS4a 24-32 150-280 Putative RNA replication

cofactor
NS4b 10-11 110-250 Unknown
NS5 91-98 900 Viral RNA dependent RNA

polymerase
36
1.5 Structural proteins of flaviviruses
1.5.1 The core protein

The highly basic core (C) protein makes up the structural backbone of the viral
nucleocapsid. The high proportion of arginine and lysine residues imparts a positive
charge on the C protein that facilitates binding to the RNA genome during virus
assembly (Rice et al., 1986). The C protein binds specifically to segments of both the 5’
and 3’ NCRs to facilitate viral encapsidation and/or RNA synthesis (Westaway and Ng,
1980; Khromykh and Westaway, 1996). Analysis of purified C protein has failed to
identify epitopes associated with antibody mediated neutralisation (Bulich and Aaskov,
1992).
1.5.2 The membrane proteins (prM and M)

Flaviviruses are assembled intracellularly in an immature form containing pre-
membrane (prM) protein, which is a glycoprotein of 165 amino acids, containing six
cysteine residues that form three disulphide bridges (Nowak and Wengler, 1987).
Shortly before the release of virus from the host cell, prM is cleaved in the trans-Golgi
network by a host cell derived proprotein convertase furin, following an acid induced
conformational change of prM, which renders cleavage sites accessible (Stadler et al.,
1997). The cleavage releases the amino-terminal of prM from the virus and leaves the
C-terminal portion anchored to the viral envelope and separated from the E protein
(Wengler and Wengler, 1989a). The remaining truncated protein containing 75 amino
acids is the membrane protein (M) of the mature virion and contains no cysteines or
glycosylation (Nowak and Wengler, 1987).
The prM protein is a chaperone protein that prevents the irreversible inactivation of the
E protein at low pH during virus maturation in the Golgi vesicles (Guirakhoo et al.,
1992). MVEV virus is 400 times more resistant to acidic conditions if prM rather than
M is present (Guirakhoo et al., 1992). The prM protein assists in the proper folding,
membrane association and assembly of the E protein (Konishi and Mason, 1993; Allison
et al., 1995b).
37
The infectivity, hemagglutination and fusion activity of tick-borne encephalitis virus
(TBEV) and MVEV decreased when prM to M cleavage was inhibited by ammonium
chloride late in the viral replication cycle (Randolph and Stollar, 1990; Guirakhoo et al.,
1992; Heinz et al., 1994). The E protein of prM-containing virus also cannot undergo
the structural rearrangements required for attachment to host cell receptors or fusion
with cell membranes (Heinz et al., 1994).
The prM protein forms heterodimeric complexes with the envelope (E) protein in the
immature virion (Allison et al., 1995a; Wengler and Wengler, 1989a) (Figure 1.2).
Pulse-chase radiolabeling of DENV infected vero cells demonstrated a rapid
interassociation of prM and E proteins, and sucrose gradient sedimentation analysis
suggested that prM-E complexes progressed from simple heteromers to more densely
sedimenting structures indicating increased multimerisation (Wang et al., 1999). prM-E
heteromers of even higher complexity were observed in virus particles, suggesting an
intracellular assembly process which results in the networking of prM-E subunits into a
lattice-like structure found in virus particles (Wang et al., 1999).
Pulse chase labelling experiments which confirmed that prM and E rapidly form a
heterodimeric complex also demonstrated differences in the folding rates of each
protein. The pre-membrane protein was shown to be a very rapidly and independently
folding protein, acquiring a native structure quickly and without any interaction with the
E protein. In contrast, the E protein folded more slowly and required co-expression of
and interaction with prM to acquire its native conformation (Lorenz et al., 2002).
The idea that prM-E interactions play an important role in the assembly of Flavivirus
particles is supported by the observation of ordered, membrane containing, icosahedrally
symmetrical recombinant subviral particles (RSPs) which resulted from the co-
expression of prM and E proteins in mammalian cells (Schalich et al., 1996; Ferlenghi et
al., 2001; Lorenz et al., 2003).
38
Cryoelectron microscopy (cryoEM) analysis of TBEV RSPs at a resolution of 19 Å
indicated that there were 30 copies of the E protein dimer arranged in a T=1 icosahedral
lattice (Ferlenghi et al., 2001). From the RSP data, it was proposed that the E proteins
were arranged on the native TBEV particle in a T=3 icosahedral lattice (Ferlenghi et al.,
2001). In recent studies, immature TBEV RSPs were generated by mutating the furin
cleavage recognition site of prM (Allison et al., 2003). This resulted in two distinct
populations of prM containing RSPs with diameters of 30 nm and 50 nm.
The structure of immature prM containing DENV, which were prepared by treating
DENV-infected cells with ammonium chloride to suppress prM cleavage, was
determined at a resolution of 16 to 25 Å using cryoEM and image reconstruction
techniques (Zhang et al., 2003a). In contrast to the mature DENV virion, the surface of
the immature DENV consisted of icosahedrally organised trimeric spikes with each
spike consisting of three prM:E heterodimers (Zhang et al., 2003a; Kuhn et al., 2002)
(Figure 1.3).
1.5.3 The envelope protein

The flavivirus envelope protein (E) is a “class II” viral fusion protein which initiates
attachment of virus to host cell receptors, mediates fusion with host cell membranes,
hemagglutinates specific erythrocytes and induces humoral and cell-mediated immune
responses (Roehrig, 1997; Roehrig, 2003). The E protein, in neutral pH conditions, lies
parallel to the surface of the viral membrane as dimeric or trimeric units, with each
monomeric unit anchored to the viral membrane by a carboxy terminal transmembrane
region (Winkler et al., 1987a; Wengler and Wengler, 1989a; Allison et al., 1995a; Rey et
al., 1995).
39
According to the structure of the mature DENV, defined by cryoEM and icosahedral
image reconstruction at a resolution of 24 Å, there are 180 copies of the E glycoprotein
and 180 copies of the M protein on the surface of DENV (Kuhn et al., 2002). The 180
copies of the E glycoprotein are organised as sets of three nearly parallel dimers that
interact to form a highly ordered outer shell with icosahedral symmetry (Figure 1.4).
There are three E monomers per icosahedral asymmetric unit. The interaction of the
three E dimers forms a “herringbone” configuration. The three, nearly parallel, dimers
form a dominant association with each other that supersedes the interactions between
individual monomers in the assembled icosahedral particle (Kuhn et al., 2002).
The image reconstruction of the mature DENV was refined to a resolution of 9.5 Å, and
provided structural details on the transmembrane regions of the E and M proteins as
well as the stem region of the E protein (Zhang et al., 2003b). It was determined that
the transmembrane regions of both E and M formed antiparallel helical hairpin
structures, that did not extend to the interior of the viral membrane, whereas the alpha
helical stem regions interact with the outer region of the viral membrane (Zhang et al.,
2003b).
The envelope (E) protein monomer consists of approximately 495 amino acids and is
glycosylated in the majority of flaviviruses, including DENV (Nowak and Wengler,
1987). The degree of glycosylation varies between the tick and mosquito-borne virions
but does not alter the antigenic structure of the E protein (Winkler et al., 1987b). The
DENV E protein has potential asparagine (N)-linked glycosylation sites at Asn-67 and
Asn-153 but it has been found that the utilisation of glycosylation sites differs amongst
serotypes (Johnson et al., 1994). The loss of a glycosylation site in a DENV-2 variant
resulted in tolerance to elevated pH and suggested that glycosylation is involved in the
conformational changes required for viral fusion (Guirakhoo et al., 1993). Lee et al.,
(1997) also reported that DENV-3 variants with changes at E155, which is adjacent to
the proposed glycosylation site in DENV-2 (E153), fused at a lower pH and had
increased infectivity in C6/36 cells.
40
A.

B.

Figure 1.4 The organisation of E protein dimer sets (circled) on the surface of a mature
DENV-2 particle (A) (Kuhn et al., 2002). The different colours displayed represent the
three domains of the monomeric subunits of the DENV-2 E protein dimer structural
model (B) (Modis et al., 2003). Domain I is red, domain II is yellow and domain III is
blue.
41
The presence of both N-linked carbohydrates on the DENV E protein also is important
for the interaction of DENV with DC-SIGN which is a C-type lectin receptor. It has
been proposed that the tetrameric structure of DC-SIGN may need to interact with more
than one sugar molecule at a time on the E protein of DENV for efficient internalisation
of the virus (Navarro-Sanchez et al., 2003). The recent structural model of the DENV-3
E protein determined that the spacing between the carbohydrate groups on Asn-67 and
Asn-153 was sufficient for the interaction of the E protein with DC-SIGN or other
oligomeric structures (Modis et al., 2005)
The alignment of the amino acid sequences of yellow fever virus (YFV) and West Nile
virus (WNV) E proteins identified 12 conserved cysteine residues, which in WNV and
tick-borne encephalitis virus (TBEV) were shown to form six intramolecular disulphide
bridges that were integral to protein antigenicity and stability (Nowak and Wengler,
1987; Wengler and Wengler, 1989b). The first model of the flavivirus E protein was
based on the primary amino acid sequences of WNV and YFV, and featured three
domains (R1, R2 and R3) connected by two loops (L1 and L2) (Nowak and Wengler,
1987). The E protein had reduced immunogenicity when it was reduced and denatured
or when disulphide bonds were disrupted or cysteine residues mutated (Wengler and
Wengler, 1989b; Lin et al., 1994; Roehrig et al., 2004).
The first antigenic model for flaviviruses was based on the E protein of TBEV. The
competitive binding of anti-TBEV MAbs for spatially related sites on the virion enabled
the identification of three non-overlapping antigenic domains (A, B and C). The precise
location of the epitopes was defined by analysing the genotype of antigenic variants of
TBEV, selected in the presence of neutralising monoclonal antibodies as well as the
reactivity of peptide fragments with the antibodies (Mandl et al., 1989). Each domain
was composed of several epitopes recognised by monoclonal antibodies exhibiting
different functional activities and different serological specificities (Heinz et al., 1983;
Mandl et al., 1989).
42
Domain A was a discontinuous structure formed by the combination of two distant
regions of the E protein (amino acids 50-125, 200-250). Antibodies against domain A
had greater neutralising and hemagglutination inhibiting properties than other
antibodies, suggesting an important role in viral function (Guirakhoo et al., 1989). The
region of domain A between amino acids 200 and 250 was unable to bind antibodies
following sodium dodecyl sulphate (SDS) treatment, suggesting the presence of epitopes
stabilised by hydrophobic interactions (Guirakhoo et al., 1989).
Domain B was a distinct region between amino acids 301 and 395 containing complex
specific epitopes. Antibodies against domain B have weaker neutralising and
hemagglutination inhibiting properties than domain A antibodies (Guirakhoo et al.,
1989). The antigenicity of domain B was destroyed by reduction and
carboxymethylation, suggesting the conformation of this domain was dependent on the
disulphide bridge linking amino acids 307 and 338. The binding of a neutralising MAb
to residue E307 in DENV-2 also was abrogated following the site directed mutagenesis
of critical cysteine residues (Lin et al., 1994). Domain C consisted of amino acids 132-
177 and was glycosylated at amino acid 154 of the TBEV E protein (Mandl et al., 1989).
The epitopes of domain C were resistant to SDS treatment and reduction, suggesting
they were conformation independent (Guirakhoo et al., 1989).
The antigenic model of the TBEV E protein provided some insight into the function of
domains A, B and C. The secondary structure of the domains and the conformational
changes required for functions such as viral-mediated fusion and host cell recognition
required a detailed three-dimensional model. The isolation of soluble envelope protein
(sE) dimers from TBEV composed of two truncated 45 kDa monomers (amino acids 1-
395) lacking the C-terminal transmembrane region (sE dimer) enabled hexagonal rod
shaped E protein crystals to be generated and studied by X-ray diffraction at a 2 Å
resolution (Heinz et al., 1991; Rey et al., 1995). The resulting structural model provided
new insights into the secondary structure and function of each domain of the flavivirus E
protein (Figure 1.5).
43
Figure 1.5 A ribbon diagram of the three dimensional structural model of the TBEV envelope protein dimer (Rey et al., 1995).
The different colours represent the three domains of the E protein monomer. Domain I, the central domain is colored red, domain II,
the dimerisation domain is colored yellow and domain III is colored blue. The attached carbohydrate CHO group is shown on domain
I. The N-termini (NH2) is shown in domain I and the C-termini (COOH) is shown in domain III.
44
1.6 Structural model of the envelope protein
The E protein of TBEV was a flat elongated head-to-tail dimer that extended in a
direction that was parallel to the viral membrane and was organised as an icosahedral
lattice (Rey et al., 1995; Ferhlenghi et al., 2001). This structure is also common to
alphaviruses but was unique when compared to other virus fusion proteins, which are
characterised by a trimeric spike structure (Rey et al., 1995; Allison et al., 1999).
Because of these structural features, the alphavirus and Flavivirus E proteins are
classified as type II fusion proteins, while most other viral fusion proteins are type I.
The ectodomain fragment of the sE dimer, representing 80% of the molecule, consisted
mostly of beta-sheet and loop structures and does not contain any long alpha helical
segments (Rey et al., 1995). It consisted of three primary domains, with significant β-
strand secondary structure: a central β barrel (domain I), an elongated dimerisation
region (domain II) and a C-terminal, immunoglobulin like module (domain III) (Rey et
al., 1995). Each domain contained two apposed layers of secondary structure. Domains
I, II and III corresponded well to domains C, A and B respectively of the antigenic
model.
1.6.1 Domain I
Domain I (amino acids 1-51, 137-189 and 285-302) was discontinuous, containing 120
amino acids in three distinct regions. It consisted of an eight-stranded up-and-down β-
barrel that forms two β-sheets (β sheet 1: A0C0D0E0F0; β sheet 2: B0I0H0G0) which
faced each other across a tightly packed hydrophobic interior (Rey et al., 1995).
Domain I also had two disulphide bridges joining cysteine residues 3-30 and 186-290
and a unique glycosylation site carrying a single carbohydrate side chain which is
attached to the E0F0 loop on the external surface of the protein (Rey et al., 1995).
1.6.2 Domain II
The two loops connecting the discontinuous segments of domain I represented domain II
(amino acids 52-136 and 190-284). It was an extended, finger-like structure, similar in
topology to a kringle domain (Stuart and Gouet, 1995). The base of this domain
consisted of antiparallel β-sheet of five short strands (gfeah sheet), with two α-helices
packed against one surface (αA,αB).
45
A narrow sandwich that consisted of a three-stranded β-sheet (bdc sheet) and β-hairpin
(ji) made up the elongated segment of the domain (Rey et al., 1995; Stuart and Gouet,
1995). The three-stranded β-sheet was cross-linked by disulphide bridges, and it is
probable that one of these bridges stabilised the “cd-loop” at the tip of domain II. The
cd-loop was a tightly folded structure that contained the internal fusion peptide
DRGWGNGCGLFGGK (amino acids 98-111), the sequence of which is highly
conserved amongst the flaviviruses (Rey et al., 1995). In the dimeric state the cd-loop
lay in a hydrophobic crevice of the E protein surrounded by hydrophilic epitopes, which
were the primary neutralisation and hemagglutinin sites of the virus (Rey et al., 1995).
It was suggested that the fusogenic potential of the flavivirus E protein resided in a
region extending from the base of domain II, in a flexible hinge-like region between the
gfeah and D0 sheets. It was suggested that at a low pH the hinge motion of this region
swings the tip of domain II above the viral membrane exposing the fusion peptide for
interaction with host cell membranes (Rey et al., 1995).
It has been reported that the fusion activity of TBEV RSPs was disrupted when changes
were made at residue E107 of the fusion peptide region (Allison et al., 2001). Mutants
with a threonine or aspartate residue substituted for leucine at residue 107 lost fusion
activity, whereas a phenylalanine change had no effect. It has also been shown that the
hydrophobic residues of the cd-loop (fusion peptide) are essential for the attachment of
TBEV soluble E ectodomains to target membranes (Allison et al., 2001).
The binding of monoclonal antibodies to epitopes of domain II was disrupted following

the low pH induced conformational change of the E protein necessary for viral mediated
fusion (Guirakhoo et al., 1989; Holzmann et al., 1995). There were also some regions of
domain II that were more accessible following treatment of virus at pH 6.0, including
amino acids 58-121 and 225-249 in DENV-2 and amino acids 221-240 in TBEV
(Holzmann et al., 1993; Roehrig et al., 1998).
46
The low pH induced conformational change was a two-step process involving the
reversible dissociation of the E dimers followed by an irreversible formation of E
trimers, which are more stable than the native dimers (Rey et al., 1995; Stiasny et al.,
1996; Stiasny et al., 2001). Thus the E protein exists in a metastable state that is
energetically poised to be converted to the fusogenic state by exposure to low pH. The
dimer-monomer equilibrium in the first step may depend on the protonation state of the
E protein. It was suggested that the protonation of the native E dimer is indispensable
for generating a monomeric intermediate structure that is required for the formation of
the energetically more stable final trimeric form (Stiasny et al., 2001).
It also was demonstrated that the stem anchor region of the TBEV E protein is required
for trimerisation (Allison et al., 1999). Full length E dimers formed trimers at low pH,
however truncated E dimers including the soluble E protein ectodomain (amino acids 1-
395) which lacked the stem anchor region, underwent reversible dissociation into
monomers without forming trimers (Stiasny et al., 1996).
TBEV that has been exposed to an acidic pH does not have fusion activity and cannot
interact with liposomes, which suggested that the E protein trimer form does not mediate
fusion (Corver et al., 2000; Stiasny et al., 2001). It was proposed that an intermediate E
protein structure generated during the dimer to trimer transition, mediated fusion.
Stiasny et al., (2002) utilised liposome co-flotation studies in conjunction with
chemically crosslinked soluble E protein dimers to demonstrate that the dimer to
monomer transition is vital for the E protein interaction with cell membranes in fusion.
In addition, it was demonstrated that membrane associated E protein formed trimers
(Stiasny et al., 2002). This was unexpected as previous studies have suggested that the
stem anchor region of the TBEV E protein was vital for trimerisation. It was concluded
that the formation of trimers is facilitated by membrane binding and trimer stability is
maintained by contacts between the ectodomains only and is not dependent on sequence
elements in the stem-anchor region, as previously assumed (Stiasny et al., 2001; Allison
et al., 1999).
47
1.6.3 Domain III
Domain III (amino acids 303-395) was located near the C-terminal of the E protein and
was characterised by an immunoglobulin-like β-barrel structure. One of the β-sheets of
domain III (ABED sheet) faces domain I and comes into contact with the cd loop of
domain II, while another β-sheet (CFG sheet) forms the outer lateral surface of the dimer
(Rey et al., 1995). This domain sat upright at a right angle to the viral membrane, so
that the C-terminus of the polypeptide chain projected downwards.
This meant that domain III would project slightly above and below domains I and II
which lay end to end along the membrane as a rigid unit (Stuart and Gouet, 1995). A
fifteen residue hinge region and disulphide bridge connecting domains I and III might
facilitate movement of domain III with respect to the rest of the molecule (Rey et al.,
1995). This flexibility and the projection of domain III from the viral surface hinted at
its possible role in attachment to host cell receptors. It has been shown that MAbs that
bind domain III are the most effective at preventing attachment of DENV to host cells
(Crill and Roehrig, 2001).
The charged residues residing on the upper lateral surface of domain III have been
implicated in the binding of flaviviruses to host cell receptors. This region of domain III
houses the RGD (Arg-Gly-Asp) motif, which is specific to some mosquito-borne
flaviviruses but absent in tick-borne flaviviruses (Lobigs et al., 1990). In addition, the
RGD motif is generally not present in DENV sequences. The RGD sequence makes up
three of the four additional amino acids that occur between the G and F sheets of the
structural model. This sequence has been implicated as an integrin binding domain,
however mutagenesis of the RGD sequence to a net positive charge enhanced virus
binding to heparin, which suggested that the region is important for GAG recognition
(Lee and Lobigs, 2002)
48
Changes to domain III residues have been shown to influence the virulence phenotype of
several flaviviruses and influence the binding of neutralising MAbs (Roehrig et al.,
2004). Amino acid changes in domain III of TBEV at E309-E311 also influenced the
tertiary structure of the TBEV E protein, which may affect E protein recognition of cell
receptors (Mandl et al., 2000).
Amino acid sequences important for the recognition of vertebrate and invertebrate cells
have also been localised on the upper lateral surface of domain III. This includes the
charged residues within amino acids E284-E310 and E386-E411, which have been
characterised as heparan sulphate (HS) binding domains (Chen et al., 1997) and residues
E380-E389 which are important in the DENV serotype-specific binding of C6/36 cells
(Hung et al., 2004). Thullier et al., (2001) also found that an expressed form of domain
III from the DENV-2 E protein interacted with highly sulphated heparan.
1.6.4 Stem anchor region

The stem anchor region represents the last 20% of the E protein primary sequence which
precedes the C terminus. This region contained potential amphipathic alpha-helical and
conserved structural elements that have been implicated in membrane anchoring,
interactions with prM during virion assembly and low pH-induced structural changes
associated with the virus fusion, specifically the trimerisation of soluble E protein
(Stiasny et al., 1996; Allison et al., 1995a).
To identify specific functional elements in the stem anchor region, a series of C-terminal
deletion mutants were constructed and the properties of the resulting truncated proteins
examined (Allison et al., 1999). The functional regions of the stem anchor region of the
TBEV E protein are depicted in Figure 1.6. The residues located in the first predicted
alpha helical region (H1pred: 401-413) of the stem anchor was essential for the
conversion of soluble protein E dimers to a homotrimeric form upon low-pH treatment,
a process resembling the transition to the fusogenic state in whole virions (Allison et al.,
1999). In addition, the H1 alpha helix contains sequences responsible for the
intracellular localization of E protein in DENV-2 (Purdy and Chang, 2005).
49
Figure 1.6 Schematic diagram of the TBEV E protein monomer including the
functional determinants of the stem-anchor region (Allison et al., 1999). The three
domains of the E protein ectodomain (E1-E400), derived following trypsin cleavage of
TBEV, are represented as shaded ovals. The locations of the trypsin cleavage site, as
well as the fusion peptide, are also indicated. The regions below the trypsin site are
designated as the stem and anchor regions. The anchor regions occur within the viral
membrane which is indicated by the two parallel lines. H1pred (E401-E413) and H2pred
(E431-E449) in the stem region represent predicted alpha helices, which are separated
by the conserved sequence (CS) element (E414-E430). TM1 (E450-E471) and TM2
(E473-E496) are transmembrane segments of the anchor region.
50
The residues located within the second alpha helical region (H2pred: amino acids 431-
449) and the first membrane-spanning region (TM1: amino acids 450-472) of the stem
anchor were found to be important for the stabilisation of the prM-E heterodimers but
not essential for prM-mediated intracellular transport and secretion of soluble E proteins
(Allison et al., 1999). TM1 was also required for the incorporation of E protein into
virus particles (Allison et al., 1999).
The second membrane spanning region (TM2: amino acids 473-496) of the stem-anchor
was a signal sequence for NS1, but was not required for virus particle formation (Allison
et al., 1999). It has been shown that the stem anchor is dispensable for the trimerisation
of E protein in the presence of cell membranes (Stiasny et al., 2002).
1.6.5 Dimeric interactions

The extended contact between monomeric units of the TBEV envelope protein left holes
of approximately 20 Å in diameter between the two proteins. Polar interactions and van
der Waals forces between hydrophilic amino acid side chains were involved in the
proximal interactions between monomers whereas the distal contacts were non-polar
(Rey et al., 1995).
1.7 Dengue virus E protein models
1.7.1 Antigenic model

Following the characterisation of the TBEV E protein, the antigenic map of the DENV-2
E protein was deduced using an extensive panel of murine-based monoclonal antibodies
to map sixteen epitopes (Roehrig et al., 1998). The map is similar to the antigenic map
of the TBEV with three domains (A, B and C) identified.
Monoclonal antibodies against domain A recognised 45 kDa and 22 kDa peptides

resulting from the tryptic digest of the DENV-2 E proteins. These peptides represented
amino acids 1-400 (45 kDa) and amino acids 1-120 (22 kDa) of the E protein. Domain
A specific MAbs were able to block fusion, inhibit hemagglutination and neutralise virus
infection in vitro (Roehrig et al., 1998).
51
Monoclonal antibodies against domain B epitopes bound to a 9 kDa peptide derived
from tryptic digestion of the DENV-2 E protein and represented amino acids 300-400.
MAbs recognising domain B neutralised DENV infection in vitro and inhibited
hemagglutination, but did not block fusion of virus with host cells. Both domains A and
B were spatially associated. MAbs recognising epitopes in domain C failed to neutralise
virus infectivity in vitro or inhibit virus hemagglutination and the epitopes in this
domain were sensitive to denaturation by SDS (Roehrig et al., 1998).
1.7.2 Structural model of DENV E protein dimer

The crystal structure of the DENV-2 E protein dimer (residues 1-394) has been solved to
enable a structural model of the DENV E protein to be predicted (Modis et al., 2003).
The domains of the DENV-2 E protein share essentially similar conformation to those of
the TBEV E protein domains. This might be expected as the primary sequence of the
envelope protein for both viruses shares 37% sequence homology and the location of
disulphide bridges are conserved. There were differences in the conformation of several
loop structures and in the orientation of the domains, which was a consequence of the
flexible hinge-like regions between domains (Modis et al., 2003).
More recently, the crystal structure of the DENV-3 E protein dimer (residues 1-391) was
solved (Modis et al., 2005). It closely resembled its homologs from DENV-2 and TBEV
in dimeric structure and in details of protein folds (Modis et al., 2005). This model also
demonstrated that epitopes involved in DENV neutralisation are clustered on the surface
of domain III, which has been implicated in attachment to host cell receptors.
This suggested that the neutralisation of DENV is targeted against blocking virus
attachment to host cells. In addition, the crystal structure of DENV-3 demonstrated that
the neighbouring glycans attached to the glycosylated asparagine residues at E67 and
E153 are positioned in such a way that oligomeric lectins such as DC-SIGN, reported to
be involved in virus attachment to cells, could bind tightly through multiple attachment
points (Modis et al., 2005).
52
1.7.3 Structural model of the DENV E protein dimer post fusion
The crystal structure of the DENV-2 E protein in its trimeric post-fusion conformation
was recently determined and this provided new insights into the mechanisms of fusion,
particularly how the fusion loop structure which contains the fusion peptide interacts
with target cell membranes (Modis et al., 2004).
The intermediate steps involved in the fusion of the Flavivirus E protein with target cell
membranes have been hypothesised to occur based on differences observed between the
crystallographic structures of the pre- and post-fusion E protein molecules. It was
proposed that the fusion loops of three E protein subunits interacted to form an aromatic
anchor structure at the tip of the newly formed trimer that was capable of inserting into
target membranes. The target cell membranes were believed to catalyse the
trimerisation reaction, leading to the formation of a pre-fusion intermediate in which the
trimer bridged host cell and viral membranes (Modis et al., 2004).
Formation of trimer contacts spread from the fusion loops at the trimer tip to domain I at
the base. Domain III shifted and rotated to create trimer contacts, causing the C-
terminal portion of E to fold back towards the fusion loop and cause the two membranes
to bend towards each other. The formation of a structure termed the ‘hemifusion stalk’
was an essential intermediate in the membrane fusion reaction. Creation of additional
trimer contacts between the stem anchor and domain II led first to hemifusion and then
to the formation of a lipidic fusion pore (Modis et al., 2004).
The folding back of domain III and the rearrangement of beta strands at the trimer
interface projected the C terminus of E towards the fusion peptide, and positioned it at
the entrance of a channel, which extends towards the fusion loops along the inter-subunit
contact between domains II. The 53 residue stem anchor region connecting the end of E
with the transmembrane anchor could easily span the length of this channel even if the
stem was alpha helical. By binding in the channel, the stem anchor would contribute
additional trimer contacts with domain II of another subunit (Modis et al., 2004).
53
1.8 Analysis of functional sites on the flavivirus E protein
The mapping of epitopes on the flavivirus E protein is important for understanding viral
function and for identifying determinants important for the design of effective vaccines.
Functional epitopes have been identified on the flavivirus E protein using monoclonal
antibodies directed against the E protein, peptides and fusion proteins representing
regions of the E protein and by analysing gene sequences of natural and selectively
modified viral variants, including infectious cDNA clone derived variants (Roehrig,
1997). More recently, phage display libraries have been used to identify peptide
sequences that mimic functional epitopes on the flavivirus E protein (Thullier et al.,
2001; Wu and Lin, 2001).
Competition between monoclonal antibodies for binding sites on the E protein has been
used to identify spatial relationships between different regions of the protein and help
define antigenic domains. Inhibition of binding of one antibody by another may occur
as a result of distant conformational changes caused by the binding of the first antibody,
or as a result of steric hindrance if the relevant epitopes are adjacent or overlapping
(Heinz et al., 1983). The characteristics of the monoclonal antibodies indicate whether
epitopes of a certain reactivity or function such as neutralisation or hemagglutination
inhibition are clustered or spread.
The results of competitive antibody binding experiments carried out with flaviviruses are
outlined in Table 1.2. It was found that epitopes form independent domains and/or
overlapping domains (Roehrig et al., 1983; Henchal et al., 1985; Kimura-Kuroda and
Yasui, 1986). The segregation of antigenic domains may depend on the number of
monoclonal antibodies used in the mapping (Tsekhanovskaya et al., 1993).
54
Table 1.2 Epitopes on the flavivirus envelope protein located using competitive binding
experiments with monoclonal antibodies.
Epitopes per
Author Virus MAbs Domains Reactivity Function
domain
Simantini DENV-1 10 5 2 Type HI, N
and 5 Type
Banerjee,
1995 1 Type
1 Complex
1 Group
Gentry et al., DENV-2 22 3 1 Type HI, N

1982 4 Type HI
17 Group HI, N
Henchal et DENV-2 8 4 4 Type and subcomplex HI, N

al., 1985 2 Type and subgroup HI, N
1 Complex
1 Group HI, N
Jianmin et DENV-2 22 4 16 Group HI, N

al., 1995 2 Type HI, N
2 Type HI, N
2 Type HI, N
Roehrig et DENV-2 13 3 10 Type, subcomplex and group HI, N

al., 1998 2 Subcomplex and subgroup HI, N
1 Subcomplex HI, N
Kimura- JEV 8 8 1 Group HI

Kuroda and 1 Subgroup HI
Yasui,
1986 1 Strain and type N
1 Subgroup N
1 Subgroup N
1 Subgroup
1 Subgroup HI, N
1 Type N
HI: Hemagglutination inhibition.
N: Neutralisation.
55
Table 1.2 cont
Epitopes per
Author Virus MAbs Domains Reactivity Function
domain
Cecelia et JEV 14 4 5 Group HI, N
al., 1988 4 Strain HI, N
2 Type N
3 Group N
Hall et MVEV 9 4 3 Complex HI, N

al., 1990 2 Subgroup and group HI
3 Group N
1 Type N
Roehrig et SLEV 8 6 1 Type

al., 1983 1 Type HI
1 Type HI, N
3 Type, subcomplex and group HI, N
1 Complex HI
1 Group
Heinz et TBEV 8 3 4 Type and complex HI, N

al., 1983 3 Subtype, subgroup and group HI
1 Subtype
Guirakhoo et al., TBEV 19 6 5 Subtype, complex, subgroup HI, N

