study of cross-reactive human antibodies against dengue virus envelope protein

167 Pages·2014·13.86 MB·English

STUDY OF CROSS-REACTIVE HUMAN ANTIBODIES AGAINST DENGUE VIRUS
ENVELOPE PROTEIN: IMPLICATION FOR DENGUE VACCINE

A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE
UNIVERSITY OF HAWAI‘I AT M(cid:406)NOA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
BIOMEDICAL SCIENCES
(TROPICAL MEDICINE)

AUGUST 2014

BY
Wen-Yang Tsai

Dissertation Committee:
Wei-Kung Wang, Chairperson
Michael Lieberman
Saguna Verma
Sandra Chang
Hongwei Li
ACKNOWLEDGEMENTS
I would like to thank my mentor, Dr. Wei-Kung Wang, for the dedication to guide
and enlighten me toward the path of research and science. I also would like to thank my
committee members, Dr. Sandra Chang, Dr. Saguna Verma, Dr. Michael Lieberman and
Dr. Hongwei Li, for their efforts to help me to complete my study and education. I also
want to thank my parents and for the endless supporting and encouraging me for going
abroad for pursuing the degree. I want to thank my wife, Sheng-Ti Hung, for
accompanying me through the entire journey and giving me all of the supports whenever
I need. I could not complete my degree without her. Finally I want to thank all of my
friends in Hawaii and in Taiwan for giving me such wonderful time in my life.

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ABSTRACT
With an estimated 390 million infections and approximately 90 million apparent
infections annually, the four serotypes of dengue virus (DENV) continue to be a global
threat with no licensed vaccine available. A better understanding of correlates between
protection and humoral immune responses is needed.
First, we characterized cross-reactive human anti-E mAbs derived from patients
after primary and secondary DENV infections. Group-reactive (GR) anti-E mAbs that
recognized four DENV serotypes and another flavivirus, West Nile virus (WNV), were
found as the predominant cross-reactive anti-E mAbs. The epitopes of 32 GR mAbs
were found at residues in the fusion loop (FL) of E protein domain II and residues
involving both FL and bc loop as a new epitope. The neutralizing potency and binding
avidity of GR mAbs derived from secondary infection were stronger than those derived
from primary infection.
Second, we investigated cross-reactive anti-E Abs in human polyclonal sera after
secondary DENV infection. Stronger Ab responses to E protein, higher binding avidity
and multitypic neutralizing pattern to both exposed and non-exposed serotypes were
found in sera after secondary DENV infection, compared with those after primary
infection. Depletion with WNV antigen resulted in a significant reduction of neutralizing
activities against four DENV serotypes, suggesting that GR Abs contribute greatly to
neutralizing activities after secondary infection.
Third, we investigated whether E protein ectodomain alone can preserve the
conformation of E protein. We found that C-terminal (cid:302)-helices domains of E protein were
involved in prM-E interaction and maintenance of the stability of prM protein. E protein
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affected the recognition of prM protein by anti-prM mAbs; moreover, E protein
ectodomain alone can be recognized well by all anti-E mAbs tested.
Taken together, our study of GR anti-E Abs at both monoclonal and polyclonal
levels suggest the weakly neutralizing GR anti-E Abs generated from primary DENV
infection become potent neutralizing against four serotypes after secondary infection.
The observations that the dengue immune status of host affects the quality of
cross-reactive Abs generated have implications for new strategies of DENV vaccines.
Moreover, E protein ectodomain alone could be a promising subunit immunogen without
inducing potentially harmful anti-prM response.

