MECHANISMS OF DENGUE VIRUS NEUTRALIZATION BY ANTIBODY Wahala Mudiyanselage ...

186 Pages·2009·3.14 MB·English

MECHANISMS OF DENGUE VIRUS NEUTRALIZATION BY ANTIBODY

Wahala Mudiyanselage Padmasriri Bandara Wahala

A dissertation submitted to the faculty of the University of North Carolina at
Chapel Hill in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in the Department of Microbiology and Immunology

Chapel Hill
2009

Approved by:
Aravinda de Silva , MPH, PhD.
Robert E Johnston, PhD.
Edward J Collins, PhD.
Raymond J Pickles, PhD.
Laura J White, PhD.
Ronald I Swanstrom, PhD.
ABSTRCT

WAHALA MUDIYANSELAGE PADAMASIRI BANDARA WAHALA:
Mechanisms of dengue virus neutralization by antibody
(under the direction of Aravinda de Silva)

Dengue virus (DENV), the causative agent of dengue, is a group of viruses consisting
of four different serotypes designated DENV1-4. Each serotype is further divided into
different genotypes. Primary DENV infection induces a life-long type-specific immune
response against the homologous DENV serotype. It is widely assumed that all the strains of
a DENV serotype are equally neutralized by type-specific antibodies irrespective of the
genetic variability within the serotype. Studies with mouse monoclonal antibodies (mAb)
have demonstrated that serotype-specific neutralization of DENV is mainly mediated by
antibody binding to epitopes on domain III of the viral Envelope protein (EDIII).
Although DENV3 has spread worldwide, neutralization mechanisms of DENV3 is
poorly studied. Results of my studies with mouse mAbs demonstrate that type-specific
neutralization of DENV3 was confined to EDIII lateral ridge as reported for other
flaviviruses. I further demonstrated that DENV3 Envelope (E) protein sequences were
variable between different genotypes of DENV3. Variable positions were located on or near
the known antibody epitopes on E protein, and natural amino acid variations of DENV3 E
protein led to complete or partial escape from antibody neutralization. These results suggest
that natural intra-serotype variation should be considered when characterizing natural and
vaccine induced immunity.
ii
The specificity and functionality of the human antibody response to DENV is poorly
characterized. It is unknown if humans also develop antibodies to EDIII epitopes
recognized by mouse mAbs. Using a panel of sera from people exposed to DENV, I
demonstrate that people develop low levels of type-specific EDIII reactive antibodies after
primary infection and low levels of serotype cross reactive EDIII antibodies after secondary
infection. I further demonstrated that these low levels of EDIII reactive antibodies only
make a minor contribution to the total neutralization potency of human immune sera. I
conclude that the EDIII epitopes identified using mouse reagents, which have been the focus
of much recent work, are not the primary target of human antibodies that neutralize DENV. I
believe these results will stimulate investigators to study previously neglected regions of the
DENV envelope to identify functionally important epitopes engaged by human antibodies.

iii
ACKNOWLADGEMENTS

I first want to thank my mentor, Aravinda de Silva, for providing me with a
wonderful opportunity to explore the world through hypothesis driven scientific research.
With his enthusiasm, his inspiration, and his great efforts to explain things clearly and
simply, he helped to make scientific research fun for me. I further want extend my gratitude
to Aravinda for providing me with an opportunity to visit his lab in 2002 as a visiting scholar,
which induced me to pursue as a graduate student. I am indebted to all of my committee
members for their continued support and guidance. I would like to thank both present and
past members of the de Silva lab whose continued support drives me to write this thesis. I
especially want to thank Anne Broadwater, lab manager of the de Silva lab, for the wonderful
support she provided throughout my graduate career.
I am grateful to the “Presidents Funds” of Sri Lanka for providing me with an
opportunity to study at UNC CH as a visiting scholar from 2002 December to 2003 June. I
would also like to thank all Sri Lankan friends in North Carolina, for their continued moral
support during my stay at UNC CH. At this juncture, I want to thank my parents for their
wonderful support throughout my life. I especially want to thank Jayath, my brother, who has
been helping me whenever I faced difficult time in my life. Finally, support from my wife
Nishanthi and my son Supun have been enormous and invaluable throughout my graduate
career. I am especially thankful to my lovely son for tolerating me and my busy schedule as a
graduate student.