1989 and group
5 Type and complex HI, N
6 Subtype and complex HI, N
1 Complex
1 Complex N
1 Type
Tsekhanovskaya TBEV 25 5 12 Type, subtype, complex and HI, N

et al., 1993 subcomplex
9 Complex and subcomplex HI, N
2 Subcomplex HI
1 Type HI
1 Subcomplex HI
56
The linear epitopes of the E protein which are immunogenic can be identified by
screening anti-flavivirus antibodies, either polyclonal or monoclonal, against peptides
representative of the E protein primary sequence (Aaskov et al., 1989; Innis et al., 1989).
The peptides can be designed as overlapping segments or as potential immunogenic hot-
spots based on hydrophilicity plots, homology analysis and secondary structure
predictions (Roehrig et al., 1989).
Peptide fragments have also been derived for MAb analysis by digesting viruses with
proteases such as trypsin and chymotrypsin (Roehrig et al., 1998) Flavivirus envelope
proteins expressed as fusion proteins in E.coli and phage display libraries also have been
used to identify regions of the envelope protein recognised by monoclonal antibodies
(Mason et al., 1990; Megret et al., 1992; Thullier et al., 2001).
The epitopes identified on the flavivirus envelope protein using peptides, fusion proteins
and phage display libraries are outlined in Table 1.3. Overall, these methods provide
useful information but can be problematic when characterising conformationally
dependent epitopes. The expression of DENV E protein fragments may present a
problem because the native structure of conformationally dependent E protein epitopes
is dependent on the appropriate disulphide bond formation and the coexpression of prM
and E proteins (Konishi and Mason, 1993; Allison et al., 1995b; Roehrig et al., 2004).
In order to maintain MAb reactivity, large DENV E protein fragments are expressed,
however, this defeats the purpose of precisely mapping an epitope location.
In order to characterise conformationally dependent functional epitopes, several other

strategies have been used. Viral variants have been isolated from flavivirus populations
by passage in vitro in cell culture or by selection in the presence of neutralising
antibodies, low pH or increased temperature. The analysis of viral variants defines
epitopes by identifying the amino acid changes indicative of a particular viral phenotype.
57
Table 1.3 Epitopes in the flavivirus envelope protein identified using linear peptides,
fusion proteins and phage display.
Author Virus Method Antibody Immunogenic regions of E

Mason et al., DENV-1 Fusion proteins Non-neutralising aa 76-93, 298-403
1990 MAb
Neutralising MAb aa 293-403
Thullier et DENV-1 Phage display Neutralising aa 306-314

al., 2001 MAb
Aaskov et al., DENV-2 Pepscan: 488 Dengue immune Rabbit (6 domains):

1989 overlapping antisera: aa 1-58, 59-297,
octapeptides human and rabbit 288-391, 392-442,
446-476, 479-495
Neutralising MAb aa 50-57,

(1B7) 127-134, 349-356
Innis et al., DENV-2 Pepscan: 490 Convalescent 124 hexapeptides

1989 overlapping antisera from 25 domains react with
hexapeptides 7 dengue patients 2 or more antisera
22 hexapeptides
7 domains react with
all 7 antisera
Roehrig et al., DENV-2 18 synthetic Mouse 9/18 peptides

1990 peptides ascites reactive
DENV-2 0/18 peptides

MAbs reactive
Antipeptide antibody aa 35-55,

neutralising DENV-2 352-368
Antipeptide antibody aa 98-110

binding low pH treated
DENV-2
aa: Amino acid
58
Table 1.3 cont
Immunogenic regions
Author Virus Method Antibody
of E
Megret et DENV-2 Fusion proteins Non aa 22-58, 60-135,
al., 1992 neutralising 60-205, 298-397,
MAb 304-332
Neutralising aa 60-135,
MAb 60-205, 298-397
Mouse aa 22-58, 60-135,

ascites 60-205, 293-397,
304-332
Trirawatanapong et DENV-2 Fusion proteins Neutralising aa 386-397

al., 1992 MAb
Mouse aa 386-397
ascites
Roehrig et al., 1998 DENV-2 Peptide fragments from Group 1 MAbs 45 kDa fragment
protease digest Neutralisation (+) aa 1-400
30 kDa, 28 kDa,
25 kDa fragment
aa 158-400
9 kDa fragment
aa 300-400
Group 2 MAbs 45 kDa fragment

Neutralisation (+) aa 1-400
30 kDa, 28 kDa,
25 kDa fragment
aa 158-400
22 kDa fragment
aa 1-120
Group 3 MAbs 45 kDa fragment

Weak or no aa 1-400
neutralisation
59
Table 1.3 cont
Immunogenic regions
Author Virus Method Antibody
of E
Roehrig et DENV-2 Synthetic peptides MAb panel aa 333-351
al., 1998
Falconar, 1999 DENV-2 Pepscan: 47 overlapping Neutralising MAb aa 274-283,

nona/decapeptides 349-359
Mason et JEV Fusion proteins 10 neutralising MAb aa 303-396

al., 1989
Mouse aa 303-396
ascites
Wu and Lin, 2001 JEV Phage display Neutralising aa 307-309,

MAb 327-333,
386-390
Roehrig et MVEV 11 synthetic peptides MVEV 9/11

al., 1989 antisera peptides
reactive
Neutralising aa 35-50
anti-peptide
antibody
Holzmann et al., TBEV 19 synthetic peptides MAb panel aa 1-22,

1993 221-240
60
The amino acid changes in the flavivirus E protein related to escape from antibody
mediated neutralisation are outlined in Table 1.4. Amino acid changes related to other
viral phenotypes are outlined in Table 1.5. To confirm the affect of specific amino acid
changes on the phenotype of flaviviruses, reverse genetics experiments have been
performed on infectious cDNA clones of different flaviviruses (Table 1.6).
Neutralisation epitopes have been identified in each domain of the DENV E proteins
using various methods. However there have been no neutralisation epitopes identified in
the DENV-4 E protein, which is the rationale behind this project. The identification of
epitopes involved in neutralisation of each DENV serotype is important for the design of
a tetravalent vaccine that induces a neutralising antibody response against each DENV
serotype, specifically in this study, a DENV chimeric E protein based vaccine.
61
Table 1.4 Epitopes on the flavivirus envelope protein involved in neutralisation identified using
monoclonal antibodies.
Author Virus Amino acid change Phenotypic changes of variant

Beasley and Aaskov, DENV-1 279 (Phe-Ser) Both changes caused HA at lower pH
2001 and decreased temperature sensitivity
293 (Thr-Ile)
Lin et al., 1994 DENV-2 307 (Lys-Glu) n.d.
Lok et al., 2001 DENV-2 69 (Thr-Iso) Both changes caused a decrease in

311 (Glu-Gly) FFWI and temperature sensitivity
Serafin and Aaskov, DENV-2 169 (Ser-Pro) Both changes caused smaller plaques,
2001 275 (Gly-Arg) HA at lower pH and decreased FFWI
DENV-3 386 (Lys-Asn) Altered cytoplasmic staining of infected cells
Cecilia and Gould, JEV 270 (Ser-Ile) Loss of HA and neurovirulence

1991 333 (Gly-Asp) Loss of HA and neurovirulence
Hasegawa et al., 1992 JEV 52 (Gln-Lys) Decreased neurovirulence

52 (Gln-Arg)
Wu et al., 1997 JEV 306 (Glu-Gly) n.d.

331 (Ser-Arg)
Morita et al., 2001 JEV 52 (Gln-Arg) n.d.

52 (Gln-Glu)
126 (Ile-Thr)
136 (Lys-Glu)
275 (Ser-Pro)
367 (Asn-Asp)
HA: Hemagglutination
FFWI: Fusion from within
n.d.: not determined
62
Table 1.4 cont.
Author Virus Amino acid change Phenotypic changes of variant
Jiang et al., 1993 LIV 308 (Asp-Asn) Loss of HA and decreased neuroinvasiveness
310 (Ser-Pro)
311 (Lys-Asn) No HA or neurological changes
Gao et LIV 308 (Asp-Asn) n.d.

al., 1994
McMinn et MVEV 126 (Ala-Glu) No virulence changes

al., 1995 128 (Arg-Ser)
128 (Arg-Lys)
274 (Phe-Val)
276 (Ser-Arg)
277 (Ser-Asn)
277 (Ser deletion)
277 (Ser-Ile) Decreased neuroinvasiveness, inhibition of HA and
decreases in vitro growth
Mandl et TBEV 67 (Ala-Val) n.d.
al., 1989 171 (Lys-Glu)
Holzmann et al., 1989 TBEV 71 (Asp-Gly) n.d.
Holzmann et al., 1990 TBEV 384 (Tyr-His) Decreased neuroinvasiveness
Holzmann et al., 1997 TBEV 123 (Ala-Lys) Decreased neuroinvasiveness

181 (Asp-Tyr)
368 (Gly-Arg)
Chambers et al., 1998 WNV 68 (Leu-Pro) Decreased neuroinvasiveness

307 (Lys-Glu)
Beasley and WNV 307 (Lys-Arg) n.d.

Barrett, 2002 307 (Lys-Asn)
330 (Thr-Ile)
332 (Lys-Thr)
Lobigs et YFV 71 (Asp-Lys) n.d.

al., 1987 71 (Asp-Tyr)
71 (Asp-His)
72 (Asp-Gly)
Ryman et al., 1997 YFV 125 (Met-Iso) n.d.

155 (Asp-Gly)
158 (Thr-Iso)
173 (Iso-Thr)
240 (Ala-Val)
LIV: Louping ill virus
63
Table 1.5. Functional determinants defined by comparing the phenotype of viral variants
Viral variant
Author Virus selection Amino acid change Phenotypic changes
Despres et al., DENV-1 HA/ fusion 96 (Phe-Val) Lower optimal pH for HA and
1993 variants 180 (Ala-Thr) FFWO assay
297 (Thr-Met)
379 (Val-Ile)
473 (Thr-Ala)
Puri et al., 1997 DENV-1 Chemical 202 (Glu-Lys) Increased temperature sensitivity,
mutagenesis and decreased plaque size and mouse
passage in neurovirulence
FRhL-2
cells
Puri et al., 1997 DENV-1 Serial passage in 293 (Thr-Ile) Increased temperature sensitivity,
PDK cells 44(Glu-Lys) human attenuation, decreased
plaque size and mouse
156 (Thr-Ile) neurovirulence
202 (Glu-Lys)
264 (Leu-Ser)
293 (Thr-Ile)
366 (Asn-Asp)
Duarte dos DENV-1 Neuroadapted in 196 (Met-Val) Increased neurovirulence

Santos et al., mice 365 (Val-Iso)
2000 405 (Thr-Iso)
Guirakhoo et DENV-2 Growth at 153 (Asn-Asp) Decreased in vitrogrowth and

al., 1993 altered pH plaque size, fusion at alkaline/
neutral pH
6 (Ile-Met) Decreased in vitro growth and
134 (Asn-Ser) plaque size, fusion at alkaline/
neutral pH
153 (Asn-Tyr)
Sanchez and DENV-2 Plaque 390 (Asp-His) Increased plaque size and
Ruiz, 1996 morphology neurovirulence
variants
FFWO: Fusion from without.

FRhL: Fetal rhesus lung.
PDK: Primary dog kidney.
64
Table 1.5 cont.
Viral variant
Author Virus selection Amino acid change Phenotypic changes
Lee et al., 1997 DENV-3 Mouse brain 18 (Ala-Ser) Increased neurovirulence and
passage 54 (Ala-Glu) FFWI occurs at lower pH
277 (Phe-Ser)
401 (Glu-Lys)
403 (Thr-Ile)
Vero cell passage 191 (Phe-Val/Leu) Increased growth in Vero cells and
202 (Lys-Arg) FFWI occurs at lower pH
266 (Thr-Ile)
268 (Ile-Thr)
268 (Ile-Ser)
268 (Ile-Val)
291 (Glu-Val)
C6/36 cell passage 155 (Thr-Ala) Increased growth and cytopathic

155 (Thr-Met) effect in C6/36 cells and FFWI
occurs at lower pH
Hasegawa et JEV Vero cell 364 (Ser-Phe) Decreased neurovirulence

al., 1992 Passage 367 (Asn-Iso) Altered virus-cell interactions
Chen et al., JEV Gamma irradiation 138 (Glu-Lys) Decreased neurovirulence and
1996b of JEV neuroinvasiveness
Ni et al., 1995 JEV Comparison of 138 (Glu-Lys) Decreased neurovirulence

high and low 176 (Iso-Val)
neurovirulence
strains
Ni and Barrett, JEV Comparison of 46 (Thr-Ile) Decreased neuroinvasiveness and

1996 high and low 76 (Met-Thr) neurovirulence
neurovirulence
strains 129 (Ala-Thr)
209 (Arg-Lys)
227 (Pro-Ser)
306 (Gly-Glu)
352 (Ala-Val)
388 (Glu-Gly)
408 (Leu-Ser)
65
Table 1.5 cont.
Viral variant
Author Virus Amino acid change Phenotypic change
selection
Ni and Barrett, JEV Mouse brain 306 (Gly-Glu) Decreased neuroinvasiveness and
1998 receptor binding 408 (Leu-Ser) neurovirulence
variants selected at
varied pH
212 (Leu-Met) Decreased neuroinvasiveness
222 (Ala-Ser)
202 (Tyr-His) Decreased neuroinvasiveness and

306 (Gly-Glu) neurovirulence
408 (Ala-Ser)
Vrati et al., JEV Compare different Decreased neurovirulence and

76 (Thr-Met)
1999 JEV isolates reduced fusion activity
Monath et al., JEV Passage in fetal 279 (Lys-Met)

2002 rhesus lung cells
Holbrook et al., LGTV Mouse and human 416 (Lys-Ala) Decreased neurovirulence
2001 brain binding 438 (His-Tyr)
variants 440 (Val-Ala)
473 (Asn-Lys)
MVEV SW13 cell 390 Decreased neuroinvasiveness

Lobigs et al.,
Adapted (Asp-Asn/Glu/Tyr)
1990
Labuda et al., TBEV Sequential tick 84 (Glu-Lys) Decreased neurovirulence and loss
1994 passage 319 (Ile-Thr) of HA activity
Wallner et al., TBEV Compare isolates 52 (Asn-Ser) Increased temperature sensitivity

1996 with differing 167 (Ile-Val) and decreased neurovirulence
neurovirulence
331 (Thr-Ser)
Chambers et al., WNV Compare isolates 68 (Leu-Pro) Decreased neuroinvasiveness

1998 with differing
neuroinvasiveness
Jennings et al., YFV Compare YFV 155 (Gly-Asp) Increased neuroinvasiveness and
1994 vaccine strains with 303 (Gln-Lys) neurovirulence
differing virulence
LGTV: Langat virus
66
Table 1.5 cont
Viral variant
Author Virus Amino acid change Phenotypic change
selection
Schlesinger et YFV Neuroadapted in 52 (Arg-Gly) Increased neurovirulence and
al., 1996 mice 173 (Ile-Thr) replication rates in mouse central
nervous system
305 (Phe-Val)
380 (Arg-Thr)
462 (Ile-Met)
305 (Ser-Phe)
Ryman et al., Attenuation of YFV
YFV 325 (Ser-Leu) Decreased neurovirulence
1998 vaccine strain
52 (Arg-Gly)
Chambers and Neuroadapted in 173 (Iso-Thr)
YFV Increased neurovirulence
Nickells, 2001 mice 326 (Lys-Gly)
380 (Arg-Thr)
67
Table 1.6. Functional determinants identified by infectious clones
Amino acid
Author Virus substitutions Functional changes
Gualano et al., DENV-2 126 (Glu-Lys) Increased neurovirulence
1998 126 (Lys-Glu) Decreased neurovirulence
Hiramatsu et al., DENV-2 (prM/E)/ 383 (Glu-Gly) No neutralisation or binding by anti-

1996 DENV-4 chimera 384 (Pro-Glu/ Asp/ DENV-2 MAb 3H5 and decreased
Asn) neurovirulence
385 (Gly- Lys/ Ser)
Bray et al., 1998 DENV-2 (prM/E)/ 71 (Glu-Asp) Increased neurovirulence

DENV-4 chimera 126 (Glu-Lys)
Chen et al., 1995 DENV-3 (prM/E)/ 406 (Glu-Lys) Increased neurovirulence

DENV-4 chimera
Kawano et al., DENV-4 intratypic 155 (Thr-Iso) Increased neurovirulence

1993 chimera 401 (Phe-Leu)
Sumiyoshi et al., JEV 138 (Glu-Lys) No neuroinvasiveness and small plaques

1995
Chambers et al., JEV (prM/E)/ YFV 107 (Leu-Phe) Decreased neurovirulence

1999 chimera 176 (Iso-Val)
138 (Glu-Lys)
279 (Lys-Met)
Arroyo et al., 2001 JEV (prM/E)/YFV 107 (Leu-Phe) Decreased neurovirulence

chimera 138 (Glu-Lys)
(ChimeriVax-JE) 279 (Lys-Met)
Campbell and LGTV 119 (Phe-Val) Decreased neuroinvasiveness

Pletnev, 2000 308 (Asp-Ala)
389 (Asn-Asp)
438 (His-Tyr)
Pletnev and LGTV 119 (Phe-Val) Decreased neuroinvasiveness

Men, 1998 (prM/E) / 285 (Gly-Ser) and neurovirulence
DENV-4 333 (Phe-Ser)
chimera 389 (Asn-Asp)
68
Table 1.6 cont.
Amino acid
Author Virus substitutions Functional changes
Lee and Lobigs, MVEV 390 (Asp-Gly/Ala/His) Decreased neurovirulence
2000
Hurrelbrink and MVEV 277 Changes in HA activity and

McMinn, 2001 (Ser/Iso/Asn/Val/Pro) decreased neuroinvasiveness
390 (Asp-Asn/Glu/Tyr) Decreased neuroinvasiveness
Mandl et al., 2000 TBEV 310 (Thr-Lys) Decreased neuroinvasiveness

and neurovirulence
Gritsun et al., TBEV 496 (His-Arg) Decreased neuroinvasiveness and virus

2001 growth
Pletnev et al., TBEV 154 (Asn-Lys) Decreased neurovirulence

1993 (prM/E) / DENV-4
chimera 384 (His-Gly) Increased in vitro growth
387 (Leu-Phe) Increased neurovirulence
434 (Asn-Leu) Decrease in vitro growth, plaque size and

neurovirulence
Guirakhoo et al., YFV/DENV-1 chimera 204 (Lys-Arg) Reduced neurovirulence and

2004 (ChimeriVax-DEN1) reduced viremia in monkeys
69
1.9 Dengue vaccine design
An ideal vaccine against dengue must be cost effective, provide long lasting protection
against all serotypes, and invoke effective humoral and cell-mediated immune responses
(Brandt, 1988; Halstead, 1988). The possibility of enhanced dengue infections in
vaccinees must also be addressed (Cardosa, 1998).
Several approaches to vaccine design have been developed. This includes the use of live
attenuated virus vaccines, inactivated whole-virion vaccines, synthetic peptides, subunit
vaccines, vector expression, recombinant live vector systems, infectious cDNA clone-
derived vaccines and naked DNA (Gubler, 1998). The most extensively trialled
vaccines have been the live attenuated dengue virus vaccines. The first live attenuated
vaccine which was developed by researchers in Thailand, and was licensed by Aventis
Pasteur produced 80-90% seroconversion rates to all four serotypes after the
administration of two doses in young children (Sabchareon et al., 2004). The second
live attenuated vaccine which was developed by the Walter Reed Army Institute of
Research (WRAIR) in the USA and was licensed by GlaxoSmithKline, had similar
seroconversion rates in adult subjects (Edelman et al., 2003).
Despite these results, the molecular basis of virus attenuation is not understood and it
was proposed that interference in replication between DENV serotypes and/or
interference in immune stimulation may lead to imbalanced immune responses resulting
in incomplete protection and enhanced disease severity (Stephenson, 2005). The
reversion to virulence through mutation or recombination between the vaccine
components or with wild viruses is also a concern (Stephenson, 2005)
70
The safety and immunogenicity of the live attenuated dengue vaccines may be improved
using infectious cDNA clones. Candidate vaccines derived from infectious cDNA
clones may also be more genetically stable and this may decrease the level of phenotypic
reversion (Trent et al., 1997). Full length cDNA clones yielding infectious viruses upon
transfection of transcribed RNA in mammalian cell lines are now available for several
flaviviruses, some of which are outlined in Table 1.6.
Using infectious cDNA clones, it is possible to identify regions of the viral genome
important in virulence. A change to these regions in the infectious cDNA clone makes it
possible to engineer highly attenuated viruses. The genetic modification of infectious
cDNA clones is a promising technique for achieving stable attenuation of flaviviruses
that can be included in the rational design of novel flavivirus live vaccines (Mandl et al.,
1998). Attempts to attenuate an infectious clone derived DENV-4 by deleting regions of
the 5’ and 3’ NCR unfortunately, resulted in mutant viruses with low attenuation,
immunogenicity and decreased replication in cell culture (Cahour et al., 1995; Men et
al., 1996).
A highly attenuated flavivirus was generated from a TBEV cDNA clone by deleting
regions of the 3’ NCR (Mandl et al., 1998). In recent studies, attenuated DENV for
each serotype were generated by deleting a 30 nucleotide region of the 3’ NCR of their
infectious clones (Durbin et al., 2001; Whitehead et al., 2003; Blaney et al., 2004a;
Blaney et al., 2004b).
Infectious cDNA clones have also been used to engineer chimeric DENV. There are
several reports of intertypic chimeric DENV where the structural proteins of one DENV
serotype have been used to replace these proteins in a parental virus from a second
DENV serotype or an attenuated flavivirus (Table 1.6).
71
If intertypic DENV are possible, then there is potential to engineer viruses with
intertypic dengue envelope proteins using infectious cDNA clones (Aaskov, 2001). A
hybrid DENV E protein was derived from a baculovirus expression system and
contained domain I and II of DENV-2 and a C-terminal truncated domain III of DENV-
3 (Bielefeldt-Ohmann et al., 1997). Immunisation of mice with the hybrid E protein
resulted in a low level of neutralising antibodies and this may have been due to the low
levels of protein expressed by baculovirus (Bielefeldt-Ohmann et al., 1997).
In a recent study, a single chimeric DENV E protein, produced using DNA shuffling
techniques, demonstrated a tetravalent neutralising antibody response against each
DENV serotype in mice (Apt et al., 2006). If a chimeric virus containing neutralising
epitopes from more than one serotype were to be constructed using infectious cDNA
clones, then knowledge of the location of these epitopes on the E protein and their amino
acid sequence would be essential (Aaskov, 2001).
72
1.10 Objectives
The primary objective of this study was to identify epitopes in the envelope protein of
DENV-4 involved in neutralisation by antibodies. In order to achieve this objective, the
following approaches were undertaken:
1) A panel of MAbs were generated against DENV-4 in BALB/c mice and

characterised using various serological and functional assays. DENV-4 specific
neutralising MAbs were used for subsequent domain and epitope mapping
studies of the DENV-4 E protein.
2) MAbs were used in competitive binding experiments to define antigenic domains

of the DENV-4 E protein involved in neutralisation. Competitive binding
experiments employing the anti-DENV-4 MAbs and sera from DENV patients
were also used to determine whether the epitopes recognised by the MAbs were
the same or spatially related to epitopes recognised by serum from DENV
patients. This is important for determining which epitopes are important for the
design of DENV vaccines.
3) Several strategies were employed to identify structural domains, peptides or

amino acid residues recognised by neutralising MAbs on the DENV-4 E protein.
By analogy with studies of other DENV and other flaviviruses, knowledge of the
epitopes in the E protein of DENV-4 may provide insight into the relationship between
the structure and function of the protein. In addition, it may provide information that
would assist development of interserotypic DENV chimeras for use as vaccines.
If epitopes involved in the neutralisation of different DENV serotype are distributed in

several domains of the E protein then it may be possible to elicit neutralising antibodies
against multiple DENV serotypes using chimeric E proteins derived from two or more
different DENV serotypes. Therefore, identifying the location of domains or epitopes
on the DENV-4 E protein involved in neutralisation by antibodies is imperative.
73
2 MATERIALS AND METHODS
2.1 Cells
Baby hamster kidney cells (BHK-21 clone 15; Diercks, 1959), Aedes albopictus
mosquito cells (C6/36; Igarashi, 1978), African green monkey cells (Vero clone E6),
porcine-equine kidney (PS-EK; Gorman et al., 1975) cells and myeloma cells (SP2/0)
were cultured in RPMI-1640 medium (Invitrogen, U.S.A) supplemented with 10% v/v
heat inactivated fetal calf serum (FCS; Invitrogen, U.S.A) and 100 units/ml penicillin,
100 µg/ml streptomycin and 300 µg/ml L-glutamine solution (Invitrogen, U.S.A) (10%
FCS-RPMI). Hybridoma cell lines were maintained in either 10% FCS-RPMI or 10%
FCS-RPMI supplemented with 2% v/v of a 50x Hypoxanthine Thymidine solution that
contained 5 mM Hypoxanthine and 0.8 mM Thymidine (50x HT; ICN Biomedical,
U.S.A) (HT-RPMI). The cells were cultured in 25 cm2, 80 cm2 and 175 cm2 tissue
culture flasks (Nunc, Denmark) using 10 ml, 20 ml and 50 ml volumes of culture
medium respectively.
The attached cell lines (BHK, C6/36, PS-EK and Vero) were passaged every 4 to 7 days.
The cell monolayer was washed with RPMI-1640 and 5 ml of trypsin-EDTA solution
was added for 5 minutes at 37oC. The trypsin-EDTA solution was prepared in sterile
phosphate buffered saline (PBS; appendix section 6.1.2) from a 10x trypsin-EDTA stock
containing 0.5% w/v Trypsin and 5.3 mM EDTA (Gibco, U.S.A). After addition of
trypsin-EDTA the flask was tapped to dislodge cells which then were resuspended in
culture medium and added to new flasks. The unattached cell lines (hybridomas and
SP2/0) were passaged every 4 to 7 days.
74
For cryopreservation, cells from three confluent 175 cm2 flasks were centrifuged at 400g
for 5 minutes and the cell pellet resuspended in 20 ml of chilled 10% FCS-RPMI
supplemented with 10% v/v Dimethyl sulphoxide (DMSO; Sigma, U.S.A). The cell
suspensions were added to 1 ml cryotubes (Nunc, Denmark) and stored in a “Mr Frosty”
Cell Freeze Box (Nunc, Denmark) at -80oC for 12 hours. The ampoules of frozen cells
then were transferred to liquid nitrogen.
For cell passaging, a single ampoule of cells was obtained from liquid nitrogen stores
and was thawed in a water bath at 37oC and the contents were decanted into 50 ml tubes
(Falcon, U.S.A) containing 5 ml of growth medium and centrifuged at 400g for 5
minutes. The supernatant was discarded and the cell pellet was resuspended in 20 ml of
culture medium and decanted into an 80 cm2 flask.
2.2 Virus
2.2.1 Preparation of working stocks

Working stocks of the prototype strains of dengue virus (DENV) serotypes 1, 2, 3, 4;
Kunjin virus (KUNV) and Murray Valley encephalitis virus (MVEV) were recovered
from the supernate of cultures of C6/36 cells infected with suckling mouse brain (SMB)
virus preparations. Additional DENV-4 isolates, from a variety of geographical
locations, were recovered from cultures of C6/36 cells infected with virus previously
grown in cultures of C6/36 cells infected with virus in patient serum. Stocks of DENV
used in the study are shown in Table 2.1. Stocks of prototype strains of virus or stocks
of DENV-4 isolates were diluted 1 in 10 in RPMI-1640 and added to C6/36 monolayers
for 2 hours to allow virus attachment and internalisation. The volume of virus inoculum
used was 2 ml for 25 cm2 flasks, 8 ml for 80 cm2 flasks and 15 ml for 175 cm2 flasks.
Following infection, RPMI-1640 was added to the flasks to give standard volumes used
for cell culture and the cells were incubated at 30°C/ 2.5% CO2 for 7 days. The virus
supernatant was then decanted from the flask into a 50 ml tube and centrifuged at 400g
for 5 minutes. The clarified supernatant was decanted into a fresh 50 ml tube and FCS
was added to produce a 30% v/v solution. Virus was stored in 1 ml cryotubes at -800C.
75
Table 2.1 Dengue viruses used in this study
Virus Origin Seed stocks

Serotype Strain Country Date of isolation Tissue Supplier
DENV-1 Hawaii Hawaii 1944 SMB YARU
DENV-2 New Guinea C New Guinea 1944 SMB YARU
DENV-3 H87 Philippines 1956 SMB YARU
DENV-4 H241 Philippines 1956 SMB YARU
DENV-4 1674 Singapore 1990 TCS WHO
DENV-4 508 Thailand 1999 TCS WHO
DENV-4 520 Thailand 1999 TCS WHO
DENV-4 083 Timor 2001 TCS WHO
DENV-4 31500 Vietnam 2000 TCS WHO
DENV-4 38201 Vietnam 2001 TCS WHO
SMB, suckling mouse brain; TCS, tissue culture supernatant.
YARU, Yale Arbovirus Reference Centre
WHO, World Health Organisation virus reference centre,
Queensland University of Technology
76
2.2.2 Concentration of DENV-4 by precipitation with polyethylene glycol
Supernate from fifty 175 cm2 flasks of C6/36 cells infected 7 days previously with
DENV-4 H-241 was decanted into 500 ml centrifuge bottles (Beckman Instruments,
U.S.A) and centrifuged at 14,300g for 1 hour at 40C in a JA-10 rotor (Beckman
Instruments, U.S.A.) in an Avanti J25I high speed centrifuge to remove cell debris. One
volume of sterile Polyethylene glycol (PEG) solution consisting of 40% w/v PEG
(molecular weight (Mw) 60 kDa; BDH Laboratory Supplies, U.K.), 15% w/v sodium
chloride (BDH Chemicals, U.K.) in distilled water was added to 4 volumes of clarified
virus supernate and stirred at 40C for 16 hours. The solution then was centrifuged at
14,300g for 2 hours to precipitate the PEG-virus complexes. The virus pellet was
resuspended in 5 ml of RPMI-1640 and stored at -80°C in 200 µl aliquots in cryotubes.
2.2.3 Preparation of lysate of DENV-4 infected, and uninfected, cells

Five ml of 10% w/v sodium dodecyl sulphate (SDS) in distilled water (appendix section
6.2.5) was added to a 175 cm2 monolayer of C6/36 cells infected with DENV-4 H241 or
to uninfected cells. The resulting cell lysate was decanted into a 50 ml tube and the
DNA was removed by winding round a glass rod. Two hundred microlitre aliquots of
the lysates were stored in cryotubes at -800C.
2.3 Production of hybridomas and anti-DENV-4 MAbs

Six to 8 week old female BALB/c mice were acquired from the University of
Queensland Central Animal Breeding House and experiments were conducted at the
Herston Animal Research Facility.
To generate hybridomas producing IgM antibodies, the mice were injected

intraperitonealy (i.p.) with 250 µl of DENV-4 H241 on day 1 and boosted with 250 µl of
the same virus intravenously (i.v.) on day 7. Spleens were harvested on day 10. To
generate hybridomas producing IgG antibodies, the mice were injected with 250 µl of
DENV-4 H241 i.p. on day 1 and day 14 and i.v. on day 21 and spleens were harvested
on day 25. SMB virus preparations with a virus titer of 5x 105 plaque forming units
(pfu) /ml were used for mouse inoculations.
77
The mice were anaesthetised in CO2 and euthanased by cervical dislocation, at the
completion of the immunisation schedule. The spleen was removed from each mouse
into 20 ml of 10% FCS-1640 and disrupted by cutting with sterile scissors and then
drawing cell clumps in and out of a 1 ml syringe. Residual clumps of cells were allowed
to settle for 10 minutes, and the supernate decanted into a 15 ml tube (Falcon, U.S.A).
An aliquot of spleen cells was diluted 1 in 10 in leucocyte dilution solution (2% v/v
acetic acid in PBS) and the concentration of leucocytes determined in a hemocytometer.
Hybridomas were prepared using a modification of the method described by Zola