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Table of Contents

Acknowledgements ........................................................................................................... ii
Abstract .............................................................................................................................. iii
List of Tables ..................................................................................................................... ix
List of Figures ....................................................................................................................x
List of Abbreviations ....................................................................................................... xii
Chapter 1: Introduction .................................................................................................... 1
Genome and virion structure ........................................................................................... 2
Replication cycle ............................................................................................................. 6
Epidemiology ................................................................................................................... 9
Clinical features of dengue ........................................................................................... 11
Pathogenesis of DHF/DSS ........................................................................................... 12
Epitope of anti-E antibodies .......................................................................................... 13
Antibody responses after DENV infection in humans .................................................. 16
Dengue vaccines ........................................................................................................... 17
Hypothesis and specific aims ........................................................................................ 19
References .................................................................................................................... 22
Chapter 2 : High-avidity and potently neutralizing cross-reactive human
monoclonal antibodies derived from secondary dengue virus infection ................ 35
Abstract ......................................................................................................................... 36
Introduction .................................................................................................................... 37
Materials and methods .................................................................................................. 39
Human mAbs and binding specificity ........................................................................ 39
Epitope mapping by dot blot assay ........................................................................... 39
Virus-like particles -capture ELISA ............................................................................ 40
Epitope analysis by structure-based modeling ......................................................... 40
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Binding avidity by virion-capture ELISA .................................................................... 41
Competitive virion-capture ELISA ............................................................................. 41
Focus reduction neutralization test ........................................................................... 42
Statistical analysis ..................................................................................................... 42
Results ........................................................................................................................... 42
Epitope residues containing FL only or both FL and bc loop of domain II
recognized by GR human anti-E mAbs ..................................................................... 42
Stronger neutralization potency of GR anti-E mAbs derived from secondary
DENV infection than those derived from primary infection ....................................... 44
Higher binding avidity of GR anti-E mAbs derived from secondary DENV
infection than those derived from primary infection .................................................. 44
Repertoire of anti-E mAbs derived from patients with primary and secondary
DENV infections ........................................................................................................ 46
Discussion ..................................................................................................................... 47
Acknowledgements ....................................................................................................... 53
References .................................................................................................................... 63
Chapter 3 : In-depth analysis of cross-reactive anti-envelope antibodies
following secondary dengue virus infection ............................................................... 68
Abstract ......................................................................................................................... 69
Introduction .................................................................................................................... 70
Materials and methods .................................................................................................. 72
Study participants ...................................................................................................... 72
Western blot analysis and dot blot assay .................................................................. 73
Virion ELISA and IgG avidity assay .......................................................................... 73
Focus reduction neutralization test ........................................................................... 74
Depletion of cross-reactive Abs ................................................................................ 74
The proportion of GR Abs and TS Abs ..................................................................... 74
Elution of GR Abs from NC membrane ..................................................................... 75
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Results ........................................................................................................................... 75
Binding specificity and neutralizing pattern of vaccinee sera ................................... 75
GR anti-E Abs contribute to neutralizing activities against all four DENV
serotypes after secondary infection in vaccinees ..................................................... 76
Higher avidity of Abs to exposed and non-exposed serotypes following
secondary DENV infection compared with primary infection .................................... 78
CR anti-E Abs also contribute to NT activities against four DENV serotypes after
secondary infection in vaccinees .............................................................................. 79
Determination of % GR Abs and % TS Abs in sera following secondary DENV
infection in vaccinees ................................................................................................ 80
GR anti-E Abs contribute greatly to neutralizing activities after natural secondary
DENV infection .......................................................................................................... 81
Discussion ..................................................................................................................... 82
References .................................................................................................................... 96
Chapter 4 : C-terminal helical domains of dengue virus type 4 E protein affect
the expression/stability of prM protein and conformation of prM and E proteins 101
Abstract ....................................................................................................................... 102
Introduction .................................................................................................................. 104
Materials and methods ................................................................................................ 106
Plasmid constructs .................................................................................................. 106
Transfection, cell lysates, Western blot analysis and dot blot assay ..................... 107
Mouse and human mAbs ........................................................................................ 108
Radioimmunoprecipitation and pulse-chase experiment ........................................ 108
Sucrose gradient sedimentation analysis ............................................................... 109
Capture ELISA ......................................................................................................... 109
Enzyme digestion .................................................................................................... 110
Results ......................................................................................................................... 110
E protein increases the expression of prM protein and EH2 domain is critical ...... 110
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EH2 domain is involved in maintaining the stability of prM protein ........................ 111
ET1 and EH2 domains are involved in prM-E interaction ....................................... 112
PrM protein and C-terminal truncation of E protein affect the recognition of
E protein by mouse anti-E mAbs ............................................................................. 114
PrM protein and C-terminal truncation of E protein affect the recognition of
E protein by human anti-E mAbs ............................................................................ 116
E protein affects the recognition of prM protein by human anti-prM mAb .............. 117
Discussion ................................................................................................................... 118
Acknowledgements ..................................................................................................... 124
References .................................................................................................................. 138
Chapter 5 : Summary and discussion ........................................................................ 142
Summary ..................................................................................................................... 143
GR Abs are the predominant Abs generated after DENV infection ........................... 144
Maturation status ..................................................................................................... 145
Temperature ............................................................................................................ 145
Antibody-binding ...................................................................................................... 146
Antigen transportation and retention ....................................................................... 146
Increase in the binding avidity of GR Abs suggests memory B cells undergo
affinity maturation after secondary DENV infection .................................................... 147
Alternative strategies for DENV vaccines ................................................................... 148
Future direction ........................................................................................................... 151
References .................................................................................................................. 152