iv
TABLE OF CONTENTS

List of Tables ...………………………………………………………………………………ix
List of Figures………………………………………………………………………………....x
List of Abbreviations.……………………………………………………………………….xiv
Chapter1 Background and significance……………………………………………………...1
1.1 Introduction………………………………………………………………………. 2
1.2 Dengue, Dengue virus, its transmission and global distribution………………….2
1.3 Classification and the structure of DENV…….…………………………………..3
1.4 Replication and life cycle of DENV………………………………………………4
1.5 Dengue: the disease and the pathogenesis…….…………………………………..5
1.6 Protective host immune response against DENV infection……………………….7
1.7 Mechanisms and theories of virus neutralization by antibody…………………….8
Aggregation and disintegration of virus particles……………………………..8
Block receptor attachment..………………………………………………….10
Neutralization of virus at a post attachment step..…………………………...11
1.8 Models of Virus neutralization..………………………………………………...13
1.9 Neutralization of Flaviviruses by antibodies..………………………………….15
Structure of the E protein...…………………………………………………..16
Conformational changes of E protein..………………………………………18
Known epitopes of Flaviviruses...……………………………………………19
Occupancy and flavivirus neutralization..…………………………………...20
v
Type specific neutralization, known epitopes and their role
against DENV infection..…………………………………………………….21

1.10 Objectives of the study…………………………………………………………23
1.11 References..……………………………………………………………………..25
Chapter 2 Phylogenetic analysis of dengue virus in Sri Lanka from 1981 to 2006
with a special reference to severe epidemic in 2004...…………………………..42
2.1 Abstract..…………………………………………………………………………43
2.2 Introduction..……………………………………………………………………..44
2.3 Materials and Methods...…………………………………………………………46
Sample Collection...………………………………………………………….46
Reverse Transcription–PCR for Detection and Serotyping of DENVs...……46
Isolation of DENV from acute serum samples..……………………………..47
Sequencing and Phylogenetic Analysis of DENV...…………………………47
2.4 Results...………………………………………………………………………….48
Circulating Dengue Serotypes in Sri Lanka...………………………………..48
Isolation of DENV from acute serum samples................................................49
Phylogeny of DENVs in Sri Lanka...………………………………………...50
Phylogeny of DENV-1 during 1983–2004..…………………………………50
Phylogeny of DENV-2 during 1981–2004..…………………………………51
Phylogeny of DENV-3 during 1983–2004..…………………………………51
Phylogeny of DENV-4 during 1978–2004..…………………………………53
2.5 Discussion..………………………………………………………………………54
2.6 References..………………………………………………………………………58

vi
Chapter 3 Development of mouse monoclonal antibody (mAb)
against dengue virus serotype...…………………………………………………..72
3.1 Abstract..…………………………………………………………………………73
3.2 Introduction...…………………………………………………………………….74
3.3 Materials and method...…………………………………………………………..75
Purification of DENV..………………………………………………………75
Immunization of mice..………………………………………………………75
Expression and purification of recombinant EDIII (rEDIII)..……………….76
Detection of DENV reactive antibody in immunized mouse
sera or hybridoma culture supernatants by ELISA..…………………………76
DENV Neutralization assays..……………………………………………….77
3.4 Results...………………………………………………………………………….78
3.5 Discussion..………………………………………………………………………79
3.6 References..………………………………………………………………………80
Chapter 4 Natural Strain Variation and Antibody Neutralization of Dengue
Serotype 3 Viruses..………………………………………………………………82