(1987). Mouse feeder cells were prepared 2 to 4 days prior to preparing hybridomas.
Feeder cells were obtained by injecting 5 ml of 10% FCS-1640 into the peritoneal cavity
of a euthanased BALB/c mouse and then recovering the cell suspension from the cavity
5 minutes later. The suspension was centrifuged at 400g for 5 minutes and the cell pellet
was resuspended in 100 ml of 10%FCS-1640. One hundred microlitres of the feeder
cells were added to each well of 20 flat bottom 96 well plates (Nunc, Denmark).
The myeloma cells from three confluent 175 cm2 tissue culture flasks were centrifuged
at 400g and the cell pellet was resuspended in 20 ml of RPMI-1640. The concentration
of myeloma cells was determined in a hemocytometer. Myeloma cells were added to the
spleen cells at a ratio of 1 myeloma cell to 10 spleen cells. The cell mixture was
centrifuged at 400g for 5 minutes and the supernate was decanted. The cell pellet was
resuspended by tapping the tube.
78
One millilitre of PEG-1500 (Boehringer-Mannheim, Germany) was added to the cell
pellet over 1 minute using a 1 ml syringe and needle. The cells were left to stand for 1
minute. One ml of RPMI-1640 was then added over 1 minute, followed by 5 ml of
RPMI-1640 over 2 minutes and 20 mls of RPMI-1640 over 3 minutes. The previous
steps were performed at 37°C. The cell suspension was then centrifuged at 400g for 5
minutes and the cell pellet was resuspended in 100 ml of RPMI-1640 supplemented with
20% v/v heat inactivated FCS and 2% v/v of a 50x HAT (5 mM Hypoxanthine, 0.02
mM Aminopterin 2H2O and 0.8 mM Thymidine [HAT; ICN Biomedicals, U.S.A])
(HAT-RPMI). One hundred microlitres of the cell suspension was added to each well of
96 well flat bottom tissue culture plates containing mouse feeder cells.
One hundred microlitres of HAT-RPMI medium was removed from each well of the 96
well plates every 3 days and replaced with 100 µl of fresh HAT-RPMI, until the cells in
the wells were ≥ 50% confluent. The supernatant from wells with ≥ 50% confluent cells
was screened for the presence of antibody against DENV-4 H241 by indirect ELISA and
indirect immunofluorescence assay (IFA) (see section 2.4.2 and 2.4.3). Hybridomas
from wells containing anti-DENV-4 antibodies detectable by both ELISA and by IFA
were either cloned in HAT-RPMI medium or transferred to 24 well plates (Nunc,
Denmark) and then to 25 cm2 flasks, 80 cm2 flasks and finally to 175 cm2 flasks in HT-
RPMI. When these 175 cm2 flasks were confluent, the cells were recovered and stored
in 1 ml cryovials in liquid nitrogen.
To clone the hybridomas, cells were resuspended in HAT-RPMI and 5 µl of the

hybridoma suspension was added to 3 ml HAT-RPMI in each well of a 6 well plate
(Nunc, Denmark) and the cells were allowed to settle for 5 minutes. Single cells from
the 6 well plates were transferred to individual wells in 96 well plates containing feeder
cells in 10% FCS-RPMI and 100 µl of HAT-RPMI then was added to each well.
79
The plates were incubated at 370C/ 5% CO2 and after incubation for 4 days; the wells
were checked for the presence of a single clump of cells, indicating a clone of cells.
When the cell cultures were ≥ 50% confluent supernate from wells containing a single
clump of cells was screened for the presence of anti-DENV-4 antibody by indirect IFA
using both uninfected and DENV-4 infected C6/36 cells. Cells from wells producing
antibody of interest were transferred to 24 well plates and then to 25 cm2 flasks, 80 cm2
flasks and finally to 175 cm2 flasks in HAT-RPMI. These cells were either stored in
liquid nitrogen or used immediately to produce stocks of MAbs.
Stocks of MAbs were prepared from cloned hybridoma cultured in standard growth
medium (10% FCS-RPMI or HT-RPMI) or specialised medium such as protein-free
hybridoma medium (PFHM-II; Invitrogen, U.S.A). The hybridomas were cultured in
175 cm2 flasks for a period of 7 to 10 days in standard medium or in PFHM-II. The
culture medium then was centrifuged at 400g for 5 minutes to remove any cells or debris
and the supernate stored at -20°C in 50 ml tubes. The isotype of each MAb was
determined using a Mouse Isotyping Kit (Roche, Germany). Reference MAbs from the
Walter Reed Army Institute of Research (WRAIR) or the Centre for Disease Control
(CDC), which recognised DENV or other flaviviruses were prepared from hybridoma
stocks cultured in 10% FCS-RPMI and PFHM-II (Table 2.2). The anti-NS1 MAb, 3D1,
was kindly provided by Dr Paul Young at the University of Queensland (Table 2.2).
The supernatant from hybridomas cultured in PFHM-II was concentrated 10-fold using
YM-50 centrifuge columns (Amicon Bioseparations, U.S.A) and stored in 200 µl lots at
-20°C. The concentration of each MAb was determined using single radial immuno-
diffusion (SRID) plates (The Binding Site, UK) specific for each mouse MAb isotype
(IgM, IgG1, IgG2a and IgG2b).
80
Table 2.2 Reference MAbs used in this study
Origin
MAb Virus Reference Source Specificity
Gentry et
4G2 DENV-2 WRAIR FLAVI anti-E
al.,1982
Roehrig et al.,
6B6C1 SLEV CDC FLAVI anti-E
1983
Henchal et
15F3 DENV-1 WRAIR DENV-1 anti-NS1
al.,1983
Gentry et
3H5 DENV-2 WRAIR DENV-2 anti-E
al.,1982
Henchal et
5D4 DENV-3 WRAIR DENV-3 anti-E
al.,1983
Henchal et
1H10 DENV-4 WRAIR DENV-4 anti-E
al.,1983
Falconar &
3D1 DENV-2 UQ DENV anti-NS1
Young, 1991
SLEV, Saint Louis encephalitis virus; FLAVI, flavivirus
E, Envelope protein; NS1, Non-structural 1 protein
CDC, Centre for Disease Control; UQ, University of Queensland
WRAIR, Walter Reed Army Institute of Research
81
2.4 Serological and Functional Assays
2.4.1 Hemagglutination and hemagglutination inhibition

Hemagglutination (HA) and hemagglutination inhibition (HI) activities were determined
in 96 well microtitre plates (Sardsedt, U.S.A) according to the micro-method of Clarke
and Casals (1958). Fifty microlitres of doubling dilutions of serum-free tissue culture
supernate (TCS) from virus-infected C6/36 cells in borate saline pH 9.0 (appendix
section 6.1.3) was added to duplicate columns of a 96 well plate (1/2-1/128). The last
row of the plate was left with only borate saline, as a negative control. Fifty µl of a 0.25
% v/v suspension of gander erythrocytes in HA buffer (pH 6.2 or pH 6.4; Queensland
Public Health Scientific Services) was added to all wells of the plate. The plates were
then incubated at 37°C for 45 minutes. The highest dilution of virus that caused
hemagglutination was recorded as the HA titre.
The HI titre of protein-free MAb preparations prepared in PFHM-II medium was

determined by first performing doubling dilutions of MAb from a 1 in 5 dilution in
borate saline (pH 9.0) in a 96 well plate (1/5-1/640). Fifty microlitres of virus (16 HA
units) was added to the 50µl diluted MAb in each well and incubated for 12 hours at
4°C. The 0.25% v/v gander cell suspension diluted in the appropriate HA buffer (pH 6.2
or pH 6.4; Queensland Public Health Scientific Services) then was added to the virus-
MAb mixtures. Different flaviviruses hemagglutinate gander erythrocytes at specific pH
values, which is why both pH 6.2 and pH 6.4 HA buffers were used. DENV-1, DENV-3
and DENV-4 prototype strains were tested at pH 6.2, whereas the DENV-2 and MVEV
prototype strains were tested at pH 6.4. The plates were incubated at 37°C for 45
minutes. The highest dilution of MAb that inhibited hemagglutination was recorded as
the HI titre.
82
2.4.2 Indirect ELISA
Fifty microlitres of DENV-4 H241 tissue culture supernate, HA 16-32, diluted 1 in 2 in
chilled borate saline (pH 9.0) or PEG-concentrated virus diluted 1 in 20 in chilled borate
saline (pH 9.0) was coated to the wells of 96 well Polysorp plates (Nunc, Denmark) for
2 hours at 4°C. The coating solution was decanted from the plates and the wells were
blocked for 1 hour with 50 µl of a 5% v/v solution of Milk Diluent Blocking Solution
Concentrate (Milk diluent; Kirkegaard and Perry Laboratories, U.S.A) diluted in
distilled water. The blocking solution was decanted from the plates and 50 µl of
undiluted hybridoma TCS or a 1 in 125 dilution of concentrated MAb was added to
duplicate wells and incubated for 45 minutes. RPMI-1640 also was added to duplicate
wells as a negative control. The wells then were washed four times in PBS containing
0.5% v/v Tween-20 (BDH Chemicals; 0.5%-PBST).
Fifty microlitres of a secondary antibody solution composed of a 1 in 1000 dilution of

horse radish peroxidase (HRP) labelled anti-mouse Ig; (Dako, Denmark) and a 1 in 4000
dilution of HRP labelled anti-mouse IgM (Southern Biotech, U.S.A) in 0.5% PBST
supplemented with 5% v/v milk diluent (0.5%-PBST/milk) was added to each well for
45 minutes. The wells then were washed in 0.5%-PBST four times. Fifty microlitres of
3, 3’, 5, 5’-Tetramethylbenzidine substrate (TMB; ELISA Systems, Australia) was
added to each well and incubated for 10 minutes. Fifty microlitres of 3 M hydrochloric
acid (HCl) (appendix section 6.1.4) was added to each well to stop the reaction. The
absorbance of each well was determined at a wavelength of 450 nm and blanked against
a wavelength of 690 nm in the Biomek plate reader (Beckman Instruments, U.S.A).
83
2.4.3 Indirect immunofluorescence assay (Indirect IFA)
The anti-DENV MAbs were tested for their ability to react with C6/36 cells infected
with the prototype strains for each DENV serotype; KUNV and MVEV. Fifty
microlitres of virus-infected cells resuspended in RPMI-1640 were added to the spots of
Teflon coated glass IFA slides (ICN Biomedicals, U.S.A) and the cells were allowed to
settle for 10 minutes. Excess liquid was aspirated from the spots by pipette and the cells
were air dried for 10 minutes and then fixed in ice-cold acetone for 1 minute, dried for 5
minutes and stored at -20°C.
Fifty microlitres of undiluted hybridoma TCS was added to each spot and incubated for
45 minutes. The slides were washed 3 times for 10 minutes each wash with PBS (pH
7.4). Fifty microlitres of a secondary antibody solution composed of a 1 in 30 dilution
of fluorescein isothiocyanate (FITC) labelled anti-mouse Ig (Dako, Denmark) and a 1 in
50 dilution of FITC-anti-mouse IgM (Southern Biotechnology, U.S.A) in PBS was
added to each spot and incubated for 45 minutes. The PBS washes were repeated and
the slides dried. Coverslips were mounted on the slides with Dabco solution (Sigma,
U.S.A) and viewed under the “Leitz Laborlux S” UV microscope (Leica, Switzerland)
using ploem illumination. The images were captured using the DXM1200 Digital
camera (Nikon, Japan). Flavivirus-specific MAbs 4G2 and 6B6C1 and DENV serotype-
specific MAbs 15F3 (DENV-1), 3H5 (DENV-2), 5D4 (DENV-3) and 1H10 (DENV-4)
from WRAIR and the CDC were used as positive controls. Uninfected C6/36 cells were
used as a negative control.
84
2.4.4 Polyacrylamide gel electrophoresis (PAGE) and immunoblotting of DENV
proteins
Both PEG-concentrated virus and DENV-4 H241 infected C6/36 cells were used as a
source of viral proteins as outlined in sections 2.2.2 and 2.2.3. Samples were disrupted
in 2x PAGE sample buffer containing 4% w/v SDS (appendix section 6.2.4) with or
without 10% v/v 2-mercaptoethanol (2ME) (Sigma, U.S.A) and heated to 95-100°C for
5 minutes before being loaded on to polyacrylamide gels in a single large well. Proteins
were separated on a discontinuous (5% stacking/10% resolving) 29:1 acrylamide: bis-
acrylamide gel using a method based on that of Laemmli (1970). Ten microlitres of
prestained low range protein Mw standards (Biorad, U.S.A) with a range from 110 kDa
to 20 kDa were loaded next to the samples. The buffers and gel recipes used for PAGE
are outlined in appendix sections 6.2 and 6.3.
Proteins were transferred from the gels to 0.2 µm pore size “Protran nitrocellulose
transfer membranes” (Schleicher and Schuell, Germany) in 3-(Cyclohexylamino)-1-
propanesulfonic acid buffer (CAPS Buffer, pH 11.0; appendix section 6.2.7) by
electrophoresis at 200 mA for 3 hours. The nitrocellulose then was washed 3 times for 5
minutes each wash in Tris-buffered saline (TBS, pH 7.4; appendix section 6.2.9)
containing 0.5% v/v Tween 20 (0.5%-TBST). The unreacted sites on the membrane
were blocked by soaking it in 3% w/v skim milk (Nestle, Australia) diluted in 0.5%-
TBST (0.5%-TBST/milk) for a minimum of 3 hours. The membrane was cut into 0.5
cm strips and placed on Nesco sealing film (Azwell Inc, Japan).
Two hundred microlitres of anti-DENV-4 MAb, either as concentrated MAb diluted 1 in

100 in 0.5%-TBST/milk or as undiluted hybridoma TCS was added to each strip. The
strips were rocked at 20 cycles per minute for 1 hour on a “Bio-line” rocking platform
(Edwards Instrument Company, Australia). MAbs 4G2 (anti-E protein), 2H2 (anti-prM
protein) and 3D1 (anti-NS1 protein) were used as positive controls. 4G2, 2H2 and 3D1
were diluted 1 in 100, 1 in 2 and 1 in 1000 respectively in 0.5%-TBST/milk.
85
The strips then were washed 3 times for 15 minutes, each wash in 0.5%-TBST at 20
cycles per minute on a Bio-line orbital shaker (Edwards Instrument Company,
Australia). Two hundred microlitres of HRP-labelled secondary antibody containing a 1
in 1000 dilution of anti-mouse IgG (Dako, Denmark) and a 1 in 4000 dilution of anti-
mouse IgM (Southern Biotechnologies, U.S.A) diluted in 0.5%-TBST/milk was added to
each strip and incubated for 30 minutes on the rocking platform. The strips were then
washed 3 times for 15 minutes, each wash in 0.5%-TBST on the orbital shaker. The
strips were rinsed briefly in TBS to remove excess Tween-20 and then were dried on
filter paper and aligned on a single transparency sheet (Marbig, Australia).
Thirty microlitres of “Lumilight plus” substrate solution (Roche, U.S.A), diluted 1 in 5

in distilled water, was added to each strip and a second transparency sheet was overlaid
so each strip was covered with substrate. The strips were placed in an X-ray film
cassette (Kodak, Australia) and incubated for 10 minutes at room temperature. The
strips then were exposed to X-ray film (Kodak, Australia) for 1 minute, 5 minutes, 30
minutes, 1 hour and overnight. The films were processed using an automatic developer
(Kodak, Australia)
2.4.5 Infectivity and neutralisation

Infectivity and neutralisation assays were based on the methods described by Morens et
al., (1985). Cell monolayers were prepared in 24 well plates by adding 1 ml of cells at a
concentration of 2.5 x 105 cells/ml to each well and incubating the plates for
approximately 24 hours at 37°C/ 5% CO2 (vertebrate cells; BHK) or 30°C/ 2.5% CO2
(invertebrate cells; C6/36). DENV-4 was diluted ten-fold (10-1 to 10-6) in RPMI-1640
and each dilution was mixed at a 1:1 ratio with MAb or RPMI and then was incubated at
37°C for 1 hour. When the cell monolayers were confluent, the culture medium was
decanted and 200 µl of virus or virus-antibody inoculum was added to the cell
monolayers for 2 hours at the appropriate conditions for the cell line (37°C/ 5% CO2 for
vertebrate cells and 30°C/ 2.5% CO2 for C6/36 cells).
86
Alternatively, the virus-MAb and virus-RPMI mixtures were added to an equal volume
of cells in suspension (2.5 x 105 cells/ml) for the 2 hour infection. Following the
infection, 1 ml of RPMI-1640 supplemented with 2.5% v/v heat inactivated FCS and
1.5% v/v carboxymethylcellulose (CMC; BDH Laboratory Supplies, U.K.) was added to
each well and the plates were incubated for 7 days at 37°C/ 5% CO2. For C6/36 cells,
1ml of RPMI-1640 supplemented with 2.5% FCS, but without CMC, was added.
For the selection of neutralisation escape mutant (n.e.m.) viruses, the virus inoculum
was decanted following the 2 hour infection step and the cell monolayer was washed
once with 1 ml of RPMI-1640. One volume of TCS containing MAb of interest was
mixed with one volume of RPMI-1640 supplemented with 5% v/v heat inactivated FCS
(RPMI-5% FCS) and 1 ml of this mixture was added to cells infected with virus in the
presence of selecting MAb (n.e.m. viruses). One ml of RPMI-1640 supplemented with
2.5% v/v heat inactivated FCS was added to cells infected with virus in the absence of
selecting MAb.
For the chemical mutagenesis of DENV-4 in BHK cells, the virus inoculum was
decanted following the 2 hour infection step and 1 ml of RPMI-5%FCS containing
different final concentrations (1 µM-1 mM) of the mutagen 5-fluorouracil (5FU, Sigma,
USA) was added to the cells.
The titre of virus in BHK cells was determined 7 days after infection by adding crystal
violet to identify viral plaques. Briefly 100 µl of crystal violet/formalin stain (appendix
section 6.1.5) was added to each cell monolayer and was incubated for 45 minutes at
room temperature. The stain was decanted and the monolayers were rinsed with water
and air dried. The viral plaques were counted and the virus titre was recorded as plaque
forming units per ml (PFU/ml) of inoculum.
87
The titre of DENV-4 virus in C6/36 cells was determined 5 days after infection using
direct foci staining and indirect IFA to identify virus infected cells. These methods also
were utilised to determine the titre of virus in BHK, PS-EK and Vero cells. For indirect
IFA, the cell monolayers were resuspended in 250 µl of PBS and added to IFA slides.
The IFA protocol described in section 2.4.3 was used to stain the cells. The tissue
culture infectious dose (TCID) was recorded as the highest dilution of virus producing
an infection in cells detectable by IFA.
The direct staining of foci in cell monolayers was based on the method described by
Blaney et al., (2001). For the direct foci staining, the culture supernatant was decanted
and the cell monolayers were washed once in 1ml of PBS. The cells then were fixed in
1 ml of 5% v/v 30% formaldehyde (Merck, U.S.A) diluted in PBS for 30 minutes at
room temperature. The fixative was decanted and the cells were washed twice with 1 ml
of PBS and then were blocked for 30 minutes with 1 ml of 3% w/v skim milk diluted in
PBS (milk-PBS).
The milk-PBS was decanted and 200 µl of HRP-labelled 6B6C1 antibody (Panbio,
Australia) diluted 1 in 5000 in milk-PBS was added to the cells and incubated for 1 hour.
The HRP-labelled 6B6C1 was decanted and the cells were washed three times each with
1 ml PBS. Two hundred µl of TMB Stabilised Substrate for HRP (Promega, U.S.A) was
added to the cells and incubated for 30 minutes in the dark. The TMB was decanted and
the wells were washed with water and air dried.
Foci of infected cells were counted and the virus titre was recorded as foci forming units
per ml (FFU/ml). Virus neutralisation was indicated by the reduction in virus titre
following the addition of MAb to cultures of virus. Neutralisation was expressed as a
Neutralisation Index (NI).
Neutralisation Index: log10 titre of virus + RPMI
titre of virus + MAb
Values greater than 1.0 were considered indicative of neutralisation
88
2.4.6 Capture ELISA
2.4.6.1 Standard capture

Five-fold dilutions of concentrated MAb (1/5- 1/15625) were prepared in chilled borate
saline (pH 9.0) and 50 µl coated to duplicate wells of a Maxisorp plate (Nunc, Denmark)
for 2 hours at 4oC. The wells were blocked for 1 hour with 50 µl of 5% v/v “Milk
Diluent Blocking Solution Concentrate” (milk diluent; Kirkegaard and Perry
Laboratories, U.S.A) diluted in distilled water. The milk diluent was decanted and 50 µl
of DENV-4 TCS diluted 1 in 2 in 0.5%-PBST with 5% v/v milk diluent (0.5%
PBST/milk) or PEG-concentrated DENV-4 diluted 1 in 20 in 0.5% PBST/milk was
added to each well and incubated for 45 minutes. Fifty microlitres of 0.5% PBST/milk
also was added to each well with no virus.
The wells then were washed 4 times with 0.5%-PBST. The HRP-labelled anti-
flavivirus MAb 6B6C1 (Panbio Ltd, Australia) was diluted 1/40000 in 0.5%-PBST/milk
and 50 µl was added to each well and incubated for 45 minutes. The wells were washed
4 times with 0.5%-PBST and TMB substrate (ELISA Systems) was added to the wells
and the reaction was stopped after 10 minutes by the addition of 50 µl of 3 M
hydrochloric acid. The absorbance of each well was determined at a wavelength of 450
nm and blanked against a wavelength of 690 nm in the Biomek plate reader (Beckman
Instruments, U.S.A).
2.4.6.2 Avidity capture

The avidity of capture MAbs for virus was tested by incorporating “avidity solution”
(Panbio Ltd, Australia) which contains urea into the standard capture ELISA protocol.
The avidity solution was diluted in 0.5% PBST/milk to produce urea concentrations of
6 M, 4 M, 2 M and 1 M. Fifty microlitres of each urea solution were added to duplicate
wells containing virus captured by 1 µg/ml capture MAb and incubated for 10 minutes.
89
Fifty microlitres of 0.5% PBST/milk was also added to wells containing virus and MAb
as a control. The urea solutions and 0.5% PBST/milk were also added to wells with
MAb and no virus, to identify any affects of urea on the attachment of capture MAbs to
the Maxisorp plates. Following the urea treatment, the wells were washed with 0.5%
PBST/milk. Fifty microlitres of HRP-labelled 6B6C1 diluted 1 in 40000 in 0.5% PBST
was added to wells containing virus and MAb for 45 minutes to detect the captured
virus.
Fifty microlitres of HRP-labelled anti-mouse kappa-specific MAb (Southern Biotech,

U.S.A), diluted 1 in 1000 in 5% milk 0.5% PBST, were added to MAb coated wells
without virus for 45 minutes to detect the amount of capture MAb coated to the
Maxisorp well. The wells were washed 4 times with 0.5% PBST and the amount of
bound HRP-labelled Ab was quantitated by the addition of TMB substrate (ELISA
systems, Australia). The TMB reaction was stopped after 10 minutes by the addition of
3 M HCl. The absorbances of each well were determined at a wavelength of 450 nm
and blanked against 690 nm in the Biomek plate reader (Beckman Instruments, U.S.A).
2.4.6.3 Capture of virus exposed to low pH

The capture of virus exposed to low pH and untreated virus was compared using the
standard capture ELISA protocol. DENV-4 H241 was exposed to a low pH using a
modification of the protocol of Holzmann et al., (1995). Briefly 1 ml of 100 mM 2-(N-
morpholino) ethanesulfonic (MES; Sigma, U.S.A) acid diluted in water was added to 4
ml of virus solution and the pH was adjusted to 6.0 with 1 M HCl. The solution was
incubated at 37°C for 15 minutes, the pH was adjusted to pH 8.0 with 1 M NaOH and
the virus was incubated at 37°C for a further 15 minutes. The “untreated” virus was
incubated at the same temperature and times at pH 7.0 and the pH then adjusted to 8.0.
The ability of MAbs to capture the low pH-treated virus and untreated virus was tested
in the standard capture ELISAs.
90
2.4.6.4 Competitive capture
Competitive capture ELISAs were performed to determine whether anti-DENV-4 MAbs
or MAbs and human serum recognised spatially related epitopes on DENV. One
volume of DENV-4 TCS was added to one volume of blocking MAb (10 µg/ml in 0.5%
PBST/milk) and the virus-MAb mixture was incubated at 37°C for 2 hours. The ability
of other MAbs to capture virus in the virus-MAb mixture was assessed in a standard
capture ELISA. Serum from DENV-4 immune (D4), flavivirus immune (JA) and
flavivirus non-immune (JD) patients was diluted 1 in 50 in 0.5%-PBST/milk and was
mixed with an equal volume of DENV-4 TCS. The virus and patient serum were
incubated at 37°C for 2 hours and the ability of MAbs to capture virus or virus in the
virus-MAb mixtures was assessed in the standard capture ELISA.
Mean absorbances, standard deviation (s.d.) and the percent inhibition of virus capture
were determined for each MAb in the ELISAs and a two-tailed equal variance student T
test was used to identify significant differences between data sets.
2.5 Molecular Biology
2.5.1 RT-PCR and sequencing

RNA was extracted from virus samples using the QIAamp MiniElute Virus Spin Kit
(Qiagen, Australia) according to the manufacturer’s instructions. The complementary
DNA (cDNA) was produced from the RNA using random hexamer primers p(dN)6
(Roche, U.S.A) and avian myeloblastosis virus reverse transcriptase (AMV-RT; Roche,
U.S.A). Briefly, 1 µl of p(dN)6 was added to 10 µl RNA in a 0.5 ml tube (Eppendorf,
Germany) and the reaction volume was made up to 12.5 µl with diethylpyrocarbonate
(DEPC) treated water (appendix section 6.4.1). The mixture was incubated at 72°C for
10 minutes in a heating block and then was placed on ice for 1 minute. Five microlitres
of 5x AMV-RT buffer (Roche, U.S.A), 2.5 µl 10 mM dNTPs (Roche, U.S.A), 0.2 µl
RNASE Inhibitor (40 U/µl; Roche, U.S.A) and 0.5 µl AMV-RT then were added to the
tube and the volume was made up to 50 µl with DEPC-water. The RT reactions were
incubated in an MJ thermocycler (Bresatec, U.S.A) at 55°C for 10 minutes, and then
45°C for 60 minutes.
91
The primers used in the polymerase chain reaction (PCR) amplification and sequencing
of the DENV-4 E gene are listed in Table 2.3. A 1.8 kb PCR product, which spanned
the entire DENV-4 E gene, was amplified from the cDNA using the oligonucleotide
primer set D4-742 and D4-CP2536 (Proligo, Australia). Five microlitres of 10x Expand
Polymerase Buffer 2 (Roche, U.S.A), 1 µl 10 mM dNTPs, 1 µl D4-742, 1 µl D4-CP2536
and 0.2 µl Expand Polymerase (Roche, U.S.A) were mixed in a 0.5 ml tube and the
volume made up to 49 µl with water. One microlitre of cDNA was added to each tube
and the PCR was performed in the MJ thermocycler using an annealing temperature of
55°C and the cycling conditions shown in Table 2.4 which were recommended in the
Expand Polymerase protocol (Roche, U.S.A).
Ten microlitres of 6x DNA sample loading buffer (appendix section 6.4.4) was added to
the completed PCR reaction and 30 µl was loaded in duplicate lanes of a gel containing
1% w/v agarose (Boehringer Mannheim, Germany) in 1 x Tris-acetate/EDTA (TAE)
buffer (appendix section 6.4.3) with 0.2 µg/ml ethidium bromide (Sigma, U.S.A). The
gel was immersed in a submarine gel chamber (Biorad, U.S.A) containing 1 x TAE and
the samples were electrophoresed for 30 minutes at 100 volts. The size of the PCR
products was confirmed by comparison with Mw markers (Mix 10; Roche, U.S.A).
Double strand cDNA of anticipated size was excised from the TAE gel and purified
using a hi-pure kit (Roche, U.S.A) according to the manufacturer’s instructions. The
DENV-4 E gene was sequenced in both directions. Two to five microlitres of purified
PCR product (10 to 40 ng) was mixed with 2 µl of Big Dye Terminator Version 2 or 2 µl
of Big Dye Terminator Version 3 (Applied Biosystems) and 3.2 pmoles of primer and
the reaction made up to 12 µl with water. The sequencing reactions were performed in
the MJ thermocycler using the cycling conditions outlined in the Australian Genome
Research Facility (AGRF) protocol: 96°C/ 30 seconds, 50°C/ 15 seconds, 60°C/ 4
minutes (25 cycles).
92
Table 2.3 Oligonucleotide primers used for PCR and DNA sequencing
a
Primers Sequence (5'-3')
D4-742 TGGGATTGGAAACAAGAGCTGAGACATGGATGTC
D4-903 TTTGTCCTAATGATGCTGGTCGCCCCATC
D4-1236 GGGTGGGGCAATGGCTGTGGCTTGTTTGG
D4-1569 CAATGGTTTTTGAATCTGCCTCTTCCATGG
D4-1916 TGAAGGTGCCGGAGCTCCGTGTAAAGTCCC
D4-2219 GTTCACATCATTGGGAAAGGCTGTGCACCA
D4- CP2536 GGGGACTCTGGTTGAAATTTGTACTGTTCTGTCCA
D4-CP2471 GCTTCCACACTTCAATTCTTTCCCTTTCTCCA
D4-CP1838 CGTGTATGACATTCCCTTGATTCTCAATTTCTCCA
D4-CP1461 ATTTGACTTCCACCGATGGTGACCTAGGAGTTAT
D4-CP1200 GGTCCTGTTCCTCTTTCAGATAAGGCTCTCCTTG
a
The number following D4 is the position of the 5' nucleotide of the primer in the
dengue 4 genome. Numbering according to Lanciotti et al.,1997. CP indicates that
the nucleotide sequence of the primer is complementary to that of the genome.
93
Table 2.4 Thermal cycling conditions for PCR
Temperature (°C) Time Cycles

92 2 min 1
92 40 sec
55 40 sec 9
68 2.5 min
92 40 sec
55 40 sec 9
68 3.0 min
92 40 sec
55 40 sec 9
68 3.5 min
92 40 sec
55 40 sec 9
68 4.0 min
68 10 min 1
94
Reaction products were precipitated at room temperature by the addition of 50 µl of
analytical reagent (AR) grade ethanol (Univar, U.S.A) and 2 µl of 3M sodium acetate
pH 5.2 (appendix section 6.4.5) to the sequencing reaction for 10 minutes. The mixture
was centrifuged in a bench top centrifuge (Eppendorf, Germany) at 16,200g for 10
minutes at room temperature, the supernatant discarded and the pellet washed with 250
µl of 70% v/v AR ethanol in distilled water. The mixture was centrifuged again at the
same speed for 10 minutes, the supernatant discarded and the pellet dried by heating at
80°C for 10 minutes in a heating block. The reactions then were submitted to AGRF at
the University of Queensland where the samples were sequenced using ABI 377
automatic DNA sequencers. The sequences were analysed using the DNASTAR
software package (DNASTAR, U.S.A). The amino acid sequences of some of the
DENV-4 isolates were kindly provided by Kym Lowry.
2.5.2 Site directed mutagenesis