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List of Tables

Chapter 2
Table 1. Patients and origins of group-reactive anti-E human MAbs in this study ...... 54
Table 2. Epitopes of group-reactive anti-E human MAbs in this study ........................ 55
Table 3. Neutralization potencies and binding avidities of group-reactive human
anti-E MAbs in this study............................................................................................... 56
Chapter 3
Table 1. Basic information and serum neutralization titers of vaccinees in this study . 86
Table 2. Reduction in FRNT50 titers (%) after depletion of GR anti-E Abs using
WNV antigens ............................................................................................................... 87
Chapter 4
Table S1. Sequences of the primers for PCR and cloning in this study .................... 125

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List of Figures

Chapter 1
Figure 1. Genome structure and E protein structure and their organizations on virus
particles ........................................................................................................................... 5
Figure 2. The replication cycle of flaviviruses ................................................................. 8
Figure 3. E protein amino acid sequence identity among flaviviruses and cross-
reactivity of anti-E Abs .................................................................................................. 15
Chapter 2
Figure 1. Epitope involving both FL and bc loop residues recognized by a GR
human mAb 753C6 ....................................................................................................... 57
Figure 2. Binding avidity and neutralization potency of GR human anti-E mAbs ........ 59
Figure 3. The % inhibition in competitive virion-capture ELISA of four DENV
serotypes by GR human anti-E mAbs .......................................................................... 60
Figure 4. Relationship between Kd and FRNT50 against four DENV serotypes
for GR mAbs .................................................................................................................. 61
Figure 5. Binding-avidity and neutralization potency of repertoire of anti-E mAbs
from patients with primary and secondary DENV infections ........................................ 62
Chapter 3
Figure 1. The binding specificity and neutralizing patterns in sera of 3 vaccinees
after primary and secondary DENV infections ............................................................. 88
Figure 2. Contribution of GR Abs to neutralizing activities after secondary DENV
infection in vaccinees .................................................................................................... 89
Figure 3. The binding avidities of sera in vaccinees after primary or secondary
DENV infections ............................................................................................................ 90
Figure 4. Epitopes and binding avidity of eluted GR Abs from vaccinee sera after
secondary DENV infection ............................................................................................ 91
Figure 5. Contribution of CR Abs to neutralizing activities after secondary DENV
infection in vaccinees .................................................................................................... 92
Figure 6. The % GR Abs and % TS Abs in sera from vaccinees after secondary
DENV infection .............................................................................................................. 93
x