4.1 Abstract..…………………………………………………………………………83
4.2 Introduction..……………………………………………………………………..84
4.3 Materials and Methods...…………………………………………………………86
Cells and Viruses...…………………………………………………………..86
Monoclonal Antibodies...…………………………………………………….86
Expression and purification of recombinant EDIII (rEDIII)..……………….87
Mutagenesis of recombinant EDIII (rEDIII)..……………………………….87
Binding ELISA with rEDIII protein...……………………………………….87
Neutralization assays..……………………………………………………….88
vii
DENV3 Sequence Analysis..……………………………………………….. 89
4.4 Results…..………………………………………………………………………. 89
Variable Amino acids on DENV3 E protein...…………………………….....89
Relationship of DENV3 E protein informative sites to
known antibody epitopes..…………………………………………………...90
Mapping of DENV3, EDIII reactive antibodies..……………………………91
Binding of mAbs to different genotypes of DENV3..……………………….92
Neutralization of DENV3 genotypes by mAbs..…………………………….94
4.5 Discussion..………………………………………………………………………94
4.6 References..………………………………………………………………………99
Chapter 5 Dengue virus neutralization by human immune sera: role of envelope
protein domain III - reactive antibody………………………………………….113
5.1 Abstract…………………………………………………………………………114
5.2 Introduction……………………………………………………………………..115
5.3 Materials and Method…………………………………………………………..117
Viruses……………………………………………………………………...117
Immune sera and Antibodies……………………………………………….117
Purification of DENV antigen for ELISA………………………………….118
Expression of the ectodomain of E protein (Es) from DENV3…………….118
Expression and purification of DENV EDIII....……………………………119
Detection of dengue reactive antibody in human immune sera
by ELISA.………………………………………………………………….119
Depletion of EDIII-reactive antibody in human immune sera.……………..121
DENV neutralization assays…………………………………………….….121
5.4 Results….……………………………………………………………………….122
viii
DENV Binding Antibodies in Human Immune Sera.………………………123
Purification and Characterization of Recombinant DENV
Envelope Protein Domain III (EDIII)………………………………………124
EDIII-reactive antibodies in human DENV immune sera………………….125
Role of EDIII-reactive Antibodies in DENV Neutralization.………………126
5.5 Discussion………………………………………………………………………128
5.6 References.……………………………………………………………………...133
Chapter 6 Discussion……………………………………………………………………….148
6.1 Clade replacement of DENV3 in Sri Lanka and possible role
in pathogenesis…………………………………………………………………149
6.2 Neutralization of DENV3 by EDIII reactive antibodies and its
role in natural DENV infections in human.……………………………………154
Strain variability and differential neutralization of DENV3..………………154
Role of EDIII reactive antibodies after natural infection in people..……….155
rEDIII as a diagnostic tool to detect DENV infection and to
discriminate primary infection from secondary infection..…………………161
6.3 References ……………………………………………………………………...164

ix
LIST OF TABLES
Table 2.1 DENV strains and the source of the sequence used in the study...……………….64
Table 3.1 Binding and neutralization properties of mouse mAb..…………………………..81
Table 4.1 Location of antigenic sites and informative sites on DENV3 E protein.………..102
Table 4.2 Source of mouse mAb used in the current study.……………………………….103
Table 4.3 Binding of mouse mAbs to mutant DENV3 EDIII proteins.……………………104
Table 4.4 Neutralization of DENV3 genotypes by EDIII mAbs.………………………….105
Table 5.1 Human DENV immune sera used in the study.…………………………………137
Table 5.2 Titer of DENV and EDIII reactive antibody in immune sera.…………………..138
Table 5.3 Binding of DENV neutralizing monoclonal antibodies to r MBP-EDIII
fusion protein.…………………………………………………………………...139

Table 5.4 DENV neutralization by immune sera depleted of EDIII reactive antibodies.…140

x