The QuikChange™ multi-site directed mutagenesis (SDM) kit (Stratagene, U.S.A) was
utilised to make nucleotide substitutions in the DENV-4 E gene in the plasmid pVAX-
D4 to identify amino acid changes in the DENV-4 E protein involved in the binding of
neutralising MAbs. The pVAX-D4 plasmid which was kindly provided by Steve Liew
is a mammalian-expression vector (pVAX; Invitrogen, U.S.A) containing the structural
proteins (C-prM-E) of DENV-4 H241. Nucleotide substitutions were made at different
sites of the DENV-4 E gene of pVAX-D4 in separate reactions using the oligonucleotide
primers in Table 2.5 and the conditions outlined in the manufacturer’s instructions.
Briefly 2.5 µl of pVAXD4 plasmid (20ng/ml) and 2.0 µl of oligonucleotide primer (50
ng/ml; Proligo, Australia) were added to a reaction mix in a 0.2 ml tube (Eppendorf,
Germany) containing 2.5 µl of 10x Reaction Buffer, 1 µl dNTP mix and 1 µl
QuikChange Multi enzyme blend (Stratagene, U.S.A). The reaction was made up to a
final volume of 25 µl with water and the mutagenesis reactions were performed in the
MJ thermocycler using the following cycling parameters: 95°C/ 1 minute (1 cycle),
95°C/ 1 minute, 55°C/ 1 minute, 65°C/ 10 minutes (30 cycles).
95
Table 2.5 Oligonucleotide primers used for site directed mutagenesis
Mutation and
amino acid
a b c
Primer Sequence (5'-3') change
SDM GAT-GCT
TACATTTGCCGGAGAGCTGTGGTAGACAGAGGGTGGGGC
95 E95(D-A)
SDM GTG-ATG
TACATTTGCCGGAGAGATATGGTAGACAGAGGGTGGGGC
96 E96(V-M)
CCC-TCC
SDM E156(P-S)
GCAGTAGGAAATGACATATCCAGCCATGGAGTGACAGCC
156157 AAC-AGC
E157(N-S)
SDM AAC-AGC
GCAGTAGGAAATGACATACCCAGCCATGGAGTGACAGCC
157 E157(N-S)
SDM AAG-ACG
CTGATGAAAATGAAAACGAAAACGTGGCTTGTGCACAAGC
203 E203(K-T)
SDM GCT-ACT
AAGTCAAGTATGAGGGTACTGGAGCTCCATGTAAAGTTCC
329 E329(A-T)
SDM CTT-TTT
AGCTCCATTGGCAAGATGTTTGAGTCCACATACAGAGGC
402 E402(L-F)
a
Number after SDM indicates the amino acid in the DENV-4 E protein being changed
b
The underlined nucleotides represents the codon (amino acid) being changed and the nucleotide in
bold is the specific change resulting in amino acid substitutions.
c
Used standard one letter amino acid code
96
After the mutagenesis reaction step, the reactions were chilled on ice for 2 minutes and
then were treated with 1.0 µl of Dpn1 (10 U/µl; Stratagene, U.S.A) for 1 hour at 37°C
using the MJ thermocycler. XL-10 Gold competent cells (Stratagene, U.S.A) were
transformed with 1.5 µl of the Dpn-1 treated reactions by heating the cells at 42°C for 30
seconds. XL-10 cells containing pVAXD4 plasmid that potentially were mutated were
selected on agar-based growth medium (appendix section 6.4.7) containing 50 µg/ml
kanamycin sulphate (Sigma, U.S.A), the resistance marker in the pVAX plasmid. Five
colonies were selected from the plates and were cultured overnight in liquid growth
medium (appendix section 6.4.6) containing 50 µg/ml kanamycin sulphate. The
pVAXD4 plasmid was purified from the cultures using miniprep columns (Qiagen,
Australia). The plasmids were sequenced with DENV-4 E gene primers (Table 2.3)
using the conditions outlined in section 2.5.1 to confirm the nucleotide changes in the E
gene.
2.5.3 DNA transfection of BHK cells

Monolayers of BHK cells grown in 6 well tissue culture plates (Nunc, Denmark) were
transfected with parental and mutated pVAXD4 plasmids as well as with the pVAX
plasmid lacking DENV structural genes (pVAX control), as a negative control.
Transfections were carried out using Lipofectamine 2000 (Invitrogen, U.S.A) according
to manufacturer’s instructions. Briefly, 4 µg of plasmid DNA was resuspended in
serum-free and antibiotic-free RPMI-1640 in a total volume of 250 µl. Lipofectamine
2000 was diluted 1 in 25 in serum-free and antibiotic-free RPMI-1640 and was
incubated at room temperature for 5 minutes. An equal volume of Lipofectamine 2000
(250 µl) was added to the plasmid DNA and incubated at room temperature for 30
minutes. The growth medium was aspirated from the 6 well plates and was replaced
with 500 µl of Lipofectamine-DNA complexes, and an additional 500 µl of serum-free
and antibiotic-free RPMI-1640. The cells were incubated for 36 hours at 37°C / 5%
CO2. The culture supernate was then discarded and the cells removed from the wells
using trypsin-EDTA and transferred to a 15 ml tube. Five ml of RPMI-1640 was added
and the tube was centrifuged at 400g for 5 minutes.
97
The cell pellet was resuspended in 5 ml of RPMI-1640 and used in IFAs with the anti-
DENV-4 MAbs F1G2, 18F5, 13H8, F2D1 and the reference MAbs 4G2 and 2H2
according to the method outlined in section 2.4.3. Digital images of the IFAs were
recorded to analyse the effect of the mutations on MAb binding and IFA fluorescence
intensity.
In addition, transfected BHK cells expressing DENV-3/DENV-4 chimeric E proteins

were kindly provided by Steve Liew. These cells were screened by IFA with the anti-
DENV-4 MAbs F1G2, 18F5, 13H8 and the reference MAbs 4G2 and 2H2 according to
section 2.4.3 to determine structural domains of the DENV-4 E protein recognised by
neutralising MAbs.
2.6 Peptide Display

The “FliTrx TM random peptide display library” (Invitrogen, U.S.A) is a bacterial library
(E.coli) which is induced in the presence of tryptophan to produce fusion proteins on the
surface of bacteria that display random peptides (Figure 2.1). The library was used in
conjunction with the reagents provided by the FliTrx Panning Kit (FPK) (Invitrogen,
U.S.A) to identify peptides that interacted with the anti-DENV-4 type specific
neutralising MAbs F1G2, 13H8 and 18F5. The growth of the FliTrx library, the
induction and expression of the FliTrx fusion protein and random peptides and the
panning of the peptides against the MAbs F1G2, 13H8 and 18F5 was carried out
according to manufacturer’s instructions.
Briefly, a 1 ml aliquot of the FliTrx library was inoculated into 50 ml of liquid medium
(IMC Medium; Invitrogen, U.S.A) containing 100 µg/ml w/v Ampicillin (Boehringer
Mannheim, Germany) and grown with shaking at 225 rpm for 16 hours at 25°C.
Expression of the FliTrx library was induced by the addition of 1 x 1010 cells of the
initial culture to 50 ml IMC Medium containing 100 µg/ml w/v Ampicillin and 100
µg/ml v/v tryptophan. The cells were induced with shaking of the culture at 225 rpm for
6 hours at 25°C.
98
Figure 2.1. Diagram of the FliTrx bacterial peptide display library (FliTrx user manual;
Invitrogen, U.S.A). The pFliTrx plasmid is induced in the presence of tryptophan to
produce flagellin-thioredoxin fusion proteins, which are displayed on the flagellin of
E.coli. Each fusion protein contains random peptides that are 12 amino acids in length.
These peptides are presented on the fusion protein as a loop-like structure that is
conformationally constrained by a disulphide bridge.
99
The panning steps involved the addition of the induced FliTrx library to anti-DENV-4
MAb immobilised on a 60 mm plate (Nunc, Denmark). One ml of anti-DENV-4 MAb
(1µg/ml) diluted in distilled water was added to the 60mm plate, and incubated on an
orbital rocker for 30 minutes at room temperature. The plate was then blocked for 30
minutes with 1 ml of 5% skim milk diluted in water. Following blocking the induced
FliTrx library was added to the plate and incubated at room temperature for 30 minutes.
The bacterial cells expressing a peptide that was recognised by the immobilised MAbs
were captured whereas the unbound cells were washed away. The cells bound to the
MAb were removed from the plate by mechanical shearing using a vortex mixer.
Bacteria from the first panning were cultured, induced and used again in a panning step.
Following the fifth panning, the captured bacteria were plated on solid medium (RM
agar medium; Invitrogen, U.S.A) containing 100 µg/ml ampicillin and grown for 16
hours at 30°C. Ten colonies were picked from the plates and each colony was cultured
in liquid medium (RM medium; Invitrogen, U.S.A) for 16 hours at 30°C. The cultures
then were induced in IMC medium containing 100 µg/ml ampicillin and 100 µg/ml
tryptophan to express the fusion protein and random peptides. The induced cultures
were lysed in PAGE sample buffer (appendix section 6.2.4) and the reactivity of the
fusion protein peptide and the MAb was determined by western blot as outlined in
section 2.4.4. The detection of the 53 kDa FliTrx fusion protein was indicative of a
positive reaction between MAb and expressed peptide.
To screen a larger number of colonies for the presence of peptides recognised by the
MAbs, a colony blot was performed. Fifty to one hundred colonies were transferred
from RM agar plates to “Protran” nitrocellulose. The colonies attached to the membrane
then were placed on another RM agar plate containing 100 µg/ml tryptophan for 6 hours
at 25°C to induce the FliTrx fusion proteins. The colonies were lysed with 20 mg/ml
lysozyme (Boehringer-Mannheim, Germany) diluted in milk/0.5% TBST, which also
blocked the membrane. The reactivity of the induced peptides with MAbs was
determined using the blot protocol outlined in section 2.4.4.
100
Plasmids were recovered from bacteria which produced peptides recognised by MAbs
using miniprep columns (Qiagen, Australia) and sequenced using the FliTrx forward and
reverse oligonucleotide primers and the sequencing protocol outlined in Section 2.5.1.
The peptide sequences were analysed using DNASTAR software to identify any
similarities between the amino acid sequences of the peptides and that of the DENV-4 E
protein.
2.7 Virus Overlay Protein Binding Assay (VOPBA)

The VOPBA was used to determine, whether DENV-4 attaches to host cell proteins and
whether the pre-treatment of DENV-4 with neutralising MAbs prevented DENV-4
attachment to host cell proteins. Uninfected C6/36 cells in an 80 cm2 flask were lysed
with 5 ml of 1% v/v Nonidet P-40 (NP-40; Sigma, U.S.A) in TBS and decanted into a 50
ml tube. The solution was mixed and incubated on ice for 30 minutes and then
transferred to 1.5 ml tubes (Eppendorf, Germany) and centrifuged at 16100g for 15
minutes. The supernate was transferred to 0.5 ml tubes (Eppendorf, Germany) and
stored in 200 µl aliquots at -80°C. PAGE and western transfer of protein to
nitrocellulose membranes was performed with the lysates as described in section 2.4.4.
Five hundred microlitres of DENV-4 TCS was mixed with 500 µl of MAb or 500 µl
RPMI-1640 and incubated on ice for 2 hours. RPMI-1640 alone was included as a
control. After 2 hours, 1 ml of chilled 3% w/v skim milk diluted in 0.5%-TBST (0.5%-
TBST/milk) was added to each tube. The tubes were mixed by inversion and the entire
2 ml volume was added to strips of nitrocellulose on to which cell lysate had been
transferred.
The strips were rocked at 20 cycles per minute for 10 hours at 4°C on a rocking platform
and then were washed three times for 15 minutes each wash in 0.5%-TBST at 20 cycles
per minute using an orbital shaker. Virus bound to blotted proteins was detected by
adding 1 ml of HRP-labelled 6B6C1 MAb diluted 1/5000 in 0.5%-TBST/milk to each
strip and mixing on a rocking platform for 30 minutes at room temperature. The wash
steps were repeated and the strips were soaked briefly in TBS.
101
The strips were dried on filter paper and aligned on a single transparency sheet (Marbig,
Australia). One hundred microlitres of “Lumilight plus” substrate solution (Roche,
USA) diluted 1/5 in distilled water was added to each strip and a second transparency
sheet was overlaid so each strip was covered with substrate. The strips were placed in
an X-ray film cassette and incubated for 10 minutes at room temperature. The strips
then were exposed to X-ray film (Kodak, Australia) for 1 minute and 5 minutes. The
films were processed using an automatic developer (Kodak, Australia).
102
3 RESULTS
3.1 Production and characterisation of monoclonal antibodies
A panel of MAbs against DENV-4 was produced to identify epitopes on the DENV-4 E
protein involved in neutralisation. To produce the MAb panel, spleen cells derived from
BALB/c mice infected with DENV-4 were fused with myeloma cells, in the presence of
PEG, to produce hybridoma cells. Hybridomas producing MAbs against DENV-4 were
identified by Indirect ELISA and Indirect IFA. The resulting MAb panel was
characterised using various serological and functional assays.
The serological assays included immunoglobulin (Ig) isotyping, an immunofluorescence

assay (IFA) for testing the virus specificity of each MAb and western blotting to identify
the virus protein recognised by each MAb as well as the conformation of the epitope
(linear or conformational). The functional assays determined the ability of MAbs to
neutralise DENV-4 infection of mammalian and mosquito cells, and the ability of MAbs
to inhibit the hemagglutination of gander erythrocytes by DENV-4. The identification
of DENV-4 specific neutralising MAbs in the panel was essential for subsequent
experiments aimed at determining antigenic domains, structural domains or specific
epitopes involved in neutralisation.
In addition to the other serological assays, the ability of MAbs to capture DENV-4 was
also tested by ELISA. The determination of MAbs which captured DENV-4 was
important for the development of competitive binding experiments which were used to
define antigenic domains on the DENV-4 E protein. MAbs that captured DENV-4 also
were used in capture ELISAs that measured MAb avidity and MAb capture of low pH
treated DENV-4, which were also important for defining the nature of each epitope.
Following four cell fusion reactions, fourteen clones of hybridomas were identified that
produced MAbs which reacted with DENV-4 in indirect ELISA and IFA assays. The
results from the serological and functional assays for each MAb are summarised in
Table 3.1. The results for reference MAbs (6B6C1, 4G2, 1H10) from the WRAIR are
also included in Table 3.1, except HI data, which was not determined.
103
Table 3.1 Characteristics of anti-DENV-4 MAbs and reference MAbs 4G2, 6B6C1 and 1H10 used in this study
Neutralisation index (log10) DENV-4
C6/36 BHK HI titre

a a Western
TCS TCS 100ug/ml
MAb Isotype IFA MAb MAb MAb blot b DENV-1 DENV-2 DENV-3 DENV-4 MVEV
F1G2 IgM DENV-4 2.0-3.0 2.0-3.0 ≥3.0 E (C) <10 <10 <10 20 <10
4B1 IgM DENV 1.0 1.0 1.0 E (C) <10 <10 ≥640 ≥640 <10
F18B10 IgM DENV, KUNV <1.0 <1.0 1.0 E (C) <10 <10 <10 160 <10
F7MF7 IgM DENV, KUNV, MVEV <1.0 <1.0 <1.0 NS1 (L) <10 <10 <10 <10 <10
17A3 IgM DENV <1.0 <1.0 1.0 E (C) <10 <10 ≥640 ≥640 <10
F2D1 IgG1 DENV, KUNV, MVEV 2.0-3.0 2.0-3.0 2.0 E (C) 160 320 ≥640 ≥640 160
13H8 IgG1 DENV-4 2.0-3.0 2.0-3.0 2.0 E (C) <10 <10 <10 <10 <10
F16B5 IgG1 DENV-2, DENV-4 <1.0 <1.0 <1.0 E (C) <10 <10 <10 20 <10
18F5 IgG2a DENV-4 2.0-3.0 2.0-3.0 2.0 E (C) <10 <10 <10 160 <10
F19F11 IgG2a DENV 1.0 <1.0 1.0 E (C) <10 <10 20 20 <10
3C9 IgG2a DENV, KUNV <1.0 <1.0 <1.0 E (C) <10 <10 20 ≥640 <10
7 E3 IgG2a DENV, MVEV <1.0 <1.0 <1.0 NS1 (L) <10 <10 <10 <10 <10
F12A3 IgG2b DENV-4, KUNV <1.0 <1.0 <1.0 NS1 (C) <10 <10 <10 <10 <10
F20F10 IgG2b DENV-4 <1.0 <1.0 1.0 E (C) <10 <10 <10 <10 <10
1H10 IgG1 DENV-4 2.0-3.0 2.0-3.0 2.0 E (C) n.d n.d n.d n.d n.d
4G2 IgG1 DENV, KUNV, MVEV 2.0-3.0 2.0-3.0 2.0 E (C) n.d n.d n.d n.d n.d
6B6C1 IgG2a DENV, KUNV, MVEV 2.0-3.0 2.0-3.0 2.0 E (C) n.d n.d n.d n.d n.d
a
TCS, tissue culture supernate from hybridomas, concentration of antibody not determined.
b
C, conformational epitope; L, Linear epitope
n.d, not determined
104
3.1.1 Serological assays
3.1.1.1 Isotyping and virus specificity

Several immunoglobulin (Ig) isotypes were identified in the MAb panel against DENV-
4. Five of the MAbs were IgM, 3 IgG1, 4 IgG2a and 2 IgG2b MAbs. The IFAs
identified differences in virus specificity of MAbs in the panel. Four of the MAbs
(F1G2, 13H8, 18F5 and F20F10) reacted only with DENV-4 infected C6/36 cells, 3
MAbs (4B1, 17A3 and F19F11) reacted with C6/36 cells infected with each dengue
virus (DENV) serotype and two MAbs (F2D1 and F7MF7) reacted with C6/36 cells
infected with each DENV serotype as well as Kunjin virus (KUNV) or Murray Valley
encephalitis virus (MVEV). MAb F12A3 reacted with C6/36 cells infected with DENV-
4 or KUNV. MAbs F18B10 and 3C9 reacted with C6/36 cells infected with all DENV
serotypes or KUNV whereas the MAb 7E3 reacted with C6/36 cells infected with all
DENV serotypes or MVEV. MAb F16B5 reacted with C6/36 cells infected with
DENV-2 or DENV-4 (Table 3.1).
3.1.1.2 Western blotting

Eleven of the 14 MAbs reacted with a non-reduced 56 kDa protein recognised by the
anti-E reference MAb 4G2 in immunoblots of either PEG-concentrated DENV-4 or of
lysate from DENV-4 infected C6/36 cells (Table 3.1, Figure 3.1 and Figure 3.2). No
binding of these MAbs to viral proteins was detected if the virus was treated with the
reducing agent 2-mercapto-ethanol prior to PAGE, thus indicating the epitopes were
conformational in nature (Figure 3.2).
In addition to the reaction with the 56 kDa putative E protein, a number of these MAbs
also reacted with larger and smaller viral proteins (Figure 3.2). The larger proteins were
most probably multimeric E proteins, such as the E protein dimer (120 kDa) or
uncleaved polyproteins (E-prM, E-NS1). The smaller proteins were most probably
breakdown products resulting from proteolytic digestion.
105
Mw (kDa)
A
110
90
51
36
29
21
4G2 F2D1 F1G2 13H8 18F5 3D1 F12A3 F7MF7
B
110
90
51
36
29
21
4G2 F2D1 F1G2 13H8 18F5 3D1 F12A3 F7MF7
Figure 3.1 Western blot analysis of selected anti-DENV-4 MAbs using lysate of (A)
DENV-4 infected C6/36 cells and (B) uninfected C6/36 cells. MAb 4G2 was included
as a positive control for the detection of the E protein and MAb 3D1 as a positive
control for the detection of the NS1 protein.
106
Mw (kDa)
110 E dimer
90
E
51
36
29
21 - + - + - + - +
4G2 13H8 18F5 F1G2
110
90
NS1 dimer
51
NS1
36
29
21 - + - + - + - +
3D1 F12A3 F7MF7 7E3
Figure 3.2 Reaction of MAbs with western blots of PEG-concentrated DENV-4 (-) or
with the same virus preparation treated with 2ME (+). MAb 4G2 was included as a
positive control for the detection of the E protein and MAb 3D1 as a positive control for
the detection of the NS1 protein. The proposed dimers for the E and NS1 proteins
recognised by MAbs have also been indicated.
107
The remaining 3 MAbs, F12A3, F7MF7 and 7E3, reacted with a 45 kDa protein
recognised by the anti-NS1 reference MAb 3D1 (Figure 3.2). In contrast to the reactions
of MAbs with the E protein, three of the 4 anti-NS1 MAbs including the reference MAb
3D1 reacted as well, or better, with NS1 treated with 2-mercapto-ethanol, thus indicating
the epitopes were linear in nature (Figure 3.2). In addition, the MAb 7E3 also reacted
with a 90kDa protein, which is most probably the NS1 dimer (Figure 3.2). None of the
MAbs reacted with immunoblots of lysate from uninfected C6/36 cells, ruling out MAb
reactivity with C6/36 cell derived proteins (Figure 3.1).
In addition, different staining patterns were observed in IFAs when DENV-4 infected
C6/36 cells were stained with anti-NS1 and anti-E MAbs. The anti-NS1 MAbs reacted
with antigen on the inside of the cell membrane of the C6/36 cells, whereas anti-E MAbs
stained the whole cell or localised regions within the cell (Figure 3.3).
108
MAb
13H8
(Anti-E)
MAb
F12A3
(Anti-NS1)
Figure 3.3 Differences between the reactivity of anti-E MAbs and anti-NS1 MAbs with
DENV-4 infected C6/36 cells in IFAs, represented by the MAbs 13H8 (anti-E) and
F12A3 (anti-NS1). The green fluorescence staining indicates that anti-NS1 MAbs
reacted with antigen on the inside of the cell membrane of the C6/36 cells, whereas anti-
E MAbs stained the whole cell or localised regions within the cell. The images of the
IFAs were taken at 200 times magnification.
109
3.1.2 Functional assays
3.1.2.1 Virus neutralisation

The MAbs F1G2, 13H8, 18F5 and F2D1 neutralised the infectivity of DENV-4 in
vertebrate cells (BHK) and invertebrate cells (C6-36) by 2.0-3.0 log10 (Table 3.1). This
is equivalent to a 100 to 1000 fold reduction in virus titre in the presence of these MAbs.
The flavivirus group-reactive MAb, F2D1, also neutralised DENV-2 infectivity in BHK
cells by 2.0 log10, MVEV infectivity in BHK cells by 1.5 log10 and DENV-1 and DENV-
3 infectivity in BHK cells by 1.0 log10. The reference MAbs 4G2, 6B6C1 and 1H10
neutralised at least 2.0 log10 of DENV-4 in each cell line.
The MAbs 4B1 and F19F11 neutralised 1.0 log10 of virus, which in the case of F19F11
was only in C6/36 cells. The remaining MAbs neutralised <1.0 log10 of DENV-4 in
either cell line.
The initial neutralisation tests were performed using supernates from cultures of
hybridomas with undefined MAb concentrations. When the tests were repeated with
BHK cells using 100 µg/ml of each MAb, four levels of neutralisation were observed
(Table 3.1). The MAb F1G2 neutralised DENV-4 infectivity of BHK cells by ≥ 3.0
log10. The MAbs 13H8, 18F5, F2D1 and the reference MAbs 1H10, 6B6C1 and 4G2
neutralised DENV-4 infectivity of BHK cells by 2.0 log10. The MAbs 4B1, F19F11,
17A3, F18B10, F20F10 neutralised DENV-4 infectivity of BHK cells by 1.0 log10. The
remaining MAbs neutralised DENV-4 infectivity of BHK cells by less than 1.0 log10.
3.1.2.2 Hemagglutination inhibition assay

Nine of the 14 MAbs inhibited the hemagglutination of gander cells by DENV-4 (Table
3.1). The HI titres of 4B1, F18B10, 17A3, F2D1, 18F5 and 3C9 were ≥ 160, whereas
the HI titre of the MAbs F1G2, F16B5 and F19F11 was 20. The remaining MAbs had
HI titres ≤ 10. The concentration of undiluted MAb used in the HI tests was 50-500
µg/ml. The MAb 4B1 which was used in the assay at a concentration of 50 µg/ml had a
high HI titre (≥ 160) indicating the concentration of MAbs used in the assay was not an
issue.
110
MAbs F1G2 and 18F5, which reacted only with DENV-4 infected cells in IFA, inhibited
agglutination of gander erythrocytes only by DENV-4. The MAb F2D1 was flavivirus
group-reactive by IFA, and inhibited agglutination of gander erythrocytes by each
DENV serotype and by MVEV. MAbs 4B1, F19F11 and 17A3 which reacted with
C6/36 cells infected with any DENV serotype inhibited the agglutination of gander
erythrocytes only by DENV-3 and DENV-4.
3.1.3 Capture of DENV-4

All the MAbs that reacted with the 56 kDa envelope protein from lysate of infected
C6/36 cells and with the PEG preparation of DENV-4 were able to capture virus if they
were first added to ELISA plates (Table 3.2). The highest absorbances were observed in
capture ELISAs with a PEG preparation of DENV-4 when MAbs were diluted 1 in 125
or 1 in 625 before coating to the plates. This dilution range was equivalent to
approximately 1 µg/ml of each MAb. Each of these MAbs and the reference MAbs
4G2, 6B6C1 and 1H10 subsequently was employed in capture ELISAs at a
concentration of 1 µg/ml.
DENV-4 tissue culture supernate (TCS) was used undiluted or diluted 1/2 as a source of
viral antigen in capture ELISAs. At these dilutions, the virus completely saturated the
capture MAb as illustrated by the MAb F1G2 in a representative experiment (Figure
3.4).
All MAbs which captured DENV-4 also reacted with virus in an indirect ELISA. The
absorbances for the indirect ELISA and capture ELISA using a 1/125 dilution of MAb
were similar (Table 3.2). There was a decrease in absorbances for indirect ELISAs
using the MAbs 4B1, F18B10, F19F11, 3C9 and F20F10 and an increase in absorbances
for indirect ELISAs using the MAb F16B5 compared to capture ELISA absorbances
(Table 3.2). The MAbs that reacted with the 45 kDa NS1 protein from cell lysate or in
virus preparations did not capture virus when they were coated to ELISA plates at 1
µg/ml. For this reason, the anti-NS1 MAb 7E3 was used in capture ELISAs as the
negative control MAb. The anti-NS1 MAbs were able to bind DENV-4 in indirect
ELISAs, but only when high concentrations of MAb were used.
111
Table 3.2 The ability of anti-DENV-4 MAbs to combine with PEG-concentrated DENV-4 in antibody
capture ELISAs and indirect ELISAs
Absorbance (mean ± 1 s.d.; n=2)
Capture ELISA Indirect ELISA
MAb 1/125 MAb 1/625 MAb 1/125
MAb Virus No Virus Virus No Virus Virus
F1G2 0.761 ± 0.024 0.020 ± 0.005 0.709 ± 0.015 0.015 ± 0.002 0.767 ± 0.013
F2D1 0.513 ± 0.045 0.021 ± 0.002 0.595 ± 0.061 0.019 ± 0.000 0.489 ± 0.07
13H8 0.794 ± 0.010 0.013 ± 0.003 0.856 ± 0.013 0.010 ± 0.000 0.719 ± 0.047
18F5 0.574 ± 0.014 0.014 ± 0.000 0.671 ± 0.021 0.014 ± 0.000 0.665 ± 0.010
4B1 0.613 ± 0.056 0.012 ± 0.000 0.626 ± 0.012 0.011 ± 0.000 0.487 ± 0.052
F18B10 0.520 ± 0.032 0.010 ± 0.002 0.494 ± 0.019 0.009 ± 0.002 0.312 ± 0.014
17A3 0.794 ± 0.041 0.011 ± 0.001 0.738 ± 0.013 0.011 ± 0.000 0.660 ± 0.083
F16B5 0.369 ± 0.026 0.015 ± 0.005 0.366 ± 0.018 0.030 ± 0.026 0.565 ± 0.021
F19F11 0.576 ± 0.024 0.015 ± 0.003 0.618 ± 0.023 0.044 ± 0.009 0.340 ± 0.024
3C9 0.797 ± 0.012 0.024 ± 0.002 0.625 ± 0.077 0.019 ± 0.003 0.518 ± 0.010
F20F10 0.358 ± 0.036 0.012 ± 0.000 0.344 ± 0.024 0.009 ± 0.000 0.158 ± 0.040
F7MF7 0.024 ± 0.000 0.010 ± 0.001 0.022 ± 0.007 0.009 ± 0.000 0.087 ± 0.002
F12A3 0.034 ± 0.007 0.010 ± 0.000 0.020 ± 0.004 0.009 ± 0.000 0.049 ± 0.004
7 E3 0.021 ± 0.000 0.022 ± 0.002 0.025 ± 0.004 0.016 ± 0.003 0.050 ± 0.011
s.d.: standard deviation
n: number of values
112
2.5
1/2
2 1
ELISA Absorbance (450nm)
1.5
1/4
1/8
0.5
1/16
0 0
0 16 8 4 2
1/2 UNDIL
1
Reciprocol dilution of DENV-4
Figure 3.4 The capture of DENV-4 at different dilutions by MAb F1G2 coated to an
ELISA plate at 1 µg/ml. The virus saturates the capture MAb when diluted 1 in 2 or
when used undiluted. A similar trend was obtained when using other MAbs that capture
DENV-4.
113
3.1.3.1 Capture of low pH treated DENV-4
MAb capture studies using TBEV and DENV-2 have identified peptides within domain I
(TBEV: E1-22) and domain II (DENV-2: E58-E121; E225-E249, TBEV: E221-E240) of
the E protein that are more accessible following low pH treatment of viruses (Roehrig et
al.,1990; Holzmann et al.,1993) . Similar capture experiments were performed in this
study to determine whether acid resistant epitopes exist in the DENV-4 E protein, and
whether these epitopes occur within similar regions. If DENV-4 was exposed to pH 6.0
and then restored to pH 8.0 prior to use in the capture ELISA, the absorbance values of
capture ELISAs employing 10 of the 12 MAbs was reduced by >80%. MAbs 13H8 and
1H10 were the only MAbs to capture similar amounts of low pH treated DENV-4 and
untreated DENV-4 (student T-test; probability value (p) >0.05) (Table 3.3).
3.1.3.2 MAb avidity

The difference in neutralisation strength between MAbs may be due to the strength of
binding of the MAb to the viral epitope, which is a measure of MAb avidity. The
avidity of the MAbs for DENV-4 was assessed by measuring the ability of urea to
dislodge virus bound to antibody in capture ELISAs. The use of 4-6 M urea in wash
buffers reduced absorbance values in the capture ELISAs by more than 50%. However,
the absorbance values for ELISAs employing the reference MAb 1H10 were not reduced
by increasing concentrations of urea. These affects are illustrated by avidity experiments
using the DENV-4 specific neutralising MAbs 18F5, 13H8, F1G2 and 1H10 (Figure
3.5). Despite having a stronger avidity for DENV-4, the MAb 1H10 still had similar
neutralisation strength to the other MAbs. 6 M urea caused a significant (Student T-test;
p≤0.05) reduction in the amount of DENV-4 captured by all MAbs except 1H10 (Table
3.4).
In addition, the reduction in absorbances was not the result of urea removing capture
MAb attached to ELISA plates. The absorbance values representing the amount of MAb
attached to the ELISA plates were reduced by <40% for all capture MAbs following the
6 M urea wash (Section 7.1).
114
Table 3.3 Capture of DENV-4 by MAbs before and after exposure of the virus to pH
6.0 for 15 minutes.
Absorbance (mean ± 1 s.d.; n=2)

virus exposed to
a b
MAb untreated virus pH 6.0 buffer Inhibition (%) p
F1G2 0.708 ± 0.013 0.086 ± 0.001 88 ≤0.05
F2D1 0.285 ± 0.027 0.023 ± 0.002 92 ≤0.05
4B1 0.776 ± 0.011 0.084 ± 0.002 89 ≤0.05
17A3 0.728 ± 0.031 0.095 ± 0.001 87 ≤0.05
F16B5 0.486 ± 0.021 0.068 ± 0.003 86 ≤0.05
F18B10 0.604 ± 0.008 0.066 ± 0.001 89 ≤0.05
18F5 0.849 ± 0.062 0.057 ± 0.004 93 ≤0.05
3C9 0.543 ± 0.052 0.147 ± 0.014 73 ≤0.05
F19F11 0.636 ± 0.030 0.054 ± 0.000 91 ≤0.05
F20F10 0.475 ± 0.036 0.063 ± 0.001 87 ≤0.05
13H8 1.011 ± 0.018 0.992 ± 0.012 2 >0.05
1H10 1.161 ± 0.062 1.234 ± 0.046 -6 >0.05
6B6C1 0.268 ± 0.021 0.107 ± 0.000 60 ≤0.05
4G2 0.139 ± 0.010 0.044 ± 0.000 69 ≤0.05
c
7E3 0.049 ± 0.007 0.038 ± 0.001
a
(absorbance of untreated virus - absorbance of virus exposed to pH 6.0 buffer) x100
absorbance of untreated virus
b
Student T-Test
c
Control MAb. Does not capture DENV-4
The shading indicates MAbs (13H8 and 1H10) which captured DENV-4 following low pH
treatment of the virus.
115
2
1.8
1.6
ELISA Absorbance (450nm)
1.4
13h8
1.2
1h10
1 18f5
0.8 f1g2
0.6
0.4
0.2
0
0 1 2 3 4 5 6
Concentration of urea [M]
Figure 3.5 Effects of different concentrations of urea on the capture of DENV-4 by

DENV-4 specific neutralising MAbs 13H8, 1H10, 18F5 and F1G2. Increasing the
concentration of urea did not affect capture of DENV-4 by the reference MAb 1H10
derived from the WRAIR. In contrast, increasing concentrations of urea reduced the
capture of DENV-4 by the other DENV-4 specific neutralising MAbs.
116
Table 3.4 The effect of 6 M urea on the capture of DENV-4 by anti-DENV-4 MAbs
Mean Abs ± 1 s.d (n=2)

a b
MAb Virus Untreated Virus + 6M Urea Inhibition (%) p
F1G2 1.347 ± 0.028 0.053 ± 0.002 96 ≤0.05
F2D1 0.719 ± 0.098 0.306 ± 0.000 57 ≤0.05
4B1 0.691 ± 0.064 0.053 ± 0.000 92 ≤0.05
17A3 0.834 ± 0.064 0.069 ± 0.004 92 ≤0.05
F16B5 0.499 ± 0.033 0.051 ± 0.000 90 ≤0.05
F18B10 0.672 ± 0.056 0.130 ± 0.019 80 ≤0.05
18F5 0.677 ± 0.023 0.153 ± 0.000 77 ≤0.05
3C9 0.531 ± 0.113 0.170 ± 0.003 68 ≤0.05
F19F11 0.349 ± 0.024 0.100 ± 0.001 71 ≤0.05
F20F10 0.299 ± 0.020 0.060 ± 0.001 80 ≤0.05
13H8 1.745 ± 0.041 0.108 ± 0.004 94 ≤0.05
1H10 1.259 ± 0.104 1.010 ± 0.044 20 >0.05
6B6C1 0.518 ± 0.051 0.123 ± 0.021 76 ≤0.05
4G2 0.566 ± 0.009 0.177 ± 0.004 69 ≤0.05
c
7E3 0.013 ± 0.001 0.010 ± 0.000
a
(absorbance of untreated virus - absorbance of virus washed with 6M urea) x100
absorbance of untreated virus
b
Student T-Test
c
Control MAb. Does not capture DENV-4
The shading indicates MAbs (1H10) that effectively capture DENV-4 in the presence
of 6M urea
117
3.2 Identification of antigenic domains on the DENV-4 envelope protein
The MAbs that effectively captured DENV-4 were used in competitive binding assays
(CBAs) to determine spatial relationships between epitopes and therefore define
antigenic domains on the DENV-4 E protein. All MAbs were tested against each other in
CBAs where one MAb, the capture MAb, was coated on an ELISA plate and then the
other MAb, the blocking MAb, was mixed with the virus and then added to the plate.
The capture of virus was detected using a HRP-labelled MAb (HRP-6B6C1) that was
specific for DENV and that resulted in a colour reaction that could be read as an
absorbance.
If the blocking and capture MAbs recognised distinct epitopes then virus capture was
detected and a color reaction occurred in the ELISA. On the other hand, if the blocking
and capture MAbs recognised similar epitopes then the level of virus capture was
reduced and the color reaction was reduced or non-existent. The competition between
different MAbs in the CBAs was represented as percentage blocking of virus capture,
which was determined by comparing MAb capture of virus alone and MAb capture of
virus in the presence of blocking MAb.
3.2.1 Competitive capture ELISAs

The absorbance values in virus-capture ELISAs employing all MAbs except 3C9,
F20F10, F19F11 and 18F5 were reduced by ≥90% if virus was pre-incubated with 10
µg/ml of homologous MAb (p≤0.05). When the virus was diluted 1 in 2, which still
saturates the 1 µg/ml capture MAb (Figure 3.4), prior to pre-incubation with 10 µg/ml of
homologous MAb, the absorbance values for capture of virus by MAbs 3C9 and F20F10
were reduced by ≥90% and the absorbance values for capture of virus by MAbs 18F5
and F19F11 were reduced by 82% and 73% respectively (p≤0.05).
The “blocking” MAb, with which virus was pre-incubated before addition to ELISA
plates containing capture MAbs, was used at a concentration of 10 µg/ml for all
subsequent competitive capture ELISAs and virus was diluted 1 in 2 when MAbs 18F5,
3C9, F19F11 or F20F10 were employed as capture or blocking MAbs.
118
Due to the amount of data that was generated in the competitive binding experiments
employing homologous or heterologous MAb mixtures, it has been provided as
supplementary data in Appendix section 7.2 of this manuscript. Each table in Section
7.2 represents the blocking affect of all MAbs against a specific capture MAb.
Using the data in Appendix 7.2, the MAbs could be grouped on the basis of the degree to
which they inhibited binding of virus to a capture MAb (Table 3.5). The competition
between MAbs was significant if there was greater than 10% inhibition of capture of
virus (Student T-test; p≤0.05).
There was significant competition between the MAbs that were cross-reactive by IFA,
indicated by the boxed regions of Table 3.5. The flavivirus group-reactive MAbs 4G2,
6B6C1 and F2D1 formed one group. The DENV group-specific MAbs 4B1 and 17A3
formed another group. The DENV group-specific MAbs 3C9 and F18B10 also formed a
group with the DENV-2 and DENV-4 reactive MAb F16B5. There was ≥80%
competition between the MAbs within the designated groups.
Capture of virus by MAbs was detected using HRP-labeled anti-flavivirus MAb 6B6C1.
Any MAbs which reacted with the same or a spatially-related epitope would block
binding of 6B6C1 and give a false impression that the capture of virus had been blocked.
The percentage inhibition of 6B6C1 capture of DENV-4 in the presence of blocking
MAbs shown in Table 3.5 was graphed to demonstrate the affect of the blocking MAbs
on the binding of HRP-6B6C1 (Figure 3.6). The binding of cross-reactive MAbs (4G2,
F2D1, 3C9, F16B5, F18B10, 4B1, and 17A3) and the DENV-4 type specific MAb 18F5
to DENV-4 had a greater affect on the binding of the HRP-6B6C1 MAb used to detect
captured virus than the other MAbs (Figure 3.6).
The DENV group-specific MAb, F19F11, and the DENV-4 type-specific MAbs 18F5,
F1G2 and F20F10 competed with the cross-reactive groups of MAbs. The MAbs 18F5
and F1G2 showed a “one way” pattern of competition for epitopes. The competition of
18F5, F20F10 and F19F11 with other MAbs provided examples of “one way” blocking.
119
Table 3.5. Inhibition of capture of DENV-4 by MAbs when virus was pre-incubated with the homologous or heterologous MAbs
Capture Blocking MAb

MAb 6B6C1 4G2 F2D1 3C9 F16B5 F18B10 4B1 17A3 F19F11 18F5 F1G2 F20F10 1H10 13H8
6B6C1 95 94 92 73 84 36 43 40 7 47 -83 9 -39 -309
4G2 92 88 92 73 83 47 54 51 25 55 -35 21 -45 -362
F2D1 98 98 97 67 85 53 55 49 26 63 -17 29 14 -134
3C9 98 97 77 97 97 91 93 89 61 34 79 29 64 -49
F16B5 97 92 56 96 98 84 82 70 47 35 35 12 34 -69
F18B10 97 96 62 97 97 94 59 42 21 30 11 19 -1 -85
4B1 98 90 63 38 49 41 99 97 62 21 13 20 -3 -64
17A3 98 90 68 45 54 42 98 97 65 28 10 16 8 -54
F19F11 90 82 83 76 90 84 98 94 73 37 81 10 62 1
18F5 96 89 92 72 82 59 66 55 20 82 96 19 63 25
F1G2 99 98 65 62 60 24 21 15 19 37 97 14 -15 -35
F20F10 98 90 89 65 74 56 63 53 18 96 79 92 52 23
1H10 97 97 50 16 20 5 4 4 3 4 -3 2 96 -7
13H8 91 86 45 34 20 4 1 -1 3 2 -2 -2 37 95
Boxed regions indicates MAbs exhibiting the strongest competition between each other.
MAbs listed in bold type neutralised DENV-4 infection by 2-3 log10.
50-100% Inhibition (p≤0.05)
<10% Inhibition (p>0.05)
120
Blocking MAbs
Cross-reactive MAbs DENV-4 type specific MAbs
11
10
1
2
6C
6B
8B
10
8
A3
9F
0F
F5
G
2
H
1
4G
3C
1H
6B
4B
F2
F1
F1
F1
F1
F2
17
18
13
100
50
-50
Inhibition (%)
-100
-150
-200
-250
-300
-350
Figure 3.6. The ability of MAbs to inhibit binding of the HRP labelled 6B6C1 detection
MAb to DENV-4 in a capture ELISA. Negative inhibition values indicate enhanced
binding of HRP-6B6C1 to virions. The majority of cross-reactive MAbs as well as the
DENV-4 specific MAb 18F5 effectively block the binding of the detection MAb. The
DENV-4 specific MAbs F1G2, 13H8 and the reference MAb 1H10 enhance binding of
the detection MAb.
121
The capture of DENV-4 by the MAbs F19F11, 18F5 and F20F10 was blocked by other
MAbs (cross-reactive group and F1G2). However in the reverse experiment, F19F11,
18F5 and F20F10 did not block the capture of DENV-4 by the other MAbs (cross-
reactive group and F1G2) as efficiently. In addition the MAbs F19F11, 18F5 and
F20F10 were less able to prevent capture of virus by homologous MAb than other MAbs
were. Interestingly, all these MAbs were the same isotype (IgG2b).
In some instances, the binding of virus to a capture MAb was enhanced by the presence
of the blocking MAb. The binding of the DENV-4 specific MAbs 13H8, 1H10 and
F1G2 to their epitopes enhanced virus capture by several MAbs, particularly the
flavivirus cross-reactive MAbs F2D1, 4G2 and 6B6C1. In particular, the binding of
13H8 to DENV-4 enhanced capture by the majority of dengue group and flavivirus
group reactive MAbs.
3.2.2 Competitive capture ELISAs with human serum

In an attempt to identify clinically relevant epitopes that are important markers for
DENV vaccine design, competitive capture ELISAs were also performed with serum
from patients infected with DENV to assess whether the anti-DENV antibodies in the
serum recognised the same or epitopes related spatially to those seen by MAbs (Table
3.6). Serum from donor JD who had no anti-flavivirus antibodies detectable by HI did
not inhibit virus capture by the MAbs (<10%; p>0.05). Serum from donor D4 who had
a DENV-4 infection inhibited capture of DENV-4 virus by all MAbs (>50%; p≤0.05)
with the exception of the neutralising DENV-4 specific MAb 1H10 (28%; p>0.05). The
DENV-4 specific neutralising MAb 13H8 also was weakly inhibited by D4 serum
(30%).
Serum from donor JA, who had a clinical DENV-3 infection as well as Japanese
encephalitis virus (JEV) and yellow fever virus (YFV) vaccinations, inhibited capture of
DENV-4 by all MAbs (p≤0.05) except MAbs 1H10 and 6B6C1 (<10%; p>0.05). The
capture of virus by the DENV-4 specific MAbs F1G2, 18F5, 13H8 was weakly inhibited
by JA serum (<20%) The capture of virus by MAb 4G2 was enhanced in the presence
of serum from donor JA (-39%).
122
Table 3.6. The ability of human serum containing anti-dengue or anti-flavivirus
antibodies to inhibit capture of DENV-4 by anti-DENV-4 MAbs
Capture MAb Human seruma Abs (mean ± 1 s.d.; n=2) Inhibition (%)
b
pc
1H10 D4 2.608 ± 0.396 28 >0.05
JA 3.334 ± 0.125 8 >0.05
JD 3.351 ± 0.498 8 >0.05
no serum 3.640 ± 0.394
4B1 D4 0.599 ± 0.002 73 ≤0.05

JA 1.763 ± 0.080 20 ≤0.05
JD 2.103 ± 0.019 5 >0.05
no serum 2.214 ± 0.074
17A3 D4 0.569 ± 0.007 75 ≤0.05

JA 1.716 ± 0.154 25 ≤0.05
JD 2.220 ± 0.226 2 >0.05
no serum 2.276 ± 0.108
F16B5 D4 0.269 ± 0.007 84 ≤0.05

JA 1.049 ± 0.058 38 ≤0.05
JD 1.728 ± 0.033 -2 >0.05
no serum 1.699 ± 0.173
F18B10 D4 0.409 ± 0.012 81 ≤0.05

JA 1.521 ± 0.106 31 ≤0.05
JD 2.039 ± 0.039 8 >0.05
no serum 2.206 ± 0.272
3C9 D4 0.215 ± 0.000 79 ≤0.05

JA 0.585 ± 0.014 43 ≤0.05
JD 1.041 ± 0.014 -2 >0.05
no serum 1.023 ± 0.069
F20F10 D4 0.396 ± 0.005 79 ≤0.05

JA 1.513 ± 0.084 22 ≤0.05
JD 1.955 ± 0.021 -1 >0.05
no serum 1.935 ± 0.015
a
D4. Patient from whom DENV-4 was recovered
JA. Clinical DENV-3 infection, JEV and YFV vaccination
JD. No anti-flavivirus antibody detected in HI test
b
(Absorbance of virus + no serum - Absorbance of virus + serum) x100
Absorbance of virus + no serum
c
Student T-Test
123
Table 3.6 cont.
a b c
Capture MAb Human serum Abs (mean ± 1 s.d.; n=2) Inhibition (%) p
F19F11 D4 0.236 ± 0.019 81 ≤0.05
JA 0.746 ± 0.003 40 ≤0.05
JD 1.303 ± 0.007 -5 >0.05
no serum 1.241 ± 0.032
F1G2 D4 1.267 ± 0.026 61 ≤0.05

JA 2.819 ± 0.036 13 ≤0.05
JD 3.075 ± 0.007 5 >0.05
no serum 3.244 ± 0.152
F2D1 D4 0.067 ± 0.001 95 ≤0.05

JA 1.019 ± 0.029 27 ≤0.05
JD 1.329 ± 0.037 4 >0.05
no serum 1.390 ± 0.026
13H8 D4 2.310 ± 0.001 30 ≤0.05

JA 2.999 ± 0.014 10 ≤0.05
JD 3.341 ± 0.477 -1 >0.05
no serum 3.324 ± 0.018
18F5 D4 0.312 ± 0.020 88 ≤0.05

JA 2.232 ± 0.046 12 ≤0.05
JD 2.597 ± 0.046 -2 >0.05
no serum 2.547 ± 0.008
4G2 D4 0.107 ± 0.004 83 ≤0.05

JA 0.898 ± 0.026 -39 ≤0.05
JD 0.668 ± 0.084 -4 >0.05
no serum 0.645 ± 0.009
6B6C1 D4 0.042 ± 0.000 89 ≤0.05

JA 0.364 ± 0.008 2 >0.05
JD 0.508 ± 0.030 -37 >0.05
no serum 0.371 ± 0.067
a
D4. Patient from whom DENV-4 was recovered
JA. Clinical DENV-3 infection, JEV and YFV vaccination
JD. No anti-flavivirus antibody detected in HI test
b
(Absorbance of virus + no serum - Absorbance of virus + serum) x100
Absorbance of virus + no serum
c
Student T-Test
124
The competitive binding assays indicated the spatial relationships between epitopes on
the DENV-4 E protein, but did not indicate the precise location of these epitopes or
identify them. A range of strategies was employed in an effort to identify epitopes
recognised by the DENV-4-specific neutralising MAbs 18F5, F1G2, 13H8 and the
Flavivirus-group specific neutralising MAb F2D1.

neutralisation
Once the antigenic domains of the DENV-4 E protein had been characterised, several
strategies were developed to identify epitopes on the DENV-4 E protein recognised by
neutralising MAbs. The traditional approach was to select DENV-4 n.e.m. viruses using
DENV-4 specific neutralising MAbs. DENV-4 variants including DENV-4
geographical isolates or chemically mutagenised DENV-4 were also screened with
neutralising MAbs to identify n.e.m. viruses. Site directed mutagenesis of the DENV-4
E protein confirmed whether amino acid changes identified in DENV-4 n.e.m.s were
essential for the binding of neutralising MAbs to an epitope. The reactivity of
neutralising MAbs with DENV chimeric E proteins and a bacterial peptide display
library were also used to identify structural domains or peptides involved in
neutralisation.
3.3.1 Selection of DENV-4 that escaped neutralisation by MAbs

The traditional approach for the identification of epitopes involved in neutralisation is to
select a virus population that escapes antibody neutralisation, termed a neutralisation
escape mutant (n.e.m.) virus. This method has been used to identify epitopes involved
in neutralisation on the envelope protein of DENV, flaviviruses and several other virus
families (Table 1.4). To select n.e.m.s, virus is cultured in cells in the presence of a
neutralising MAb until there is no reduction in the titre of virus in the presence of
neutralising MAb, in comparison to virus cultured without neutralising MAb. The E
protein gene sequences of the n.e.m. and wildtype viruses are compared to identify
genotypic changes involved in neutralisation escape and therefore potential epitope
locations.
125
The DENV-4 prototype strain H241 was passaged four times in C6/36 cells in the
presence of neutralising MAbs F1G2, 18F5, F2D1 or 13H8. The ability of the selecting
MAb to neutralise virus grown in the presence or absence of the MAb was quantitated
after each passage. No viruses were identified that were resistant to neutralisation by the
selecting MAb.
However, the amount of virus neutralised by the MAb decreased from 3.0 log10 with
wildtype virus to 1.0 log10 with virus passaged four times in the presence of the selecting
MAb. The deduced amino acid sequences of the E genes of the wildtype virus and the
viruses grown in the presence or absence of the selecting MAbs were the same. The
same results were obtained when selection was attempted with DENV-4 H241 and BHK
cells instead of C6/36 cells as the host cells.
Further selection experiments were undertaken in C6/36 cells using DENV-4 H241 and
MAbs at concentrations from 200-600 µg/ml. Increasing the concentration of MAb
resulted in the neutralisation of 10 to 100 times more virus. Even so, no viruses were
selected that were resistant to neutralisation by the selecting MAb. However, the
amount of virus neutralised decreased from 4.0-5.0 log10 with wildtype virus to 1.0 log10
with virus passaged four times in the presence of selecting MAbs. The deduced amino
acid sequences of the E genes of the wildtype virus and the viruses grown in the
presence or absence of the selecting concentrated MAbs were the same.
The procedures outlined above were repeated on a number of occasions, always with the
outcomes described.
126
3.3.2 Chemical mutagenesis of DENV-4 and selection of neutralisation escape
mutant viruses
The failure to select neutralisation escape mutant (n.e.m.) viruses may have been a
consequence of the original DENV-4 populations lacking genetic diversity. DENV-4
H241 therefore was cultured in BHK cells in the presence of the mutagen 5-Fluorouracil
(5FU) at concentrations from 1 µM to 10 mM, based on the protocol of Blaney et al.,
(2001). No virus was produced in the presence of 10 mM or 1 mM 5FU. The titre of
virus produced in the presence of 100 µM 5FU was 100-fold less than that of virus
produced by BHK cells not treated with 5FU (from 102 to 104 foci forming units (FFU)
/ml). However, the titre of viable virus from the 5FU treated cultures was too low to use
for the selection of n.e.m. viruses.
At 10 µM 5FU, the virus titre was reduced by 10-fold (from 104 to 103 FFU/ml) and the
surviving virus was used in an attempt to select n.e.m. viruses. No deduced amino acid
changes were detected between the E proteins of DENV-4 H241 passaged with 10µM
5FU (W10) and DENV-4 H241 passaged in parallel with no 5FU mutagen (NM) (Table
3.7). However, nucleotide sequence chromatograms from DENV-4 treated with 5FU
did have multiple nucleotide peaks in codons for amino acid residues E95, E156, E157
and E402 (Figure 3.7). The effects of 5FU treatment and BHK cell passage on the
DENV-4 H241 population are illustrated in a chromatogram of the nucleotide sequence
of the E gene of the DENV-4 (Figure 3.7).
The chromatogram shows an A at nucleotide position 284 in the NM virus which was
grown in the absence of 5FU, a mixture of C and A at nucleotide 284 in the gene of W10
virus which was grown in the presence of 5FU and a C at nucleotide 284 of the E gene
of DENV-4 n.e.m viruses which was derived from the passage of W10 virus in BHK
cells. The A-C change at nucleotide 284 was responsible for the Asp-Ala amino acid
change at E95 in the n.e.m. DENV-4. Similar affects were observed at nucleotides 466,
470 and 1204 that encoded amino acid changes at E156, E157 and E402 respectively.
127
Table 3.7. Genotypic and phenotypic properties of DENV-4 derived by treatment with 10µM 5FU and BHK cell passage
Position in E protein of Neutralisation index (log10)

Virus amino acid changes in BHK cells Virus titre (FFU/ml)
E95 E156 E157 E402 F1G2 F2D1 13H8 BHK C636
a 5 6
NM D P N L 2.0 2.4 2.4 1x10 1x10
b g 4 4
W10 D/A P/S N/S L/F 1.6 1.6 1.5 2x10 1x10
c 6 3
W10-P2 A S S F <1.0 <1.0 <1.0 1.5x10 7.5x10
d 4 1
W10-F1G2 A S S F <1.0 <1.0 <1.0 5x10 5x10
e 5 2
W10-F2D1 A S S F <1.0 <1.0 <1.0 5x10 5x10
f 4 2
W10-13H8 A S S F <1.0 <1.0 <1.0 1x10 5x10
a
NM is DENV-4 H241 grown without 5FU
b
W10 is DENV-4 H241 grown in the presence of 10uM 5FU.
c
W10-P2 is W10 virus passaged twice in BHK cells
d
W10-F1G2 is W10 virus passaged twice in BHK cells in the presence of MAb F1G2
e
W10-F2D1 is W10 virus passaged twice in BHK cells in the presence of MAb F2D1
f
W10-13H8 is W10 virus passaged twice in BHK cells in the presence of MAb 13H8
g
The W10 virus had multiple nucleotide sequences in the E gene encoding amino acids at E95, E156, E157 and E402.
The multiple nucleotides (A/C) at position 248 of the E gene encodes the D/A amino acids at E95 (Figure 3.7).
128
A
Figure 3.7 The potential affects of 5FU treatment on the genetic diversity of a DENV-
4 population and selection of DENV-4 n.e.m.s, demonstrated by a chromatogram of the
nucleotide sequence of (A) DENV-4 NM (no 5FU treatment, A at nucleotide 284), (B)
DENV-4 W10 (10 µM 5FU treatment, A/C at nucleotide 284] and (C) DENV-4 n.e.m.
[DENV-4 W10 passaged in BHK cells, C at nucleotide 284]. It was evident that the
5FU treatment of DENV-4 selected for an additional virus population to the wildtype
indicated by multiple nucleotide peaks in (B), and that subsequent cell passage favoured
selection of the new virus population (C). The A-C nucleotide change corresponded to
the D-A change at E95 in DENV-4 n.e.m. Similar changes also occurred at nucleotides
encoding other amino acid changes identified in the DENV-4 n.e.m at E156, E157 and
E402
129
DENV-4 treated with 10 µM 5FU (W10) was passaged twice in BHK cells, either in the
presence or absence of selecting MAbs F1G2, F2D1 and 13H8. The resulting viruses
were resistant to neutralisation by the corresponding selecting MAbs. These n.e.m.
viruses cultured in the presence of MAb (W10-F1G2, W10-F2D1, W10-13H8) or
without MAb (W10-P2) had the same E protein amino acid sequences. The n.e.m.
viruses had amino acid changes at E95 (Asp-Ala), E156 (Pro-Ser), E157 (Asn-Ser) and
E402 (Phe-Leu) when compared to unpassaged 5FU treated DENV-4 (W10) and
wildtype DENV-4 (NM), which were neutralised by these MAbs (Table 3.7). In
addition, the 5FU treated DENV-4 (W10), which had a mixed virus population of
wildtype and n.e.m. as indicated by the chromatogram in Figure 3.7, was neutralised by
the MAbs to a lesser extent than the DENV-4 wildtype (NM) (Table 3.7). No virus was
recovered following passage of DENV-4 W10 in the presence of MAb 18F5.
The titres of the n.e.m. viruses grown in C6/36 cells were 10-100 fold less than the titres
of these viruses grown in BHK cells, while the titres of the wildtype DENV-4 (NM) and
the 5FU treated DENV-4 (W10) in C6/36 and in BHK cells were the same (Table 3.7).
The MAbs 13H8, F2D1 and F1G2 reacted with similar intensities in IFAs with C6/36
cells infected with the wildtype DENV-4 (NM) and 5FU treated DENV-4 (W10). The
MAb 13H8 also reacted with the same intensity in IFAs with C6/36 cells infected with
the DENV-4 n.e.m. as it did with cells infected with wildtype DENV-4 (NM) (Figure
3.8). However, the proportion of C6/36 cells infected with the DENV-4 n.e.m.
recognised by the MAb 13H8 was less than the proportion of cells infected with the
DENV-4 (NM) recognised by this MAb. This data may have been a reflection of the
decreased titre of the DENV-4 n.e.m. observed in C6/36 cells (Table 3.7). The MAb
F2D1 reacted weakly (+/-) with C6/36 cells infected with n.e.m. viruses and the MAb
F1G2 did not react with C6/36 cells infected with n.e.m. viruses at all (Figure 3.8).
Further selection of DENV-4 n.e.m.s was attempted by screening DENV-4 geographical

isolates with the MAbs (Section 3.3.3).
130
C636 cells infected with wildtype C6-36 cells infected with DENV-4
DENV-4 (NM) n.e.m
MAb
13H8
IFA + Neutralisation + IFA+ Neutralisation -
MAb
F2D1
IFA + Neutralisation + IFA +/- Neutralisation -
MAb
F1G2
IFA + Neutralisation + IFA - Neutralisation -
Figure 3.8. Reactivity of MAbs 13H8, F2D1 and F1G2 with C6/36 cells infected with
wildtype DENV-4 (NM) or DENV-4 n.e.m. IFA +: MAb reacts with cells infected
with the virus indicated. IFA -: MAb does not react with cells infected with the virus
indicated. Neutralisation +: MAb neutralised the virus used to infect the cells.
Neutralisation -: MAb did not neutralise virus used to infect the cells. Each MAb reacts
with the DENV-4 wildtype and MAb 13H8 also reacts with DENV-4 n.e.m. The MAb
F2D1 reacts weakly with the DENV-4 n.e.m, whereas the MAb F1G2 does not react
with the DENV-4 n.e.m.
131
3.3.3 Ability of anti-DENV-4 MAbs to recognise different strains of DENV-4 virus
C6/36 cells infected with a panel of DENV-4 were screened by IFA to identify any
naturally occurring virus populations that might not be recognised by the anti-DENV-4
MAbs. The DENV-4 specific neutralising MAb 18F5 did not react with DENV-4
strains 31500 and 38201 isolated from patients in Vietnam or DENV-4 strains 508 and
520 isolated from patients in Thailand (Table 3.8).
A comparison of the deduced amino acid sequences of the DENV-4 used to infect C6/36
cells for the IFAs identified three amino acid changes in the ectodomain (amino acids 1-
400) of the DENV-4 E protein which were characteristic of the DENV-4 from Thailand
(508, 520) and Vietnam (31500, 31582) (Table 3.9). These changes were located at E96
(T-M), E203 (K-T) and E329 (A-T). The DENV-4 isolate 31500 from Vietnam also had
a unique change at E160 (V-E).
The infection of C6/36 cells by DENV-4 31500 and 508 was not neutralised by MAb
18F5 (<1.0 log10) but it was neutralised by the MAb F1G2 (2.0-3.0 log10) (Table 3.10).
In contrast, DENV-4 H241 and the DENV-4 isolates 1453 and 8976 were neutralised by
MAbs 18F5 and F1G2 (2.0-3.0 log10) (Table 3.10). The absorbance values representing
the capture of DENV-4 31500 and 508 by MAb 18F5 were reduced by >45% (p ≤ 0.05)
when compared to capture of these viruses by MAb F1G2. The absorbance values
representing the capture of DENV-4 H241, 1453 and 8976 by MAb 18F5 were similar to
those for capture of these viruses by MAb F1G2 (p>0.05) (Table 3.10).
The titre of DENV-4 00508, 1674 and 8976 in BHK cells was 1.0 log10 less than in
C6/36 cells. The titre of DENV-4 31500 in BHK cells was >3.0 log10 less than in C6/36
cells (Table 3.10). In addition the titre of DENV-4 31500 was also reduced in Vero cells
by >3.0 log10, however in PS-EK cells the titre was the same as in C6/36 cells. It is
probable that the decreased infectivity of DENV-4 31500 in BHK and Vero cells is due
to the unique change at E160 (V-E) (Table 3.9).
132
Table 3.8. Reaction of anti-DENV-4 MAbs with C6/36 cells infected with DENV-4 isolated from different geographical regions
in an IFA test
DENV-4 IFA test results with MAbs undiluted
Strain Country Year 4G2 6B6C1 2H2 1H10 13H8 18F5 F2D1 F1G2 4B1 F19F11 F18B10 F16B5 F20F10 3C9 17A3
H241 Phillipines 1956 + + + + + + + + + + + + + + +
31500 Vietnam 2000 + + + + + - + + + + + + + + +
38201 Vietnam 2001 + + + + + - + + + + + + + + +
508 Thailand 1999 + + + + + - + + + + + + + + +

520 Thailand 1999 + + + + + - + + + + + + + + +
4553 Singapore 2001 + + + + + + + + + + + + + + +
8976 Singapore 1995 + + + + + + + + + + + + + + +
1674 Singapore 1990 + + + + + + + + + + + + + + +
83 Timor 2001 + + + + + + + + + + + + + + +
89 Timor 2001 + + + + + + + + + + + + + + +
99 Timor 2001 + + + + + + + + + + + + + + +
133
Table 3.9. Variation in the amino acid sequences of the E proteins of the DENV-4 strains used in the IFA in Table 3.8.
DENV-4 Amino acid Position

Strain Country 46 68 82 95 96 120 160 163 200 203 209 220 227 233 290 322 329 351 357 358 384 424 429 455 461 478 494
H241 Phillipines I I L D V S V T K K H W S Y E V A I F A D S L V F T H
31500 Vietnam I I L D M S E T K T H W S H E V T I F A D S L V L S H
38201 Vietnam I I L D M S V T K T H W S H E V T I F A D S L V L S H
508 Thailand I I L D M S V T K T H W L H E V T I F A D S L V L S H
520 Thailand I I L D M S V T K T Q W L H E V T I F A D S L V L S H
4553 Singapore T I L D V L V T K K H W S H E V A I F A D S F V F T Q
8976 Singapore T I L N V S V T E K H L S Y E V A V L A N S F I F T Q
1674 Singapore T V L D V S V T K E H W L Y K V A V L A N S F V F T Q
83 Timor T I L D V L V T K K H W S Y E V A I F A D S F I F T Q
89 Timor T I F D V L V T K K H W S Y E A A I F V D S F V F T Q
99 Timor T I L D V L V G K K H W S Y E V A I F A D F F V F T Q
Boxes represent amino acid changes that differ from the DENV-4 prototype strain H241.
Shaded boxes represent amino acid changes characteristic of DENV-4 31500, 38201, 508 and 520 which were not recognised by MAb 18F5 in IFA
134
Table 3.10. Comparison of the genotypic and phenotypic properties of DENV-4 strains recognised by MAb 18F5 with those not
recognised by this MAb.
IFA Capture ELISA Neutralisation index Amino acid

DENV-4 C6/36 cells Absorbance (mean ± 1 s.d.; n=2) (log10) C6/36 cells Virus titre (FFU/ml) change
a
Strain Country 18F5 F1G2 18F5 F1G2 p 18F5 F1G2 C6/36 BHK 96 203 329
6 5
H241 Phillipines + + 2.529 ± 0.033 2.613 ± 0.024 >0.05 2.0-3.0 2.0-3.0 1x10 1x10 V K A
7 6
1674 Singapore + + 2.915 ± 0.099 3.112 ± 0.039 >0.05 2.0-3.0 2.0-3.0 2x10 1.5x10 V E A
7 5
8976 Singapore + + 3.019 ± 0.005 3.059 ± 0.008 >0.05 2.0-3.0 2.0-3.0 3.5x10 1x10 V K A
6 1
31500 Vietnam - + 1.313 ± 0.057 2.576 ± 0.030 ≤ 0.05 <1.0 2.0-3.0 1x10 <1x10 M T T
5 4
508 Thailand - + 1.408 ± 0.041 2.664 ± 0.011 ≤ 0.05 <1.0 2.0-3.0 7x10 4x10 M T T
a
Student T-test
135
3.3.4 Analysis of MAb binding sites using site directed mutagenesis of the DENV-4
E protein and chimeric DENV E proteins.
Site directed mutagenesis of a DENV-4 E gene in the plasmid pVAXD4 was employed
to produce E proteins with amino acid changes at sites believed to be involved in the
escape of DENV-4 from neutralisation by MAbs. The amino acid changes made were
based on the amino acid changes identified in the DENV-4 n.e.m. selected with 5FU
(Table 3.7: E95, E156, E157 and E402) and the DENV-4 geographical isolates that
were resistant to neutralisation by the MAb 18F5 (Table 3.10: E96, E203 and E329).
Structural domains of the DENV-3 and DENV-4 E proteins also were combined in
different orientations in the pVAX plasmid to produce chimeric E proteins that enabled
coarse mapping of domains of the E protein recognised by anti-DENV-4 MAbs.
Indirect IFAs were performed with a panel of MAbs and the BHK cells transfected with
these pVAX constructs to analyse MAb binding sites (Table 3.11). The MAb 13H8
reacted with a chimeric E protein construct which contained DENV-4 E protein from
amino acid residues 1-300 but not with a similar construct containing DENV-4 amino
acid residues 301-495. The MAb 18F5 reacted with the region E301-E495 of DENV-4
but not with E1-300. Furthermore, a change from Ala-Thr at E329 abolished this
reactivity. Amino acid changes at E95, 96, 156, 157, 203 and 402 of the DENV-4 E
protein had no effect on the binding of this MAb (Table 3.11).
The results with MAb F1G2 appeared contradictory. The MAb failed to bind to DENV
chimeric E protein containing the region of the DENV-4 E protein from amino acid
residues 1-300, yet a change at E95 from Asp-Ala abolished the binding of the MAb
(Table 3.11; Figure 3.9).
136
Table 3.11. Indirect IFA using selected MAbs as primary antibody and BHK cells
transfected with plasmids containing wildtype, chimeric and mutagenised DENV-4 E
genes.
MAb
pVAX plasmid 4G2 2H2 13H8 18F5 F1G2 F2D1
no insert - - - - - -
DEN4-C-prM-E + + + + + +
DEN4-C-prM-E
+ + + + - +
E95 (D-A)
DEN4-C-prM-E
+ + + + + +
E96 (V-M)
DEN4-C-prM-E
+ + + + + +
E156 (P-S)
DEN4-C-prM-E
E156 (P-S) + + + + + +
E157 (N-S)
DEN4-C-prM-E
+ + + + + +
E203 (K-T)
DEN4-C-prM-E
+ + + - + +
E329 (A-T)
DEN4-C-prM-E
+ + + + + +
E402 (L-F)
a
DEN4-C-prM
DEN4 E (1-300) + + + - - n.d
DEN3 E (301-495)
a
DEN4-C-prM
DEN3 E (1-300) + + - + + n.d
DEN4 E (301-495)
n.d. not determined
a
Chimeras: plasmids sharing E protein domains from DENV-3 and DENV-4.
137
A.
B.
Figure 3.9. The reactivity of MAb F1G2 with BHK cells transfected with (A) pVAX
DENV-4-C-prM-E/ E95 (Asp) and (B) pVAX DENV-4-C-prM-E/ E95 (Asp-Ala). The
green fluorescent staining pattern in (A) indicates the reactivity of F1G2 with the
DENV-4 E protein, whereas the lack of green fluorescence in (B) indicates that site
directed mutagenesis of E95 (Asp-Ala) stops binding of F1G2 to the DENV-4 E
protein.
138
3.3.5 Bacterial peptide display library
To confirm the location of DENV-4 epitopes identified using other epitope mapping
methods, an E.coli library displaying random 12 mer peptides (pFLiTrx) was screened
for reactivity with the DENV-4 specific neutralising MAbs F1G2, 13H8 and 18F5.
Bacterial clones displaying peptides that interacted with MAbs were enriched from the
initial library by panning with MAb immobilised on a tissue culture dish. The panning
process was repeated five times, individual colonies of bacteria were isolated and the
peptide-MAb reactivity was confirmed by western blot. Plasmid DNA from the
bacterial clones expressing peptides recognised by MAbs in western blots was
sequenced and the amino acid sequence of the random peptide determined. The amino
acid sequences of the peptides displayed by clones recognised by MAb F1G2 are shown
in Figure 3.10. These shared a common amino acid residue motif K/RWGG. No clones
were identified expressing peptides recognised by the MAbs 18F5 and 13H8. The
amino acid sequences of the peptides recognised by MAb F1G2 were aligned with the
primary sequence of the DENV-4 E protein. The K/RWGG sequence common to all
peptides aligns with DENV-4 E protein sequence RWGG at residues E98 (R), E101
(W), E102 (G) and E104 (G). There are also similarities between the peptide sequences
and the residues adjacent to E98-E102 in the DENV-4 E protein (Figure 3.10).
3.3.6 Virus Overlay Protein Binding Assay (VOPBA)

The virus overlay protein binding assay (VOPBA) was developed as a preliminary
method to investigate potential mechanisms used by the DENV-4 specific MAbs F1G2,
18F5 and 13H8 to neutralise DENV-4 infection. It was evident from the VOPBA, that
DENV-4 attached to a 40 kDa protein in the C6/36 cell lysate (+C), which was not
detected when RPMI-1640 medium was substituted for virus (-C). The binding of
DENV-4 to the 40 kDa protein was inhibited following pre-treatment of DENV-4 with
the MAbs F1G2 and 18F5, but not by the MAb 13H8 (Figure 3.11).
139
A.
Peptide 1 MRVRCATGKWGG
Peptide 2 FALVDTRWGGTY
Peptide 3 SEWIKWGGFGAG
Peptide 4 DNNQARWGGVVN
Peptide 5 DEDRVRWGGCGE
Peptide 6 EGQEDRWWPSGL
Peptide 7 GSQKWGGVENAG
B.
91 112
DENV-4E ICRRDVVDRGWGNGCGLFGKGG
PEP1 MRVRCATGK-WG-G--------
PEP2 --FALVDTR-WG-G--TY----
PEP3 ----SEWIK-WG-G---FGAG-
PEP4 ---DNNQAR-WG-G---VVN--
PEP5 ---DEDRVR-WG-G---CGE--
PEP6 ---EGQEDR-W------WPSGL
PEP7 -----GSQK-WG-G---VENAG
Figure 3.10. The amino acid sequences of (A) the seven peptides recognised by the
MAb F1G2 in the bacterial peptide display library and the (B) alignments of these
peptides with the DENV-4 H241 E protein sequence between residues E91 and E112.
The bolded and underlined amino acid residues in (A) represent the conserved motif
K/RWGG recognised by F1G2. The bolded residues in (B) are residues that share
sequence homology with the DENV-4 E protein sequence, which is shaded in grey. The
boxed residues represent aromatic amino acids with similar structures.
140
Mw (kDa)
110
90
51
40 kDa
C6/36
36 protein
29
21
+C -C F1G2 18F5 13H8
Figure 3.11. The binding of DENV-4 to a 40 kDa protein from uninfected C6/36 cell
lysate (+C) in the VOPBA. This binding still occurs if DENV-4 is pre-treated with the
DENV-4 specific MAb 13H8, but does not occur if DENV-4 is pre-treated with the
DENV-4 specific MAbs F1G2 or 18F5. There is no 40 kDa protein band detected if no
virus is present (-C).
141
4 DISCUSSION
The design of a chimeric DENV E protein for use as a tetravalent vaccine effective
against each DENV serotype requires an understanding of epitopes involved in
neutralisation of each DENV serotype. Epitopes involved in the antibody mediated
neutralisation have been identified on the E protein of each DENV serotype, with the
exception of DENV-4. Therefore, in this study, a panel of MAbs against DENV-4 was
generated and used in conjunction with different epitope mapping strategies to identify
epitopes involved in neutralisation of DENV-4.
4.1 Production and Characterisation of MAbs against DENV-4

The majority of MAbs generated in BALB/c mice against DENV-4 recognised the E
protein, which is expected as the E protein is the primary antigenic site of the virus.
MAb panels generated in BALB/c mice against the other DENV serotypes were also
primarily against the E protein (Jianmin et al., 1995; Beasley and Aaskov, 2001; Serafin
and Aaskov, 2001). In addition, several MAbs targeted the NS1 protein of DENV-4.
The NS1 protein is localised within host cells, but has been identified as discrete foci on
the surface of virus infected cells (Westaway and Goodman, 1987; this study, Figure
3.3), which suggested that NS1 would be accessible to an antibody response.
The MAbs against the DENV-4 E protein were mostly virus cross-reactive and
recognised conformationally dependent epitopes, which were surface accessible as
indicated by the ability of the MAbs to capture virus (Table 3.1; Table 3.2). A high
proportion of MAbs against the DENV-4 E protein neutralised DENV-4 infection (Table
3.1). The DENV-4 specific MAbs 13H8, F1G2 and 18F5 demonstrated the highest
neutralisation activity of the MAb panel and were subsequently used to identify epitopes
involved in the neutralisation of DENV-4 using strategies outlined in Section 4.2.
142
4.2 Strategies for the identification of epitopes on the DENV-4 envelope protein
involved in neutralisation
Epitopes on the flavivirus E protein involved in neutralisation have been characterised
using a variety of approaches, some of which were adopted in this study to characterise
epitopes involved in the neutralisation of DENV-4. Linear epitopes have been identified
on the flavivirus E protein using peptide mapping, E protein fragment mapping and
phage display libraries (Table 1.3). However, these methods were not suitable for
mapping conformationally dependent epitopes which require disulphide bridging and the
coexpression of the prM protein with the E protein to maintain their native structure
(Konishi and Mason, 1993; Roehrig et al., 2004).
The majority of anti-flavivirus MAbs, against the E protein, that neutralise infection
recognise conformational epitopes (Mandl et al., 1989; Jianmin et al., 1995; Roehrig et
al., 1998; Beasley and Aaskov, 2001; Serafin and Aaskov, 2001; this study). It has been
shown that the disruption of the disulphide bridges in the flavivirus E protein abrogated
the binding of neutralising MAbs (Wengler and Wengler, 1989b; Lin et al., 1994;
Roehrig et al., 2004). Likewise, the degree of binding of neutralising MAbs to
recombinant protein fragments or peptides representing the DENV E protein depended
on the conformation of the epitope, which relies on proper folding of structural domains
within the E protein (Megret et al., 1992, Roehrig et al., 1998).
Conformation dependent epitopes on the E protein of DENV and other flaviviruses

involved in neutralisation have been identified by the selection of neutralisation escape
mutant (n.e.m.) virus populations and the comparison of the deduced amino acid
sequences of the wildtype and n.e.m viruses (Table 1.4).
143
No DENV-4 n.e.m.s could be selected with the MAbs employed in this study and similar
problems have been reported when attempts were made to select DENV-1 and DENV-3
n.e.m. populations with neutralising MAbs (Beasley and Aaskov, 2001; Serafin and
Aaskov, 2001). These authors speculated that failure to select n.e.m.s could have been
due to the low frequency of genetic variants in the virus population, or to amino acid
changes in the epitope being lethal to the virus.
Several approaches to identifying domains or epitopes in the DENV-4 E protein

involved in neutralisation were used as an alternative to the direct selection of DENV-4
n.e.m.s.
In the first approach, MAbs were screened in a range of serological assays to identify
strains of DENV-4 with which they did not react (natural neutralisation escape mutants).
A comparison of the deduced amino acid sequences of DENV-4 recognised by the
MAbs, with those not recognised, allowed potential epitopes to be identified. In the
event that the natural n.e.m.s could not be identified, they were created by mutagenising
a DENV-4 population with 5FU. Site directed mutagenesis of an E protein gene
expressed in BHK cells was used to confirm the site of the epitopes of interest.
The second approach used employed constructs of chimeric DENV E proteins,

composed of domains I and II of the E protein of one DENV serotype and domain III of
the E protein of a second DENV serotype, to identify the domain to which the
neutralising MAbs attached. This had the advantage of not being a functional assay and
so could be employed with non-neutralising antibodies. The disadvantage was that it
could not be utilised if a MAb reacted with multiple DENV serotypes. Because of the
complexity of the protein folding in domains I and II of the E protein, it was not possible
to construct chimeric E proteins with domain I and II derived from different DENV
serotypes.
144
The third approach, which was used to confirm the location of epitopes identified by
other methods, was a bacterial peptide expression system in which the peptide of interest
was constrained by a disulphide bridge, and thus presented as a conformational loop
structure. This approach was adopted because all the anti DENV-4 MAbs which
neutralised infection recognised conformational epitopes and so it was unlikely the
epitopes recognised by them could be identified using linear peptides (Geysen et al.,
1984).
Competitive binding experiments were used in conjunction with these strategies to

confirm the spatial relationships between the epitopes recognised by the MAbs and
define antigenic domains involved in neutralisation (Section 4.3)
4.3 Identification of antigenic domains on the DENV-4 envelope protein involved

in neutralisation
Competitive binding experiments with pairs of anti-DENV-4 MAbs identified two
spatially distinct domains (D4E1 and D4E2) on the DENV-4 E protein involved in
neutralisation of DENV-4 by antibody. These domains were assigned based on the
degree of blocking between MAbs, indicated in Table 3.5, and represented the first
antigenic model to be developed for the DENV-4 E protein (Figure 4.1). In addition to
competing with one another for epitopes, the MAbs assigned to each domain shared
similar functional characteristics as outlined in Figure 4.1.
Domain, D4E1, contained the epitopes recognised by the serotype-specific neutralising

MAbs 13H8 and the reference MAb 1H10 from WRAIR. Both MAbs bound epitopes
on the DENV-4 E protein after it had been exposed to low pH (Table 3.3); both
enhanced capture of virus by other MAbs including the HRP-labelled MAb (HRP-
6B6C1; Figure 3.6) when bound to DENV-4; both captured virus pre-treated with MAbs
assigned to epitopes in domain D4E2 and neither inhibited hemagglutination by DENV-
4 (Table 3.1; Table 3.5). The failure of either to inhibit binding of the other to DENV-4
suggested they recognised discrete epitopes within domain D4E1.
145
1H10
(WRAIR) 13H8
Legend N* N*
N: Neutralisation (1 log) pH pH
N*: Neutralisation (2-3 log)
DI/II DI/II
HI: Hemagglutination (20)
HI*: Hemagglutination (>20)
pH: Capture of low pH treated DENV-4
DI/II: Domain I/Domain II
D4E1
DIII: Domain III
D4E2
F1G2
18F5 HI
F20F10 HI* N*
N F19F11 N* DIII
HI DIII
N
17A3
HI* 4B1
N HI* F18B10
N HI*
N
3C9
HI*
F2D1
HI* 6B6C1
F16B5
N* (WRAIR)
HI
N*
4G2
(WRAIR)
N*
Figure 4.1. Antigenic model of the DENV-4 E protein derived from competitive
binding assays. The competition between the MAbs for epitopes on the DENV-4 E
protein grouped them into two distinct domains designated as D4E1 and D4E2. The
overlapping circles are MAbs that compete for similar epitopes. The shaded circles are
epitopes recognised by flavivirus and DENV group specific MAbs. The clear circles
are epitopes recognised by DENV-4 specific MAbs. The results from functional assays
and domain assignment of MAbs using chimeric E proteins are indicated by the legend.
146
The D4E2 domain consisted predominately of spatially related and in some cases
overlapping (>90% inhibition of capture by a second MAb; Table 3.5) flavivirus and
DENV group-reactive epitopes, including the epitopes recognised by the flavivirus
group-reactive MAbs 4G2 (Gentry et al., 1982) and 6B6C1 (Roehrig et al., 1983)
derived from WRAIR.
The epitope recognised by the DENV-4 type-specific neutralising MAb 18F5 was
spatially related to the cross-reactive epitopes and grouped within domain D4E2 (Table
3.5). Further evidence for the grouping of epitopes into D4E2 was the demonstration
that the binding of the cross-reactive MAbs (4G2, F2D1, 3C9, F18B10, F16B5, 4B1 and
17A3) and the DENV-4 type specific MAb 18F5 to DENV-4 inhibited the binding of the
detection MAb (HRP-6B6C1) to virions (Figure 3.6).
Some epitopes, such as that recognised by the MAb F1G2, shared characteristics of both
D4E1 and D4E2 domains but ultimately, were assigned to a specific domain based on
competition with MAbs for which epitopes have been assigned more reliably. The MAb
F1G2 recognised an epitope which was related spatially to the epitope recognised by the
MAb 18F5, and therefore was grouped into D4E2 (Table 3.5). However, there was
limited competition between MAb F1G2 and the cross-reactive MAbs in D4E2 (Table
3.5), which made the localisation of the F1G2 epitope difficult. The majority of epitopes
identified in domain D4E2 were associated with the neutralisation of DENV-4 infection
and hemagglutination by DENV-4.
Competitive binding experiments employing the anti-DENV-4 MAbs and sera from
DENV patients suggested that the epitopes in domain D4E2, identified using the MAbs,
were the same, or related spatially to, epitopes recognised by serum from DENV patients
(Table 3.6). In contrast, both the MAbs against epitopes of D4E1 (13H8 and 1H10) did
not compete with serum from DENV patients (Table 3.6)
147
This was evidence that the MAbs employed in this study were important for the
identification of epitopes involved in the neutralisation of DENV-4 infection in humans.
The knowledge of clinically relevant epitopes is important for the design of DENV
vaccines. However, this study needs to be expanded to include more patients with
virologically confirmed infections with DENV-4 and the other DENV serotypes.
Several antigenic models of the DENV E protein have been derived from competitive
binding experiments with MAbs and share similarities with the antigenic model of the
DENV-4 E protein derived from the competitive binding experiments (Figure 4.1).
However, the definition of these domains has been limited by the number of MAbs
available and the procedures employed to identify epitopes (Tsekhanovskaya et al.,
1993).
The antigenic model of the DENV-2 E protein proposed by Henchal et al. (1985) and
expanded by Roehrig et al. (1998) was composed of three separate domains (A, B and
C) respectively. Domains C, A and B correspond to domains I, II and III respectively of
the flavivirus E protein identified from crystallographic and cryo-EM studies (Rey et al.,
1995; Modis et al., 2004). Domains C and B (I and III) contained mostly sub-complex
and type-specific epitopes, whereas Domain A (II) contained flavivirus and DENV
group-reactive epitopes (Roehrig et al., 1998). Epitopes involved in HI and in
neutralisation of virus were in domains A and B but not in C (Roehrig et al., 1998).
Further studies using the same panel of MAbs demonstrated that MAbs directed against
domain B (III) epitopes blocked adsorption of DENV-2 to Vero cells more efficiently
than MAbs against domain B (II) epitopes (Crill and Roehrig, 2001). Epitopes involved
in the neutralisation of DENV-2 were also identified on three overlapping domains using
a panel of DENV-2 type specific IgM MAbs (Jianmin et al., 1995). The antigenic model
of the DENV-1 E protein contained two distinct neutralising domains (E1 and 4G2)
separated by a non-functional domain (E2) (Beasley and Aaskov, 2001). The
neutralising epitopes in the E1 domain were recognised by DENV-1 type-specific MAbs
and were distinct from that recognised by the cross-reactive MAb 4G2.
148
The antigenic model of the DENV-3 E protein contained two overlapping domains
(Serafin and Aaskov, 2001). The first domain contained neutralising epitopes
recognised by DENV-3 type specific MAbs as well as flavivirus cross-reactive MAbs
(4G2 and 6B6C1). The other domain was recognised by non-neutralising MAbs.
The antigenic models proposed for each DENV serotype, including DENV-4, contained
at least two domains that are involved in neutralisation. One of these domains contained
epitopes recognised by Flavivirus group-reactive MAbs such as the well characterised
4G2 (Gentry et al., 1982) and 6B6C1 (Roehrig et al., 1983), whereas the other domains
were recognised by serotype-specific MAbs.
In the DENV-4 E protein, the majority of epitopes clustered in one domain, in contrast
to the antigenic models for other DENV serotypes, such as the DENV-2 E protein
(Roehrig et al., 1998) where the epitopes were distributed between multiple domains.
However, a larger panel of anti-DENV-4 E protein MAbs may have identified epitopes
spread more widely on the DENV-4 E protein (Tsekhanovskaya et al., 1993).
In addition, all MAbs were generated from hybridomas produced with B lymphocytes
from inbred BALB/c mice, so it is possible there could be some genetic restriction of
antibody repertoire. This has previously been observed when comparing the immune
responses generated in BALB/c mice against the different DENV serotypes. More than
80% of the anti-DENV-4 E protein MAbs generated in this study neutralised DENV-4
infection (Table 3.1). Similarly more than 40% of anti-DENV-2 MAbs has been
reported to neutralise DENV infection (Gentry et al., 1982; Henchal et al., 1985; Jianmin
et al., 1995; Roehrig et al., 1998). In contrast, less than 20% of anti-DENV-1 and anti-
DENV-3 MAbs were reported to neutralise infection (Simantini and Banerjee, 1995;
Beasley and Aaskov, 2001; Serafin and Aaskov, 2001).
149
It has been suggested that the restricted major histocompatibility complex (MHC)
background of inbred mice may affect the immune responses of these mice against
different DENV antigens (Roehrig et al., 1990). Indeed, preliminary data from our
laboratory suggests that the proportion of neutralising MAbs generated against DENV-3
in a MAb panel varies between different BALB strains of mice (unpublished data). It
also is probable that differences in the immunisation schedules and antigen preparations
used to prepare these MAbs may have influenced the immune responses. Given the
development of dengue vaccines based on single virus genotypes (i.e. a limited
repertoire of antigens), the influence of the MHC background of the host on the immune
response to DENV should be explored.

neutralisation.
The antigenic model for the DENV-4 E protein, derived by CBAs, predicted the spatial
relationships between different epitopes. A more accurate assignment of epitopes within
this model, specifically epitopes recognised by DENV-4 specific neutralising MAbs,
was determined employing the strategies previously outlined (Section 4.2).
The DENV-4 specific MAbs (18F5, F1G2 and 13H8) were assigned to structural
domains of the DENV-4 E protein using chimeric E proteins (Table 3.11). 13H8
recognised domains I and II whereas F1G2 and 18F5 recognised domain III. This data
confirmed the spatial relationships between these MAbs observed in CBAs (Table 3.5),
and suggested that the antigenic domain D4E1 corresponded to the structural domain I
and II of the DENV-4 E protein and that antigenic domain D4E2 corresponded to
structural domain III of the DENV-4 E protein. The analysis of DENV-4 variants, site
directed mutagenesis of the DENV-4 E protein and bacterial peptide display identified
epitopes recognised by the neutralising MAb F1G2 at amino acid residues E95 and E99-
E104 and MAb 18F5 at amino acid residue E329. No epitopes were identified using
these methods and the MAb 13H8. The epitopes for the MAbs 18F5 and F1G2 were
further analysed using a structural model of the DENV-4 E protein.
150
As the three dimensional (3D) structure of the DENV-4 E protein has not been
determined, a structural model of the DENV-4 E protein in pre-fusion conformation was
predicted using the Swiss-Model component of the Deepview Swiss PdbViewer
program (Guex and Peitsch, 1997) and the co-ordinates from the DENV-2 E protein in a
pre-fusion conformation derived by X-ray crystallography (2.75A resolution; pdb
file:1OAN; Modis et al., 2003).
A comparison of the structure of the DENV-2 and DENV-4 E proteins using the
Deepview Swiss PdbViewer program (Guex and Peitsch, 1997) suggested they were
similar and therefore, discussion of structural features of the DENV-4 E protein are
based on this assumption. The nomenclature used by Rey et al., 1995 to identify regions
of the TBEV E protein has been employed to identify regions of the DENV-4 E protein.
4.4.1Identification of an epitope involved in neutralisation by the DENV-4 specific

MAb F1G2.
The DENV-4 specific MAb F1G2 was initially assigned, based on the chimeric E
protein data (Table 3.11), to an epitope in domain III however, this result was not
confirmed using other epitope mapping methods. Site directed mutagenesis
demonstrated that an Asp-Ala change at residue E95 in domain II of the DENV-4 E
protein stopped binding of the MAb F1G2 (Table 3.11). This change at E95 was
initially identified in 5-fluorouracil (5FU) mutagenised DENV-4 populations that
escaped neutralisation by F1G2 (Table 3.7; Figure 3.8). In addition to the E95 change,
the DENV-4 n.e.m. also had amino acid changes at E156 (Pro-Ser), E157 (Asn-Ser) and
E402 (Phe-Leu) (Table 3.7). These changes did not prevent the binding of MAbs to the
DENV-4 E protein but may have contributed to the decreased infectivity of the DENV-4
n.e.m. in C6/36 cells (Table 3.7).
It was evident that 5FU mutagenesis of DENV-4 was a useful technique for increasing
the initial genetic variation within a virus population to facilitate the selection of virus
variants. This was based on the polymorphisms identified in codons in the E gene of
DENV-4 treated with 5FU that corresponded to the amino acid changes at E95, E156,
E157 and E402 (Figure 3.7).
151
However, the addition of neutralising MAbs to 5FU-treated DENV-4 during passage in
vitro in BHK cells did not play a role in the selection of the variant viruses because 5FU
treated virus passaged without MAb had the same genotype. This suggested that the
amino acid changes that occurred in the DENV-4 n.e.m. genotype may be the result of
virus adaptation to growth in mammalian cells. This would explain the reduced
infectivity of DENV-4 n.e.m.s in C6/36 cells, if BHK and C6/36 cells employ different
receptors for DENV-4.
It has been shown that the repeated passage of flaviviruses in mammalian cells selects
for amino acid changes that increase the net positive charge of the E protein, which
favours virus interaction with heparan sulphate receptors (Lee and Lobigs, 2000; Mandl
et al., 2001; Lee and Lobigs, 2002; Lee et al., 2004). Similarly in this study, the amino
acid changes in the DENV-4 n.e.m. increased the net positive charge of the E protein
which may have improved virus affinity for heparan sulphate receptors on BHK cells
and improved virus infectivity in BHK cells as opposed to C6/36 cells. Several amino
acid changes associated with changes in virus infectivity have been identified in regions
of the flavivirus E protein, which are spatially related to the E95, E156, E157 and E402
residues identified in the DENV-4 n.e.m. (Table 1.5). Further study of the effect of such
mutations on the infectivity of the DENV-4 n.e.m. is warranted.
Further evidence for the binding of MAb F1G2 to an epitope associated with residue
E95 was obtained using a bacterial peptide display library (Figure 3.10). MAb F1G2
reacted with peptides containing the conserved motif K/RWGG, which was homologous
with the RWGG motif within residues E99-E104 of the DENV-4 E protein primary
amino acid sequence (Figure 3.10). In addition to the sequence homology, the bacterial
peptides and the RWGG sequence of the DENV-4 E protein, recognised by F1G2, both
presumably formed loop-like structures that were conformationally constrained by a
single disulphide bridge. This implied that the peptide display library could effectively
present conformational epitopes. However, the inability to identify epitopes for MAbs
18F5 and 13H8 using this method suggested that conformation of the peptides is still
limited and probably more suited to the identification of linear epitopes.
152
Based on the data from the different epitope mapping strategies, F1G2 was assigned to
domain II and domain III.
The epitopes at E95 and E99-E104 associated with the binding of F1G2 are located on
the c β-sheet at the tip of Domain II in the DENV-4 E protein and are on or adjacent to
the cd-loop structure which bridges the c and d β-sheets and contains the flavivirus
conserved fusion peptide sequence (E98-E111) (Figure 4.2). This implied that the
binding of F1G2 to its epitope may neutralise DENV-4 infection by blocking fusion.
The analysis of amino acid residues adjacent to the E95 residue is important for
determining how F1G2 binds to its epitope. The alignment of the deduced amino acid
sequences of the E protein of the DENV-4 isolates used in this study and of other DENV
serotypes (Figure 4.3) indicated that the aspartate residue at E95, which was critical for
the binding of F1G2, was DENV-4 specific. In addition, the glutamine residue at E89
and the isoleucine residue at E91 were also DENV-4 serotype specific (Figure 4.3). The
glutamine residue at E88 and the tyrosine residue at E90 were found only in DENV-3
and DENV-4, and the arginine residues at E93 and E94 were found only in DENV-1 and
DENV-4.
The uniqueness of the amino acid sequence in this region of the DENV-4 E protein
supports the assignment of the epitope of the anti-DENV-4 specific MAb, F1G2, to this
region. In contrast, the RWGG sequence (E99-E104), which was also recognised by
MAb F1G2 (Figure 3.10), is within the fusion peptide sequence (E98-E111) conserved
in all flaviviruses, suggesting that this region does not have a role in the specificity of
F1G2.
Studies of antigen-antibody interactions have shown that highly exposed hydrophilic

residues and buried hydrophobic residues are both important in the binding of antibodies
(Geysen et al., 1985; Getzoff et al., 1987). The hydrophilic residues are necessary for
contact between antigen and antibody, whereas the hydrophobic residues are required
for maintaining protein conformation at the binding site (Fieser et al., 1987).
153
A.
DOMAIN III
C
E95 cd loop
(Asp)
N
DOMAIN II
DOMAIN I
B.
Figure 4.2 Location of the epitope involved in neutralisation by the DENV-4

specific MAb F1G2 at amino acid residue E95 on an (A) overhead and (B) side view
of the DENV-4 E protein in its pre-fusion conformation (model derived from DENV-
2 E protein model; 2.75A resolution; pdb file:1OAN; Modis et al., 2003). Each
domain of the E protein monomer is coloured; domain I is red, domain II is yellow
and domain III is blue. The location of the N and C termini is indicated in (A). The
cd-loop, which contains the fusion peptide and residues E99-E104 which are also
recognised by MAb F1G2, is depicted in (A). The substitution of the aspartate
residue at E95 (blue) in domain II, with an alanine residue prevented the binding of
the DENV-4 specific neutralising MAb F1G2 to the DENV-4 E protein.
154
85 105
DENV-4.Philippines.H241/1956 EQDQQYICRRDVVDRGWGNGC
DENV-4.H241 n.e.m. (MAb.F1G2) ..........A..........
DENV-4.Singapore.1674/1990 .....................
DENV-4.Singapore.8976/1995 ..........N..........
DENV-4.Singapore.4553/2001 .....................
DENV-4.Thailand.508/1999 ...........M.........
DENV-4.Thailand.520/1999 ...........M.........
DENV-4.Timor.083/2001 .....................
DENV-4.Timor.089/2001 .....................
DENV-4.Timor.099/2001 .....................
DENV-4.Vietnam.31500/2000 ...........M.........
DENV-4.Vietnam.38201/2001 ...........M.........
DENV-1.Hawaii/1944 ...ANFV...TF.........
DENV-2.New Guinea.NGC/1944 ...KRFV.KHSM.........
DENV-3.Philippines.H87/1956 ....N.V.KHTY.........
Figure 4.3 The alignment of amino acid residues of the E protein of different DENV-4
isolates and the prototype strains for each DENV serotype, associated with the E95
residue, involved in DENV-4 neutralisation by the MAb F1G2. The Asp residue at E95
and the RWGG motif at E99-E104 which are recognised by F1G2 are boxed. The
residues bolded are the amino acid changes that have been identified at residue E95 in
different DENV-4 and other DENV serotypes. Amino acid changes at residues adjacent
to E95, in this case only E96, have also been shown. The charged residues in this amino
acid sequence are shown in red print. The hydrophobic residues in this amino acid
sequence are shown in blue print.
155
It was suggested that the hydrophilic residues located at E88-E96 of the DENV-4 E
protein (indicated by red print in Figure 4.3), such as the Asp at E95, facilitate the initial
binding of the neutralising MAb F1G2. This was confirmed by the Asp to Ala change at
E95 in the DENV-4 n.e.m. that abrogated binding of the neutralising MAb F1G2. This
was a non conservative change that resulted in the loss of a negatively charged residue
and increased the hydrophobicity of the residue from -0.567 (Asp) to 0.022 (Ala) based
on the hydropathy index determined for each amino acid by Kyte and Doolittle (1982)
(Table 4.1). The hydrophobicity at residue E95 also increased when threonine or serine
residues representing the E95 residue in other DENV serotypes, which do not react with
MAb F1G2, were substituted for the Asp residue at E95 in the DENV-4 E protein In
contrast, the aspartate to asparagine change at E95 in DENV-4. 8976, which reacted
with F1G2, did not affect the hydrophobicity (Table 4.1)
It is proposed that the bulky non polar side chains of amino acids such as alanine, serine
and threonine promote hydrophobic interactions within the DENV-4 E protein, which
may have reduced the antibody binding capacity of the epitope. A comparison of the
different residues that occur at E95 in DENV-4, demonstrates that the hydrophobic
alanine in the DENV-4 n.e.m. is slightly less exposed on the surface of the E protein, in
comparison to the hydrophilic aspartate and asparagine residues which occur in DENV-
4. H241 and DENV-4. 8976 (Figure 4.4).
In contrast, the hydrophobic residues, such as the phenylalanine (Phe) at E90 and the
tryptophan (Trp) at E101 (blue print in Figure 4.3) may be important in maintaining the
conformation of the F1G2 epitope. This is supported by the structural models of the
TBEV and DENV E protein, which have shown that the Trp at E101 is important in the
stabilisation of the E protein dimer structure (Rey et al., 1995; Modis et al., 2003). The
role of these hydrophobic residues may be particularly important, if F1G2 recognises an
interdimeric epitope, which is the next topic of discussion. In addition the change at E95
may have abolished F1G2 binding by altering the conformation of adjacent hydrophobic
residues such as Trp at E101 and subsequently destabilising dimeric interactions of the
DENV-4 E protein.
156
Table 4.1 Characteristics of amino acids at residue E95 in different DENV-4
and other DENV serotypes.
F1G2 Hydrophobicity
a
Virus reactivity aa at E95 value
DENV-4.H241 + Aspartate (D) -0.567
DENV-4.nem
- Alanine (A) 0.022
(MAb F1G2)
DENV-4.8976 + Asparagine (N) -0.567
DENV-1.Hawaii - Threonine (T) -0.256
DENV-2.NGC - Serine (S) -0.267
DENV-3.H87 - Threonine (T) -0.7

a
According to method of Kyte and Doolittle, (1982)
aa. amino acid
157
cd loop
β-sheets
E95 (D)
c d
DENV-4.H241: E95(D) F1G2 +
E95 (N)
DENV-4.8976: E95(N) F1G2 +
E95 (A)
DENV-4. n.e.m. (F1G2): E95(A) F1G2 -
Figure 4.4 The amino acid substitutions occurring at residue E95 of the E protein in
different DENV-4 and the affects on the surface exposure of the residue and potential
reactivity with the MAb F1G2. The MAb F1G2 recognises an aspartate (D) or
asparagine (N) residue at E95 but does not recognise an alanine (A) residue at E95.
158
In addition to the domain II epitopes recognised by F1G2 (E95 and E99-E104), the
chimeric E protein data indicated that the MAb F1G2 also recognised domain III (Table
3.11), however no specific epitopes were identified. The Swiss Pdb viewer program was
used to identify the nearest neighbouring residues of the aspartate residue at E95 and the
RWGG residues within E99-E104 on the structural model of the DENV-4 E protein
dimer, which was derived from the DENV-2 E protein dimer model (2.75A resolution;
pdb file:1OAN; Modis et al., 2003). The RWGG residues were within a molecular
distance of 4 Å from the residues E310, E312, E313, E321-E323 located on domain III
of the opposite E protein monomer (Figure 4.5). The interactions between these residues
suggest that F1G2 recognises an interdimeric epitope consisting of domain II and
domain III regions.
The interactions that occur in the structural models of the DENV and TBEV E protein
dimers support the case for an interdimeric epitope consisting of domain II and domain
III. The E protein dimer is stabilised by contact between the cd loop of domain II of one
E subunit and the domain I and III interface of the other, where the tryptophan residue at
residue 101 fits into the hydrophobic crevice, where domain I and III come together
(Rey et al., 1995; Modis et al., 2003). This interaction is stabilised by the sugar
molecule attached to domain I, which lies over the groove that receives the cd loop (Rey
et al., 1995).
The role of residues in domain III of the DENV-4 E protein in the binding of F1G2 must
be identified to confirm the presence of an interdimeric epitope. However, from other
studies, there is evidence of discontinuous epitopes in the DENV E protein similar to the
proposed F1G2 epitope. Peptide mapping studies determined that the DENV-2 specific
MAb 1B7 recognised a discontinuous epitope, consisting of domain II (E50-E57, E127-
E134) and domain III regions (E349-E356) (Aaskov et al., 1989). The analysis of
DENV-2 antigenic variants using the chimpanzee Fab antibody 1A5 also identified a
discontinuous epitope consisting of domain II (E106) and domain III (E317) regions
(Goncalvez et al., 2004).
159
A
DOMAIN II
DOMAIN III E311

E104
E310 E313
E99
RWGG
E323 E101
E321
E322 E102
E366
DOMAIN I
Figure 4.5 (A) The potential interdimeric epitope for the MAb F1G2, formed by
interactions between domain II and domain III residues located on opposite subunits of
the DENV-4 E protein dimer. (B) The RWGG sequence within residues E99-E104 of
domain II which was recognised by F1G2 using a peptide display library, is a molecular
distance of 4 Å from residues E310, E311, E313, E321-E323 and E366 on domain III of
the opposite E protein subunit.
160
Goncalvez et al. (2004) proposed that E106 from one E monomer and E317 on the
opposite E monomer were spatially close enough to form an epitope. Flavivirus group
reactive MAbs, such as 4G2, also have been mapped to discontinuous regions consisting
of domain II and domain III epitopes (Megret et al., 1992; Falconar, 1999; Crill and
Chang, 2004). Crill and Chang, (2004) used site directed mutagenesis to determine that
the flavivirus cross-reactive MAb 4G2, like F1G2, recognises residues associated with
the fusion peptide (E104, E106, and E107).
MAb 4G2 also recognised the residue E231 which was a molecular distance of 50 Å
from the fusion peptide residues in a single E protein dimer, which was considered too
distant to allow the binding of a MAb (Crill and Chang, 2004). In contrast, the distance
between E231 in one dimer to the fusion peptide residues in a neighbouring dimer on the
viral E protein lattice surface was only 25 Å, which can be spanned by a single IgG
molecule. Crill and Chang, (2004) thus proposed an epitope that spans between opposite
E dimers.
Overall, the nature of the discontinuous epitopes defined by other studies and the
interactions between domains II and III that occur in the native E protein dimer, suggest
that the interdimeric epitope recognised by F1G2 (Figure 4.5) is possible.
The binding of F1G2 to an epitope consisting of domains II and III also suggests several
mechanisms by which F1G2 neutralises DENV-4 infection. The binding of MAb F1G2
to the E95 epitope and to the RWGG sequence may block direct interaction of the fusion
peptide with host cell membranes, which has been observed in WNV studies of
neutralisation (Gollins and Porterfield, 1984). Alternatively F1G2 binding to domain II
and domain III regions of an interdimeric epitope, may stabilise dimeric interactions and
prevent the disruption of the dimer, that according to the studies of TBEV (Stiasny et al.,
2001) is necessary for fusion to occur.
161
However, the binding of F1G2 to domain II (E95, E99-E104) cannot directly interfere
with the fusion processes that occur once the virus has entered the cell and the low pH
environment of the endosome, because F1G2 fails to bind to DENV-4 treated at pH 6.0
(Table 3.3). Therefore the binding of F1G2 to virus would presumably occur prior to
exposure of the virus to low pH during entry into the cell.
Several other studies have also reported the inability of MAbs to bind to domain II
epitopes following the low pH induced conformational changes of the E protein
necessary for virus mediated fusion (Guirakhoo et al., 1989; Holzmann et al., 1995).
The loss of MAb binding to flaviviruses treated at low pH is supported by studies on the
DENV-2 and TBEV E protein post-fusion crystal structures which indicate that at a
lower pH the E protein changes from a horizontal, antiparallel dimeric conformation to a
vertical trimer where the subunits have a parallel organisation (Bressanelli et al., 2004).
The structure of the three domains in the E protein is maintained following trimerisation
but their relative orientation changes (Bressanelli et al., 2004; Modis et al., 2004).
It was suggested that the binding of F1G2 to domain III, observed in the chimeric
DENV E proteins, may be necessary for the neutralisation of DENV-4. The specific
residues on domain III involved in F1G2 binding were not identified in this study
however CBA results suggest that F1G2 is spatially related to the E329 epitope
recognised by 18F5 (Table 3.5). Therefore, the proposed neutralisation mechanisms
resulting from 18F5 binding to domain III (E329), specifically the prevention of virus
attachment to host cell receptors, which will be discussed in Section 4.4.2, may also
apply to F1G2 mediated neutralisation.
162
4.4.2 Identification of an epitope involved in neutralisation by the DENV-4 specific
MAb 18F5.
The DENV-4 specific neutralising MAb 18F5 was assigned to an epitope in domain III
of the DENV-4 E protein, based on the chimeric E protein data (Table 3.11). This was
confirmed by site directed mutagenesis which demonstrated that an Ala-Thr change at
residue E329 in domain III of the DENV-4 E protein stopped binding of the MAb 18F5
(Table 3.11).
The Ala-Thr change at E329 which prevented MAb binding in IFA and capture assays
and allowed DENV-4 escape from neutralisation by the MAb 18F5 was identified in
naturally occurring DENV-4 n.e.m.s isolated from Thailand (DENV-4.508/1999,
DENV-4.520/1999) and Vietnam (DENV-4.31500/2000, DENV-4.38201/2001) (Table
3.10). The other changes observed in these DENV-4 strains at E96 (V-M) and E203 (K-
T) did not affect the binding of 18F5 to the DENV-4 E protein (Table 3.11). In addition,
a unique change at E160 (V-E) may have caused the reduced virus titre of DENV-4
31500 in BHK and Vero cells (Table 3.10).
The isolates resistant to 18F5 neutralisation grouped within genotype I of DENV-4

(Figure 4.6). The Ala-Thr change at E329 has only been previously identified in the E
protein sequences of DENV-4.215/1975, a sylvatic strain from Malaysia and in DENV-
4.D84-024/1984, an isolate from Thailand (Lanciotti et al., 1997; AbuBakar et al.,
2002). This suggested that screening DENV populations for naturally occurring n.e.m.s
is an effective means of identifying epitopes involved in neutralisation and a good
alternative strategy if traditional n.e.m. selection methods are unsuccessful.
The E329 epitope is located on the outer lateral surface of the DENV-4 E protein on the
loop that connects the B and C sheets (BC loop) of the ABED β-sheets of domain III
(Figure 4.7). The alanine residue at E329 is DENV-4 specific and adjacent to a cluster of
positively charged residues (Figure 4.8). Charged residues on the lateral surface of
domain III have been identified as sites involved in virus attachment to cell receptors
(Chen et al., 1997) and therefore are potential sites for neutralising MAbs.
163
DEN-4.Malaysia.215/1975 (sylvatic)
DENV-4.Singapore.4553/2001
DENV-4.Thailand.D84-024/1984 (I)
DENV-4.Vietnam.38201/2001
DENV-4.Vietnam.31500/2000 18F5
n.e.m.s
DENV-4.Thailand.520/1999
DENV-4.Thailand.508/1999
DENV-4.Philippines.12123/1984 (I)
DENV-4.Philippines.H-241/1956 (I)
DENV-4.Malaysia.22713/2001 (IIA)
DENV-4.Timor.099/2001
DENV-4.Indonesia.30153/1973 (IIA)
DENV-4.New Caledonia.5489/1984
DENV-4.El Salvador.BC6494/1994
0.01
Figure 4.6 Phylogenetic analysis by maximum parsimony of the E protein of DENV-4.

The DENV-4 isolates from Vietnam and Thailand, depicted in red, were not neutralised
by the DENV-4 specific MAb 18F5. The DENV-4 isolates from Singapore and Timor
as well as the prototype strain DENV-4.Philippines.H-241/1956 were neutralised by
18F5. The DENV-4 isolates from El Salvador, Malaysia and New Caledonia were not
used in this study. Genotypes I and IIA are indicated in brackets.
164
A. DOMAIN III
C
E329
(Ala)
N
DOMAIN II
DOMAIN I
B.
Figure 4.7 The location of the epitope recognised by the DENV-4 specific MAb 18F5
at residue E329, coloured pink, on the (A) overhead and (B) side views of the DENV-4
E protein (pdb file 1OAN). Each domain of the E protein is represented as a different
colour; domain I is red, domain II is yellow and domain III is blue. The N and C termini
of the E protein are depicted in (A). The substitution of the alanine residue at E329
(pink) in domain III, with a threonine residue prevents binding of the MAb 18F5.
165
320 340
DENV-4.Philippines.H241/1956 TVVKVKYEGAGAPCKVPIEIR
DENV-4.Singapore.1674/1990 .....................
DENV-4.Singapore.8976/1995 .....................
DENV-4.Singapore.4553/2001 .....................
DENV-4.Thailand.508/1999 .........T...........
DENV-4.Thailand.520/1999 .........T...........
DENV-4.Timor.083/2001 .....................
DENV-4.Timor.089/2001 .....................
DENV-4.Timor.099/2001 .....................
DENV-4.Vietnam.31500/2000 .........T...........
DENV-4.Vietnam.38201/2001 .........T...........
DENV-1.Hawaii/1944 VL.Q.....TD....I.FSTQ
DENV-2.New Guinea.NGC/1944 I.IR.Q...D.S...I.F..M
DENV-3.Philippines.H87/1956 ILI..E.K.ED....I.FSTE
Figure 4.8 The alignment of amino acid residues of the E protein of different DENV-4
isolates and the prototype strains for each DENV serotype, associated with the E329
residue, involved in DENV-4 neutralisation by the MAb 18F5. The charged residues
are shown in red print.
166
Several epitopes recognised by neutralising MAbs have been identified on domain III of
the flavivirus E protein by determining the amino acid changes that occur in n.e.m.s
(Table 4.2). These changes have been grouped into four clusters (A, B, C and D) based
on their relative location on domain III of the DENV-4 E protein (Figure 4.9). Cluster A
consists of the region E306-E311 (E303-E307 in DENV-4), cluster B consists of the
region E329-E333 (E326-E329 in DENV-4), cluster C is E367 and E368 (E362 and
E363 in DENV-4) and cluster D is E384 and E389 (E378 and E387 in DENV-4).
The epitope identified at E329 in the DENV-4 E protein in this study was grouped on
the structural model of DENV-4 with epitopes in cluster B, identified in the JEV E
protein at E331 and E333 (Cecilia and Gould, 1991; Wu et al., 1997) and the WNV E
protein at E330 and E332 (Beasley and Barrett, 2002; Nybakken et al., 2005). The E329
epitope also was closely associated with residues E306-E308 of cluster A.
The importance of domain III epitopes in antibody mediated neutralisation, specifically

those grouped into cluster B in Figure 4.9, was recently examined by a study
determining the crystal structure of a Fab fragment of a neutralising MAb in complex
with domain III of WNV at a 2.5 Å resolution (Nybakken et al., 2005). It was shown
that the Fab engages a total of 16 residues in four discontinuous segments of the WNV
domain III. This included the amino-terminal region (residues E302-309) and three
loops that connect beta strands of domain III: BC loop (E330-E333), DE loop (E365-
E368) and the FG loop (E389-E391).
Similar studies using solution structures of domain III for JEV and WNV, determined by
nuclear magnetic resonance (NMR), have also shown that neutralising MAbs bind to a
similar epitope in domain III (Wu et al., 2003; Volk et al., 2004).
Yeast surface display epitope mapping determined that the four residues E306, E307,
E330 and E332 were critical for binding of the Fab to domain III (Nybakken et al.,
2005). Amino acid changes at residues E306 (Ser-Leu), E307 (Lys-Glu) and E330 (Thr-
Iso) and E332 (Thr-Met) reduced the binding of the Fab to domain III of WNV.
167
Table 4.2 Neutralisation epitope clusters in Domain III of the Flavivirus E protein.
Epitope Relative position in
Cluster Virus Amino acid change DENV-4 E protein Reference
JEV E306 (Glu-Gly) E304 Wu et al., 1997
WNV E306 (Ser-Leu) E303 Nybakken et al.,
E307 (Lys-Glu) E304 2005
WNV E307 (Lys-Arg/Asn) E304 Beasley and
Barrett, 2002
A
DENV-2 E307 (Lys-Glu) E307 Lin et al., 1994
LIV E308 (Asp-Asn) E303 Jiang et al., 1993
E310 (Ser-Pro) E306 Gao et al., 1994
E311 (Lys-Asn) E307
DENV-2 E311 (Glu-Gly) E307 Lok et al., 2001
DENV-4 E329 (Ala-Thr) E329 this study

WNV E330 (Thr-Iso) E326 Beasley and
Barrett, 2002
WNV E330 (Thr-Iso) E326 Nybakken et al.,
B
E332 (Thr-Met) E328 2005
JEV E331 (Ser-Arg) E326 Wu et al., 1997
JEV E333 (Gly-Asp) E328 Cecilia and
Gould, 1991
JEV E367 (Asn-Asp) E362 Morita et al.,

2001
C
TBEV E368 (Gly-Arg) E363 Holzmann et al.,
1997
TBEV E384 (Tyr-His) E378 Holzmann et al.,

1997
D
DENV-3 E386 (Lys-Asn) E387 Serafin and
Aaskov, 2001
TBEV E389 (Ser-Arg) E387 Mandl et al., 1989
168
E387 E307
E306
E378
E304 E328 E326

E362
E303 E363
E329
Figure 4.9 Relative position of DENV and Flavivirus neutralisation epitopes on the
overhead view of domain III of the DENV-4 E protein structural model. The coloured
residues indicate the different neutralisation epitope clusters. Yellow is cluster A, Red is
cluster B, Green is cluster C and grey is cluster D. The pink molecule is the E329
epitope recognised by the MAb 18F5 which was identified in the DENV-4 E protein in
this study.
169
The decreased binding associated with changes at E306, E307 and E332 was most likely
due to a loss of hydrogen bonding potential. Whereas the residue at E330 is important
for stabilising the N-terminal strand conformation of domain III and provides numerous
van der Waals contacts with the Fab (Nybakken et al., 2005).
It is proposed that the Ala-Thr change at residue E329 in the DENV-4 E protein
disrupted binding of the DENV-4 specific MAb 18F5 to domain III in a similar fashion
to the changes in the WNV determined by Nybakken et al., (2005).
It is proposed that the DENV-4 specific MAb 18F5 may neutralise infection of cells by
DENV-4 by attaching to the lateral surface of domain III and blocking virus attachment
to host cells. Indeed several studies have demonstrated that the binding of neutralising
MAbs to epitopes on domain III of the E protein, prevent virus attachment to host cell
receptors (Roehrig et al., 1998; Crill and Roehrig, 2001; Nybakken et al., 2005). In
addition, the MAb 18F5 inhibits DENV-4 attachment to erythrocytes in the HI assays
(Table 3.1).
The involvement of domain III in mediating the binding of flaviviruses to host cell
receptors has been suggested by several lines of evidence. This includes the
immunoglobulin like fold of domain III which is characteristic of proteins with binding
function, the high proportion of charged surface residues located on the lateral surface,
the presence of motifs specific for cell receptors and the occurrence of mutations that
influence virulence and neutralisation (Mandl et al., 2000).
Several studies have confirmed the role of domain III of the flavivirus E protein in the
attachment of virus to host cells. Heparin sulphate was identified as the first putative
receptor for DENV-2 on CHO cells, and the positively charged amino acid residues at
E294-E310 and E386-E411 in domain III were the likely HS binding sites on the virion
(Chen et al., 1997). Hung et al. (2004) also showed that the region E380-E389 of domain
III of DENV-2 was critical for the attachment of DENV-2 to C6/36 cells.
170
Bhardwaj et al. (2001) demonstrated the binding of a recombinant fusion protein of
domain III of Langat virus (LGTV) to Vero cells, and that the binding of Domain III,
inhibited LGTV infection of Vero cells. Chu et al. (2005) reported that a recombinant
WNV domain III protein inhibited attachment of DENV-2 to Vero cells and completely
blocked attachment of DENV-2 to C6/36 cells.
The neutralisation epitope identified at E329 in DENV-4 was located in close proximity
to the regions of domain III that are proposed to interact with HS and C6/36 receptors
(Figure 4.10). As previously discussed, the E320-E340 region of the DENV-4 E protein
had a high degree of charged residues, which is a common feature of the lateral surface
of domain III, suggesting that strong ionic interactions between virus and potential
docking partners is plausible. It was therefore possible that binding of an antibody to
this region could interfere with cell attachment.
These highly charged residues are also important for stabilisation of the proteins tertiary
structure, to enable adequate interaction with receptors. This was proven in a study by
Mandl et al. (2000) where the mutation of residues E309-E311, which are involved in
salt bridge formation on the lateral surface of domain III, was shown to destabilise
protein structure, which inturn resulted in TBEV with reduced neuroinvasiveness in
mice.
Several studies have also identified amino acid changes in domain III that affect
virulence (Table 4.3). The residues affecting virulence were grouped into four regions
of domain III of the DENV-4 E protein, which overlap with the residues involved in
neutralisation (Figure 4.10). Based on the location of the neutralisation and virulence
epitopes, the amino acid regions E303-E311, E319-E333, E365-E368 and E383-E390
were considered the primary sites of domain III likely to be involved in flavivirus
infection. Overall, the E329 epitope identified in the DENV-4 E protein resides in close
proximity to the functional epitopes and cell binding domains previously discussed that
lie on the lateral and exposed surface of domain III. This suggested that MAb binding to
this region potentially blocks sites involved in virus attachment to cell receptors.
171
E329 E329
Neutralisation Virulence
epitopes Determinants
E329 E329
HS binding sites Putative C6/36 binding site

(Lateral loop region)
Figure 4.10 The relative position of functional epitopes identified in DENV or

flaviviruses on the overhead view of Domain III of the DENV-4 E protein structural
model. The amino acid residues involved in virus neutralisation and virulence were
grouped into four clusters, based on their location on the E protein primary sequence.
Each cluster was differentiated by colour: cluster A: yellow, cluster B: red, cluster C:
green, cluster D: grey. The orange molecules represent the positively charged residues
in domain III that are proposed to represent heparin sulphate binding sites (Chen et al.,
1997). The blue molecules represent a region that is important in DENV-2 binding to
mosquito cells (Hung et al., 2004).
172
Table 4.3 Virulence determinants in Domain III of the Flavivirus E protein
Relative position
Virulence in DENV-4 E
Cluster Virus Amino acid change protein Reference
JEV E306 (Gly-Glu) E304 Ni and Barrett, 1998
LGTV E308 (Asp-Ala) E303 Campbell and Pletnev,
2000
TBEV E308-E311 several changes E303-E306 Mandl et al., 2000
A
WNV E307 (Lys-Glu) E304 Chambers et al., 1998
YFV E303 (Gln-Lys) E305 Jennings et al., 1994
YFV E305 (Val/Ser-Phe) E307 Schlesinger et al., 1996;
Ryman et al., 1998
DENV-4 E329 (Ala-Thr) E329 this study

LGTV E331 (Phe-Ser) E326 Pletnev and Men, 1998
TBEV E319 (Ile-Thr) E313 Labuda et al., 1994
TBEV E331 (Thr-Ser) E326 Wallner et al., 1996
B
YFV E326 (Lys-Gly) E329 Chambers and Nickells,
2001; Nickells and
Chambers, 2003
YFV E325 (Ser-Leu) E328 Ryman et al., 1997
DENV-1 E365 (Val-Iso) E364 Duarte dos Santos et al.,

2000
C
JEV E364 (Ser-Phe) E361 Hasegawa et al., 1992
E367 (Asn-Iso) E364
DENV-2 E390 (His-Asn) E390 Sanchez and Ruiz, 1996

DENV-2 E383-E385 E383-E385 Hiramatsu et al., 1996
several changes
LGTV E389 (Asn-Asp) E386 Pletnev and Men, 1998;
Campbell and Pletnev,
D
2000
MVEV E390 E382 Lobigs et al., 1990; Lee
several changes and Lobigs, 2000;
Hurrelbrink and
McMinn, 2001
173
4.4.3 Domain I and II epitopes recognised by the DENV-4 specific neutralising
MAbs 13H8 and 1H10.
The epitopes recognised by the neutralising MAbs 13H8 and 1H10 could not be
identified. However, the epitopes were located in regions of domains I and II that were
still accessible following low pH-induced conformational changes to the E protein
(Table 3.3; Table 3.11). This suggested that the MAbs 13H8 and 1H10 may neutralise
DENV-4 infection by blocking virus fusion, which occurs at low pH conditions.
Previous studies with DENV-2 and TBEV identified acid-resistant epitopes within
domains I and II of the flavivirus E protein and Roehrig et al. 1990 reported that the
amino acid residues E58-E121 and E225-249 were more accessible following low pH-
treatment of DENV-2. The conserved internal fusion peptide (E98-E110) in flaviviruses
was included in the low pH accessible regions identified (Roehrig et al., 1990).
Holzmann et al. (1993) has also reported that MAbs that recognised amino acid residues
E1-E22 and E221-240 were more reactive with low pH treated TBEV than with native
virions.
These acid-resistant epitopes represented potential binding sites for the MAbs 13H8 and
1H10. Further analysis of similar regions in the DENV-4 E protein is necessary to
identify the amino acid residues involved in the binding of MAbs 13H8 and 1H10.
The binding of MAb 13H8 to domain I or II of DENV-4 also enhanced the binding of
MAbs to domain III of the DENV-4 E protein (Table 3.5). The enhancement of binding
of one MAb by prior combination of the virion with a different MAb has been reported
previously for MAbs against DENV-2 (Henchal et al., 1985) and TBEV (Heinz et al.,
1983). It was suggested that the enhancement of virus capture by a MAb results when
the binding of a second MAb induces a change in protein conformation (Henchal et al.,
1985).
174
4.4.4 DENV and Flavivirus group-reactive epitopes
The epitopes recognised by the DENV and Flavivirus group-reactive MAbs grouped in
domain D4E2 of the antigenic model were not identified. The competition of these
MAbs with the MAb 18F5, which was mapped to residue E329 on domain III of the
DENV-4 E protein, suggested that these MAbs also recognised epitopes in domain III
epitopes. The Flavivirus group-reactive MAbs 4G2, 6B6C1 and F2D1 neutralised
DENV-4 to similar levels as the type-specific MAb 18F5, but more efficiently than the
DENV group-reactive MAbs. The lower level neutralisation displayed by the DENV
group-reactive MAbs was due neither to differences in MAb concentration (Table 3.1)
nor to differences in MAb avidity (Table 3.4; Figure 3.5).
From these results, it was proposed that the Flavivirus group reactive MAbs bind
domain III epitopes directly involved in virus function, whereas the DENV group-
reactive MAbs bind to epitopes adjacent to a functional epitope. The identification of
specific epitopes recognised by the cross-reactive MAbs is required to confirm this
theory.
Failure to identify the epitopes recognised by the cross-reactive MAbs adds to the
confusion surrounding the location of epitopes recognised by cross-reactive MAbs on
the E protein of DENV. Falconar (1999) and Serafin and Aaskov (2001) identified a
putative epitope for the anti-flavivirus MAb 4G2 at, or around, E275 of the DENV-2 E
protein using peptide mapping and the selection of n.e.m.s respectively.
However Crill and Chang (2004) were able to abrogate binding of MAb 4G2 by
changing residues E102, E104, E106 and E231 of the DENV-2 E protein by site directed
mutagenesis. A second flavivirus-reactive MAb 6B6C1, which reacts with an epitope at
or near that recognised by MAb 4G2, recognises an epitope in the regions E1-E128 and
E158-E300 of the DENV-2 E protein. This is not incompatible with these epitopes
being at or near E275 (Falconar, 1999; Serafin and Aaskov, 2001).
175
4.5 Proposed neutralisation mechanisms used by DENV-4 specific MAbs.
The success of a chimeric DENV E protein as a future vaccine requires an improved
understanding of the mechanisms involved in antibody mediated neutralisation.
According to Dimmock (1993) antibodies neutralise viruses by either blocking virus
attachment to host cell receptors, blocking virus entry of host cells (fusion or receptor
mediated endocytosis) or blocking virus uncoating within the host cell. As previously
outlined in Section 4.4, it was proposed that the DENV-4 specific MAb 18F5, which
recognises residue E329 in domain III epitope, blocks the attachment of virus to host cell
receptors. A similar neutralisation mechanism was proposed for the MAb F1G2 which
recognised domain II and III epitopes, based on close spatial association with MAb 18F5
defined by CBAs (Table 3.5). In contrast, it was proposed that the MAb 13H8, which
recognised acid resistant epitopes in domains I and II, blocked virus fusion. In addition,
the MAbs F1G2 and 18F5 blocked DENV-4 attachment to erythrocytes in HI assays,
whereas MAb 13H8 did not.
The proposed difference in neutralisation mechanisms used by the MAbs was confirmed
by the virus overlay protein binding assay (VOPBA; Section 3.3.6). This assay
evaluated the attachment of whole DENV-4 and DENV-4-MAb complexes to proteins
derived from uninfected mosquito cells (C6/36 cells). C6/36 proteins were adopted in
the VOPBA, because DENV readily infects C6/36 cells, and surface proteins have been
previously identified on C6/36 cells that bind DENV-4 (Salas-Benito and del Angel,
1997). In addition, the attachment of virus-Ab complexes to Fc receptors, which occurs
on macrophages during antibody dependent enhancement (ADE) of DENV infection,
does not occur in C6/36 cells.
It was evident from the VOPBA that the binding of MAb 18F5 to a domain III epitope
(E329) on the DENV-4 E protein blocked virus attachment to a 40 kDa C6/36 cell
protein and the binding of MAb F1G2 to a proposed domain II-III epitope of the DENV-
4 E protein also blocked attachment to the C6/36 cell protein (Figure 3.11). In contrast,
the neutralising MAb 13H8 which recognises domains I and II of the DENV-4 E protein
did not prevent DENV-4 attachment to the C6/36 cell protein (Figure 3.11).
176
Previous studies by Salas-Benito and del Angel (1997) also identified a 40 kDa protein
in C6/36 cells recognised by DENV-4 using a VOPBA, however this is the first study to
demonstrate antibody mediated inhibition of DENV-4 binding to cell proteins. This was
preliminary evidence that neutralising MAbs recognising different structural domains of
the DENV-4 E protein utilise different mechanisms to prevent virus infection. It was
apparent that recognition of domain III by MAbs is important for blocking DENV-4
binding to host cell proteins.
This suggested that chimeric DENV E proteins incorporating different domains of the E
protein would be more effective against different stages of the virus life cycle; as
opposed to a domain III based vaccine, which would only be effective at blocking virus
attachment to host cells. To confirm the neutralisation mechanisms proposed for the
DENV-4 specific MAbs used in this study, and the nature of the epitopes involved in
neutralisation, a structural analysis of the MAb-DENV-4 complexes is required.
4.6 Epitopes involved in the neutralisation of DENV

Overall, the epitope mapping methods used in this study determined that amino acid
residues in domain II (E95) and domain III (E329) of the DENV-4 E protein are
necessary for the binding of DENV-4 specific neutralising MAbs and therefore are
important in the neutralisation of DENV-4 infection. Based on the n.e.m. data for the
other DENV serotypes, amino acid residues involved in neutralisation were distributed
in each domain of the DENV E protein, but were more predominate in domain III
(Figure 4.11).
Epitopes involved in DENV neutralisation were identified using n.e.m.s in domains I

(E293) and II (E279) of the DENV-1 E protein (Beasley and Aaskov, 2001), domain II
(E69) and domain III (E307, E311) of the DENV-2 E protein (Lin et al., 1994; Lok et
al., 2001) and domain III (E386) of the DENV-3 E protein (Serafin and Aaskov, 2001)
(Figure 4.11).
177
The knowledge of amino acid residues involved in neutralisation (Figure 4.11) and
observations from our laboratory (Bielefeldt-Ohmann et al., 1997; unpublished; this
study, Table 3.11) that chimeric DENV E proteins composed of domains I and II from
one DENV serotype and domain III from a second serotype fold into functional proteins
and are immunogenic, suggests that it might be possible to elicit a protective immune
response to all four DENV serotypes using two vaccines composed of domain I and II /
domain III chimeric E proteins. Indeed a recent study by Apt et al. (2006) has
confirmed that a single chimeric DENV E protein generated using DNA shuffling
techniques was able to induce a tetravalent neutralising antibody response against all
four DENV serotypes in mice.
178
(DENV-2) E311
(DENV-3) E307 (DENV-2)
E386 C
(DENV-4)
(DENV-1) E95
E329 N E279
(DENV-4)
E69
(DENV-2)
E293
(DENV-1)
Figure 4.11 Location of amino acid residues involved in the neutralisation of DENV
on the structural model of the DENV-4 E protein. DENV-4 residues identified in this
study are colored pink, DENV-1 residues are colored aqua, DENV-2 residues are
colored green and the DENV-3 residue is colored red. The ribbon backbone of the
structural model is colored to indicate the three domains of the DENV-4 E protein
monomer. Domain I is red, domain II is yellow and domain III is blue. The N and C
termini are indicated on the model.
179
5 Conclusion
There were several outcomes resulting from the research performed.
• In contrast to previous studies with DENV-1 and DENV-3 (Beasley and Aaskov,
2001; Serafin and Aaskov, 2001) the majority of anti-DENV-4 MAbs neutralised
infection by this virus in vitro. The neutralising MAbs were grouped into two
distinct antigenic domains using CBAs. Preliminary evidence suggested these MAbs
recognised similar, or spatially related, epitopes to those seen by antibodies from
dengue patients. Therefore, the epitopes identified in this study are useful markers
for the design of DENV vaccines.
• The traditional epitope mapping method, which involved the selection of n.e.m.
viruses was unsuccessful, so several alternative strategies were adopted. Where
large panels of viruses are available, identification of “natural” escape mutants
followed by site directed mutagenesis of an E protein construct may be a useful
approach to the identification of epitopes recognised by either neutralising or non-
neutralising MAbs. In addition, the mutagenesis of virus populations with agents
like 5-fluorouracil to produce greater genetic diversity may be a useful approach for
generating neutralisation escape mutant virus populations and warrants further
evaluation.
• The MAb screening of chimeric DENV E proteins also proved a useful strategy for
the course mapping of structural domains of the DENV-4 E protein involved in
neutralisation. The peptide display approach confirmed the location of an epitope
which was identified using other methods. Unfortunately, only one neutralising
epitope was identified using this technique, and this may have been due to the
conformation of the peptides not properly reflecting the conformation of neutralising
epitopes in the virus. Despite the inefficiency of the technique, peptide display may
be more suited to linear epitope identification.
180
• Epitopes recognised by DENV-4 specific neutralising MAbs were assigned to
domains I, II and to domain III of the DENV-4 E protein. Specific epitopes involved
in the binding of neutralising MAbs to the DENV-4 E protein were assigned to
residues E95 in domain II and E329 in domain III. The binding of a MAb to an
interdimeric epitope consisting of domains II and III was also proposed.
• Preliminary evidence suggests that the binding of neutralising MAbs to domain III of
the DENV-4 E protein prevents DENV-4 attachment to a host cell protein in C6/36
cells. In contrast, the binding of a neutralising MAb to domain I and II did not
prevent attachment. MAbs binding to domain III epitopes also inhibited the binding
of DENV-4 to erythrocytes, whereas MAbs against domain I and II did not.
• The distribution of epitopes involved in DENV neutralisation on several domains of

the DENV E protein suggested that chimeric E proteins consisting of domains I and
II of one DENV serotype and domain III of a different DENV serotype would be
suitable vaccine candidates.
181
6 APPENDIX A: SOLUTIONS
6.1 Solutions used for serological assays
6.1.1 25 x PBS (Phosphate buffered saline) pH 7.4

• The following components were dissolved in 700 ml distilled water in a 1 litre glass
beaker using a stirring bar and magnetic stirrer.
• 200g NaCl (BDH Chemicals, U.K).
• 28.75g Na2HPO4. (BDH Chemicals, U.K)
• 5g KH2PO4. (BDH Chemicals, U.K)
• 5g KCl. (BDH, Chemicals, U.K)
• The solution was made up to 900 ml with distilled water and adjusted to pH 7.4
using hydrochloric acid (HCl; Univar, U.S.A).
• The solution was made up to a final volume of 1 litre with distilled water (Milli-Q
water), transferred to a 1 litre schott bottle and was autoclaved at 121°C for 20
minutes.
6.1.2 1 x PBS pH 7.4

• Forty millilitres of 25 x PBS was added to a 2 litre schott bottle and was made up to
2 litres with distilled water.
6.1.3 Borate saline pH 9.0

• The following components were added to a 1 litre glass beaker
• 100 ml 0.5 M Boric acid
• 24 ml 1 M NaOH
• 80 ml 1.5 M NaCl
• The solution was made up to 900 ml with distilled water, mixed with a stir bar and
magnetic stirrer and was then adjusted to pH 9.0 with 5 M NaOH.
• The solution was made up to a final volume of 1 litre with distilled water and
transferred to a 1 litre schott bottle.
182
6.1.4 3 M Hydrochloric acid (HCl)
• 800 ml of distilled water was added to a glass beaker and 200ml of concentrated HCl
(15 M; Univar, U.S.A) was carefully added and mixed with a stir bar and magnetic
stirrer.
6.1.5 Crystal violet-formalin stain solution

• The following components were added to a 100 ml glass beaker and mixed with a
stir bar and magnetic stirrer.
• 75 ml 1 x PBS (section 6.1.2)
• 25 ml formalin (Merck, Australia)
• 5g crystal violet (Sigma, U.S.A)
• The solution was filtered through filter paper (Whatman, U.K) before use.
6.2 Solutions used for PAGE and western blotting
6.2.1 Resolving buffer (1.5 M Tris pH 8.8)

• 181.5g Tris base (BDH Chemicals, U.K) was dissolved in 800 ml of distilled water
in a 1 litre glass beaker using a stir bar and magnetic stirrer.
• The pH of the solution was adjusted to 8.8 with HCl and the final volume of the
solution was made up to 1 litre.
• The solution was transferred to a 1 litre schott bottle
6.2.2 Stacking buffer (1.0 M Tris pH 6.8).

• 121.0g Tris base was dissolved in 800ml of distilled water in a 1 litre glass beaker
using a stir bar and magnetic stirrer.
• The pH of the solution was adjusted to 6.8 with HCl and the final volume of the
solution was made up to 1 litre.
• The solution was transferred to a 1 litre schott bottle
6.2.3 10% ammonium persulfate

• 0.1g of ammonium persulfate (Sigma, U.S.A) was dissolved in 1ml of distilled water
in a 1.5 ml tube
• The tube was vortexed to mix
183
6.2.4 2 x PAGE sample buffer
• The following components were added to 10 ml of distilled water in a 20 ml glass
beaker and mixed with a stir bar and magnetic stirrer.
• 2.0 ml stacking buffer (Section 6.2.2)
• 8.0g sodium dodecyl sulphate (SDS; Sigma, U.S.A)
• 0.4g bromophenol blue (Sigma, U.S.A)
• 2.0 ml glycerol (BDH Chemicals, U.K)
• The solution was adjusted to 20 ml final volume with distilled water and was
aliquoted in 1 ml lots and stored at -20°C.
6.2.5 10% SDS solution

• 10.0g SDS was added to 100 ml of distilled water in a glass beaker and mixed using
a stir bar and magnetic stirrer.
6.2.6 5 x PAGE Running Buffer

• The following components were added to 800 ml of distilled water in a 1 litre glass
beaker and mixed using a stir bar and magnetic stirrer.
• 15.1g Tris Base
• 94.0g glycine (Sigma, U.S.A)
• 50 ml of 10% SDS (section 6.2.5)
• The final volume was adjusted to 1 litre with distilled water
6.2.7 CAPS transfer buffer

• 2.21g CAPS (Sigma, U.S.A) was dissolved in 800 ml of distilled water in a 1 litre
glass beaker using a stir bar and magnetic stirrer.
• The pH of solution was adjusted to 11.0 with NaOH.
• 100 ml Methanol (Merck, U.S.A) and 100 µl of 10% SDS was added to the solution
and the volume adjusted to a final volume of 1 litre with distilled water.
• The solution was stored at 4°C, and was prepared fresh for each use.
184
6.2.8 10 x Tris-buffered saline (TBS)
• The following components were dissolved in 800 ml of distilled water in a 1 litre
glass beaker using a stir bar and magnetic stirrer.
• 24.2g Tris Base
• 80.0g NaCl
• The pH was adjusted to 7.6 with HCl and the final volume was made up to 1 litre
with distilled water
6.2.9 1 x TBS
• 100 ml of 10 x TBS was added to 900 ml of distilled water
6.3 Recipes for Polyacrylamide Gels
6.3.1 10% resolving polyacrylamide gel

• A 10 ml solution for 10% resolving polyacrylamide gels was made by adding the
following components to a 15 ml tube (Falcon, U.S.A).
• 4.8 ml of distilled water
• 2.5 ml 40% polyacrylamide mix (29 : 1 acrylamide:bis-acrylamide; BioRad, U.S.A)
• 2.5 ml resolving buffer (appendix section 6.2.1)
• 100 μl 10% SDS (appendix section 6.2.5)
• 100 μl 10% ammonium persulfate (appendix section 6.2.3).
• The solution was mixed with a 1 ml plastic pipette (Copan, U.S.A)
• 4 μl N,N,N’,N’-tetramethylethylenediamine (TEMED; Sigma, U.S.A) was added to
the solution, which was mixed by vortex and then added to MiniProtean II Gel
apparatus (BioRad, U.S.A).
• 1 ml of butanol (Merck, U.S.A) was overlaid on the resolving gel
• When the resolving gel was set, the butanol was soaked up with filter paper and
replaced with the 5% stacking gel solution (appendix section 6.3.2).
185
6.3.2 5% stacking polyacrylamide gel
• A 5 ml solution for 5% stacking polyacrylamide gels was made by adding the
following components to a 15 ml tube
• 3.6 ml distilled water
• 630 μl 40% polyacrylamide mix (29 : 1 acrylamide:bis-acrylamide; BioRad, U.S.A)
• 630 μl Stacking Buffer (appendix section 6.2.2)
• 50 μl 10% SDS (appendix section 6.2.5)
• 50 μl 10% ammonium persulfate (appendix section 6.2.3)
• 5 μl TEMED was added to the solution, which was mixed by vortex and then
overlaid on the 10% resolving gel.
• The comb piece (Biorad, U.S.A) for marking the sample wells for PAGE was placed
in the stacking gel.
6.4 Molecular Biology
6.4.1 DEPC-treated water

• 1 ml of diethyl pyrocarbonate (DEPC; Sigma) was added to 1 litre of distilled water
and was incubated for 16 hours at 37°C
• Following incubation, the solution was autoclaved at 121°C for 20 minutes.
6.4.2 50 x Tris acetate EDTA (TAE) buffer

• 242g of Tris Base was dissolved in 800 ml of distilled water in a 1 litre glass beaker
using a stir bar and magnetic stirrer
• 57.1 ml glacial acetic acid (Merck, U.S.A), 100 ml 0.5M EDTA (pH 8.0) was added
and the final volume of the solution was adjusted to 1 litre with distilled water.
6.4.3 1 x TAE buffer

• 20 ml of 50 x TAE solution was added to 980 ml of distilled water in a 1 litre Schott
bottle.
186
6.4.4 6 x DNA loading dye
• 3 ml of glycerol was combined with 7 ml of distilled water in a 15 ml tube
• 25mg bromophenol blue and 25mg xylene cyanol (Sigma, U.S.A) was added to the
10ml and dissolved using a vortex.
6.4.5 3 M Sodium acetate pH 5.2

• 40.8g of sodium acetate (BDH Chemicals, UK) was added to 100 ml of distilled
water and the pH was adjusted to 5.2
6.4.6 Luria broth (LB) medium

• 10.0g of bacto-tryptone (Oxoid, U.S.A), 5.0g of yeast extract (Oxoid, U.S.A) and
10.0g of sodium chloride was added to 800 ml of distilled water in a 1 litre glass
beaker and mixed with a stir bar on a magnetic stirrer.
• The solution was made to a final volume of 1 litre and the pH adjusted to 7.0 using 1
M NaOH
• The solution was transferred to schott bottles and was autoclaved at 121°C for 20
minutes.
6.4.7 Luria broth agar (LBA)

• 5.0g of bacto-agar (Oxoid, U.S.A) was added to 300 ml lots of LB medium (section
6.4.6)
• The solution was autoclaved at 1210C for 20 minutes.
187
7 APPENDIX B: DATA
7.1 Affect of 6 M urea treatment on MAb adsorption to ELISA plates

Mean Abs ± 1 s.d (n=2)
MAb Untreated 6M Urea Inhibition (%)
F1G2 3.421 ± 0.083 2.316 ± 0.005 32
F2D1 2.895 ± 0.073 2.813 ± 0.041 3
4B1 2.957 ± 0.002 2.370 ± 0.010 20
17A3 3.119 ± 0.390 2.974 ± 0.065 5
F16B5 1.533 ± 0.046 1.498 ± 0.017 3
F18B10 3.318 ± 0.070 2.235 ± 0.055 33
18F5 1.899 ± 0.026 1.989 ± 0.018 -5
3C9 0.916 ± 0.028 0.668 ± 0.016 27
F19F11 0.900 ± 0.081 0.933 ± 0.063 -3
F20F10 2.888 ± 0.059 2.561 ± 0.036 11
13H8 2.569 ± 0.213 2.667 ± 0.019 -4
1H10 2.838 ± 0.166 2.634 ± 0.050 7
a
absorbance of untreated- absorbance of 6M urea treated
absorbance of untreated
188
7.2 Competitive binding assay results
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by 6B6C1

Virus Blocking Ab Mean Abs ± s.d. Blocking (%) p
1/2 F1G2 0.389 ± 0.047 -83 ≤0.05
F2D1 0.018 ± 0.002 92 ≤0.05
4B1 0.120 ± 0.003 43 ≤0.05
17A3 0.128 ± 0.002 40 ≤0.05
13H8 0.870 ± 0.043 -309 ≤0.05
F16B5 0.033 ± 0.002 84 ≤0.05
F18B10 0.136 ± 0.007 36 ≤0.05
1H10 0.296 ± 0.018 -39 ≤0.05
4G2 0.038 ± 0.004 82 ≤0.05
6B6C1 0.022 ± 0.004 90 ≤0.05
7 E3 0.212 ± 0.009
1/4 18F5 0.204 ± 0.004 47 ≤0.05

3C9 0.104 ± 0.021 73 ≤0.05
F19F11 0.355 ± 0.015 7 >0.05
F20F10 0.350 ± 0.015 9 >0.05
7 E3 0.383 ± 0.011
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by 4G2

1/2 F1G2 0.381 ± 0.021 -35 ≤0.05
F2D1 0.022 ± 0.001 92 ≤0.05
4B1 0.129 ± 0.002 54 ≤0.05
17A3 0.139 ± 0.004 51 ≤0.05
13H8 1.303 ± 0.022 -362 ≤0.05
F16B5 0.048 ± 0.004 83 ≤0.05
F18B10 0.150 ± 0.000 47 ≤0.05
1H10 0.409 ± 0.020 -45 ≤0.05
4G2 0.018 ± 0.001 94 ≤0.05
6B6C1 0.015 ± 0.000 95 ≤0.05
7 E3 0.282 ± 0.018
1/4 18F5 0.282 ± 0.002 55 ≤0.05

3C9 0.169 ± 0.004 73 ≤0.05
F19F11 0.473 ± 0.000 25 ≤0.05
F20F10 0.498 ± 0.012 21 ≤0.05
7 E3 0.383 ± 0.011
189
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by F2D1
1/2 F1G2 0.848 ± 0.033 -17 ≤0.05
F2D1 0.019 ± 0.000 97 ≤0.05
4B1 0.328 ± 0.000 55 ≤0.05
17A3 0.368 ± 0.004 49 ≤0.05
13H8 1.694 ± 0.023 -134 ≤0.05
F16B5 0.106 ± 0.007 85 ≤0.05
F18B10 0.342 ± 0.002 53 ≤0.05
1H10 0.622 ± 0.000 14 ≤0.05
4G2 0.015 ± 0.000 98 ≤0.05
6B6C1 0.012 ± 0.000 98 ≤0.05
7 E3 0.725 ± 0.011
1/4 18F5 0.284 ± 0.011 63 ≤0.05

3C9 0.251 ± 0.004 67 ≤0.05
F19F11 0.574 ± 0.005 26 ≤0.05
F20F10 0.550 ± 0.048 29 ≤0.05
7 E3 0.772 ± 0.015
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by 3C9

1/4 F1G2 0.123 ± 0.001 79 ≤0.05
F2D1 0.135 ± 0.000 77 ≤0.05
4B1 0.042 ± 0.001 93 ≤0.05
17A3 0.064 ± 0.013 89 ≤0.05
13H8 0.876 ± 0.012 -49 ≤0.05
F16B5 0.019 ± 0.001 97 ≤0.05
F18B10 0.051 ± 0.001 91 ≤0.05
1H10 0.210 ± 0.043 64 ≤0.05
18F5 0.385 ± 0.010 34 ≤0.05
3C9 0.016 ± 0.000 97 ≤0.05
F19F11 0.230 ± 0.024 61 ≤0.05
F20F10 0.417 ± 0.043 29 ≤0.05
4G2 0.018 ± 0.000 97 ≤0.05
6B6C1 0.014 ± 0.006 98 ≤0.05
7 E3 0.588 ± 0.014
190
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by F18B10
1/2 F1G2 1.252 ± 0.019 11 ≤0.05
F2D1 0.539 ± 0.004 62 ≤0.05
4B1 0.577 ± 0.027 59 ≤0.05
17A3 0.813 ± 0.001 42 ≤0.05
13H8 2.605 ± 0.026 -85 ≤0.05
F16B5 0.041 ± 0.000 97 ≤0.05
F18B10 0.077 ± 0.000 94 ≤0.05
1H10 1.428 ± 0.009 -1 >0.05
4G2 0.150 ± 0.008 89 ≤0.05
6B6C1 0.055 ± 0.000 96 ≤0.05
7 E3 1.408 ± 0.000
1/4 18F5 0.991 ± 0.019 30 ≤0.05

3C9 0.036 ± 0.002 97 ≤0.05
F19F11 1.115 ± 0.030 21 ≤0.05
F20F10 1.154 ± 0.022 19 ≤0.05
7 E3 1.418 ± 0.028
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by F16B5

1/2 F1G2 0.792 ± 0.051 35 ≤0.05
F2D1 0.533 ± 0.003 56 ≤0.05
4B1 0.215 ± 0.002 82 ≤0.05
17A3 0.362 ± 0.003 70 ≤0.05
13H8 2.059 ± 0.021 -69 ≤0.05
F16B5 0.023 ± 0.001 98 ≤0.05
F18B10 0.199 ± 0.002 84 ≤0.05
1H10 0.804 ± 0.008 34 ≤0.05
4G2 0.102 ± 0.010 92 ≤0.05
6B6C1 0.032 ± 0.000 97 ≤0.05
7 E3 1.216 ± 0.031
1/4 18F5 0.906 ± 0.014 35 ≤0.05

3C9 0.049 ± 0.000 96 ≤0.05
F19F11 0.745 ± 0.009 47 ≤0.05
F20F10 1.231 ± 0.048 12 ≤0.05
7 E3 1.405 ± 0.001
191
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by 4B1
1/2 F1G2 1.416 ± 0.009 13 ≤0.05
F2D1 0.599 ± 0.002 63 ≤0.05
4B1 0.023 ± 0.000 99 ≤0.05
17A3 0.051 ± 0.001 97 ≤0.05
13H8 2.666 ± 0.062 -64 ≤0.05
F16B5 0.824 ± 0.021 49 ≤0.05
F18B10 0.968 ± 0.036 41 ≤0.05
1H10 1.684 ± 0.012 -3 >0.05
4G2 0.157 ± 0.009 90 ≤0.05
6B6C1 0.037 ± 0.005 98 ≤0.05
7 E3 1.629 ± 0.038
1/4 18F5 0.693 ± 0.020 21 ≤0.05

3C9 0.545 ± 0.031 38 ≤0.05
F19F11 0.338 ± 0.007 62 ≤0.05
F20F10 0.701 ± 0.016 20 ≤0.05
7 E3 0.880 ± 0.019
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by 17A3

1/2 F1G2 1.521 ± 0.069 10 >0.05
F2D1 0.541 ± 0.014 68 ≤0.05
4B1 0.027 ± 0.000 98 ≤0.05
17A3 0.043 ± 0.000 97 ≤0.05
13H8 2.616 ± 0.045 -54 ≤0.05
F16B5 0.783 ± 0.099 54 ≤0.05
F18B10 0.978 ± 0.024 42 ≤0.05
1H10 1.563 ± 0.055 8 >0.05
4G2 0.177 ± 0.009 90 ≤0.05
6B6C1 0.034 ± 0.000 98 ≤0.05
7 E3 1.695 ± 0.031
1/4 18F5 1.460 ± 0.004 28 ≤0.05

3C9 1.115 ± 0.017 45 ≤0.05
F19F11 0.711 ± 0.053 65 ≤0.05
F20F10 1.689 ± 0.061 16 ≤0.05
7 E3 2.016 ± 0.049
192
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by 18F5
1/4 F1G2 0.059 ± 0.000 96 ≤0.05
F2D1 0.108 ± 0.002 92 ≤0.05
4B1 0.459 ± 0.014 66 ≤0.05
17A3 0.602 ± 0.044 55 ≤0.05
13H8 1.004 ± 0.005 25 ≤0.05
F16B5 0.240 ± 0.003 82 ≤0.05
F18B10 0.546 ± 0.006 59 ≤0.05
1H10 0.490 ± 0.010 63 ≤0.05
18F5 0.241 ± 0.036 82 ≤0.05
3C9 0.371 ± 0.000 82 ≤0.05
F19F11 1.067 ± 0.006 20 ≤0.05
F20F10 1.078 ± 0.015 19 ≤0.05
4G2 0.028 ± 0.000 98 ≤0.05
6B6C1 0.018 ± 0.003 99 ≤0.05
7 E3 1.333 ± 0.003
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by F19F11

1/4 F1G2 0.139 ± 0.007 81 ≤0.05
F2D1 0.130 ± 0.006 83 ≤0.05
4B1 0.016 ± 0.002 98 ≤0.05
17A3 0.046 ± 0.014 94 ≤0.05
13H8 0.744 ± 0.021 1 >0.05
F16B5 0.077 ± 0.007 90 ≤0.05
F18B10 0.117 ± 0.003 84 ≤0.05
1H10 0.286 ± 0.000 62 ≤0.05
18F5 0.470 ± 0.007 37 ≤0.05
3C9 0.183 ± 0.006 76 ≤0.05
F19F11 0.204 ± 0.013 73 ≤0.05
F20F10 0.676 ± 0.040 10 >0.05
4G2 0.029 ± 0.009 96 ≤0.05
6B6C1 0.023 ± 0.008 97 ≤0.05
7 E3 0.751 ± 0.031
193
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by F20F10
1/4 F1G2 0.222 ± 0.007 79 ≤0.05
F2D1 0.109 ± 0.013 89 ≤0.05
4B1 0.380 ± 0.013 63 ≤0.05
17A3 0.487 ± 0.019 53 ≤0.05
13H8 0.799 ± 0.047 23 ≤0.05
F16B5 0.266 ± 0.007 74 ≤0.05
F18B10 0.452 ± 0.021 56 ≤0.05
1H10 0.495 ± 0.008 52 ≤0.05
18F5 0.045 ± 0.002 96 ≤0.05
3C9 0.367 ± 0.007 65 ≤0.05
F19F11 0.856 ± 0.004 18 ≤0.05
F20F10 0.078 ± 0.002 92 ≤0.05
4G2 0.027 ± 0.000 97 ≤0.05
6B6C1 0.026 ± 0.000 97 ≤0.05
7 E3 1.038 ± 0.022
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by F1G2

1/2 F1G2 0.063 ± 0.006 97 ≤0.05
F2D1 0.662 ± 0.010 65 ≤0.05
4B1 1.497 ± 0.007 21 ≤0.05
17A3 1.606 ± 0.032 15 ≤0.05
13H8 2.547 ± 0.048 -35 ≤0.05
F16B5 0.766 ± 0.029 60 ≤0.05
F18B10 1.443 ± 0.015 24 ≤0.05
1H10 2.184 ± 0.043 -15 ≤0.05
4G2 0.184 ± 0.000 90 ≤0.05
6B6C1 0.047 ± 0.002 98 ≤0.05
7 E3 1.892 ± 0.007
1/4 18F5 1.591 ± 0.026 37 ≤0.05

3C9 0.949 ± 0.043 62 ≤0.05
F19F11 2.046 ± 0.026 19 ≤0.05
F20F10 2.181 ± 0.055 14 ≤0.05
7 E3 2.522 ± 0.010
194
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by 1H10
1/2 F1G2 2.543 ± 0.001 -3 >0.05
F2D1 1.243 ± 0.031 50 ≤0.05
4B1 2.380 ± 0.078 4 >0.05
17A3 2.381 ± 0.039 4 >0.05
13H8 2.650 ± 0.119 -7 >0.05
F16B5 1.965 ± 0.068 20 ≤0.05
F18B10 2.334 ± 0.015 5 >0.05
1H10 0.100 ± 0.003 96 ≤0.05
4G2 0.350 ± 0.016 86 ≤0.05
6B6C1 0.225 ± 0.000 91 ≤0.05
7 E3 2.469 ± 0.068
1/4 18F5 3.015 ± 0.007 4 >0.05

3C9 2.619 ± 0.063 16 ≤0.05
F19F11 3.031 ± 0.084 3 >0.05
F20F10 3.075 ± 0.008 2 >0.05
7 E3 3.125 ± 0.048
The effects of anti-DENV-4 MAbs on the capture of DENV-4 by 13H8

1/2 F1G2 2.763 ± 0.014 -2 >0.05
F2D1 1.491 ± 0.039 45 ≤0.05
4B1 2.678 ± 0.026 1 >0.05
17A3 2.742 ± 0.057 -1 >0.05
13H8 0.142 ± 0.006 95 ≤0.05
F16B5 2.171 ± 0.034 20 ≤0.05
F18B10 2.606 ± 0.032 4 >0.05
1H10 1.714 ± 0.041 37 ≤0.05
4G2 0.328 ± 0.004 88 ≤0.05
6B6C1 0.212 ± 0.009 92 ≤0.05
7 E3 2.705 ± 0.026
1/4 18F5 1.755 ± 0.003 2 >0.05

3C9 1.176 ± 0.032 34 ≤0.05
F19F11 1.739 ± 0.000 3 >0.05
F20F10 1.825 ± 0.016 -2 >0.05
7 E3 1.789 ± 0.046
195
7.3 Amino acid changes in DENV-4 n.e.m.s E protein sequences
7.3.1 Wildtype DENV-4:

DENV-4 H241, DENV-4 NM, DENV-4 W10.
MRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELIKTTAKEVALLRTYC
IEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFS
CSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDIPNHGVTATITPRSPSVEVKLPDYGE
LTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWAAGADTSEVHWNYKERMVTF
KVPHAKRQDVTVLGSQEGAMHSALTGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYT
MCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPFAEY
TNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMLESTYRGAKRMAILGETAW
DFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMTCI
AVGGITLFLGFTVHA
7.3.2 DENV-4 5FU induced n.e.m.s

IEASISNITTATRCPTQGEPYLKEEQDQQYICRRAVVDRGWGNGCGLFGKGGVVTCAKFS
CSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDISSHGVTATITPRSPSVEVKLPDYGE
LTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWAAGADTSEVHWNYKERMVTF
MCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPFAEY
TNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAW
AVGGITLFLGFTVHA
7.3.3 DENV-4 natural n.e.m.s

IEASISNITTATRCPTQGEPYLKEEQDQQYICRRDMVDRGWGNGCGLFGKGGVVTCAKFS
CSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDIPNHGETATITPRSPSVEVKLPDYGE
LTLDCEPRSGIDFNEMILMKMKTKTWLVHKQWFLDLPLPWAAGADTSEVHWNHKERMVTF
MCSGKFSIDKEMAETQHGTTVVKVKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPFAEY
TNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMLESTYRGAKRMAILGETAW
AVGGITLFLGFTVHA
196
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Identification of Epitopes on the

Dengue Virus Type 4 Envelope

A thesis submitted in 2006

Christopher Bruce Howard

Centre for Molecular Biotechnology

DENV-4 variants including DENV-4 isolates from different geographical locations or

Table 1.6. Functional determinants identified by infectious clones_____________________________ 68

Table 2.1 Dengue viruses used in this study _______________________________________________ 76

Table 2.2 Reference MAbs used in this study______________________________________________ 81

Table 2.4 Thermal cycling conditions for PCR ____________________________________________ 94

DHF is characterised by a high fever, haemorrhage, increased vascular permeability,

1.1 The pathogenesis of DHF/DSS

Several host cells are permissive to DENV infection, particularly mononuclear

Following attachment to the cell surface, the flaviviruses appear to be internalised to an

A primary infection with DENV-1, DENV-3 or DENV-4 followed by a secondary

1.3 Viral virulence

Flaviviruses are spherical particles approximately 40-60 nm in diameter consisting of a

Cryoelectron microscopy (cryoEM) and image reconstruction techniques indicated that

This figure is not available online.

This figure is not available online.

Protein Molecular Weight Number of Function

Pre-Membrane 19-23 166 Chaperone protein for E;

Membrane 8-8.5 75 Component of viral

Envelope 51-60 495 Major envelope protein

NS1 44-49 350-410 Putative RNA replication

NS2a 16-21 150-210 Putative RNA replication

NS2b 12-15 130 Cofactor for serine protease

NS3 67-76 615 Viral protease; RNA

NS4a 24-32 150-280 Putative RNA replication

NS4b 10-11 110-250 Unknown

NS5 91-98 900 Viral RNA dependent RNA

1.5.1 The core protein

1.5.2 The membrane proteins (prM and M)

1.5.3 The envelope protein

This figure is not available online.

This figure is not available online.

The binding of monoclonal antibodies to epitopes of domain II was disrupted following

1.6.4 Stem anchor region

1.6.5 Dimeric interactions

1.7 Dengue virus E protein models

1.7.1 Antigenic model

Monoclonal antibodies against domain A recognised 45 kDa and 22 kDa peptides

1.7.2 Structural model of DENV E protein dimer

Gentry et al., DENV-2 22 3 1 Type HI, N

Henchal et DENV-2 8 4 4 Type and subcomplex HI, N

Jianmin et DENV-2 22 4 16 Group HI, N

Roehrig et DENV-2 13 3 10 Type, subcomplex and group HI, N

Kimura- JEV 8 8 1 Group HI

Hall et MVEV 9 4 3 Complex HI, N

Roehrig et SLEV 8 6 1 Type

Heinz et TBEV 8 3 4 Type and complex HI, N

Guirakhoo et al., TBEV 19 6 5 Subtype, complex, subgroup HI, N

Tsekhanovskaya TBEV 25 5 12 Type, subtype, complex and HI, N

In order to characterise conformationally dependent functional epitopes, several other

Author Virus Method Antibody Immunogenic regions of E

Neutralising MAb aa 293-403

Thullier et DENV-1 Phage display Neutralising aa 306-314

Aaskov et al., DENV-2 Pepscan: 488 Dengue immune Rabbit (6 domains):

Neutralising MAb aa 50-57,

Innis et al., DENV-2 Pepscan: 490 Convalescent 124 hexapeptides

Roehrig et al., DENV-2 18 synthetic Mouse 9/18 peptides

DENV-2 0/18 peptides

Antipeptide antibody aa 35-55,