Antibody mediated immune response against Flavivirus ...

Antibody mediated immune response against

Flavivirus infection in West Australian

travellers

KRITU PANTA

Master of Science in Medical Microbiology

Tribhuvan University, Nepal

This thesis is presented for the degree of Doctor of Philosophy of The University of

Western Australia

School of Biomedical Sciences

2018

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THESIS DECLARATION

I, Kritu Panta, certify that:

This thesis has been substantially accomplished during enrolment in the degree.

This thesis does not contain material which has been accepted for the award of any

other degree or diploma in my name, in any university or other tertiary institution.

No part of this work will, in the future, be used in a submission in my name, for any

other degree or diploma in any university or other tertiary institution without the prior

approval of The University of Western Australia and where applicable, any partner

institution responsible for the joint-award of this degree.

This thesis does not contain any material previously published or written by another

person, except where due reference has been made in the text.

The work(s) are not in any way a violation or infringement of any copyright, trademark,

patent, or other rights whatsoever of any person.

The research involving human data reported in this thesis was assessed and approved

by The University of Western Australia Human Research Ethics Committee.

Approval #: RA/4/1/5420

Written patient consent has been received and archived for the research involving

patient data reported in this thesis.

This thesis does not contain work that I have published, nor work under review for

publication.

Signature

Date: 22-06-2018

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Abstract

This study assessed the immunological significance of Flavivirus diversity by analysing

longitudinal antibody-mediated immune responses in West Australian (WA) travellers

with dengue and Zika virus infection. The majority of study volunteers had never lived

in dengue and Zika endemic or epidemic countries and had well-defined monotypic

infection with DENV or ZIKV; some of them had received YFV vaccination. Dengue

and Zika do not occur in WA as the mosquito vectors are not present. Magnitude of

neutralising antibody, measured by focus reduction neutralisation test (FRNT); total

antibody, measured by haemagglutination inhibition (HI) test; as well as antibody

mediated enhancement (ADE) was assessed for antisera collected from individuals who

were sampled during acute phase (< 2months) and up to 6 years after presentation for

febrile illness. 35 antisera were tested against a collection of DENV isolates derived

from travellers presenting with febrile illness and who were diagnosed with DENV

infection; the virus collection includes representatives of all four serotypes collected

between 2010 – 2015. This collection is dependent on DENV circulating in the Asia

Pacific region at the time and thus is biased towards contemporaneous endemic or

epidemic isolates, predominantly DENV-1 and DENV-2. Phylogenetic analysis

identified genotypes and lineages, and representative isolates were used as targets.

Anti-DENV responses were highly cross-reactive during acute phase and neutralized

heterologous DENV at high magnitude. Over time and up to six years after infection

responses became increasingly serotype-specific - this could be visualised on antigenic

maps - however heterologous cross-neutralisation was maintained in all individuals to

some degree, even at 6 years, and magnitude of neutralisation was virus- and patient-

specific. Virus-specific neutralisation was not genotype or lineage dependent; individual

isolates were differentially neutralised by antisera from individuals collected at the

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same time point after infection. Antigenic mapping showed serotype clustering could be

distinguished after 1 year. However, individual viruses were as close to heterologous

serotype clusters as they were to homologous clusters and this pattern persisted at 6

years. A second major observation was that the magnitude of responses to homologous

DENV in these monotypic travellers also declined over time, including responses to

autologous virus, and this was true for all 4 serotypes. The magnitude of ZIKV-specific

neutralising antibody responses declined over time in the small number (16) of study

volunteers with monotypic ZIKV infection. By 13 and 20 months after infection,

FRNT90 titres were below the limits of detection (<40 as per WHO criteria for

serodiagnosis of ZIKV infection). However, ZIKV-infected individuals with previous

flavivirus infection showed prolonged anti-ZIKV antibody responses up to 13 months

post onset of illness. This study demonstrated that antibody binding to ZIKV

capsid/prM protein can differentiate recent ZIKV infection in individuals with previous

DENV infection. Two ZIKV strains were assessed and showed a high degree of

differential cross-neutralisation, with high magnitude responses directed against the

prototype ZIKV MR766 isolate but not the contemporaneous ZIKV PRVABC59 in

acute phase monotypic DENV infection, highlighting the importance of strain selection

in ZIKV diagnostic and research approaches. ZIKV replication was enhanced by acute

phase (< 2months) antisera only.

Future work includes assessment of additional antisera against viruses which will

include those DENV that have circulated more recently in the region, including

serotypes 3 and 4 which have not circulated widely, and which were under-represented

in the present study; and assessment of the flavivirus-specific B cell repertoire.

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Acknowledgements

I am incredibly grateful to my Principal Supervisor A/Prof Allison Imrie for her

guidance, patience and encouragement throughout my PhD. She has been a great

mentor constantly guiding me on how to progress my research and how to focus my

thinking so as to make logical and direct decisions. I am greatly indebted to her for the

efforts she has made to shaping me and my research throughout the past four years. I

sincerely thank all my co-supervisors: Clin/Prof. David Smith, Prof Nicholas De Klerk

and Dr. Alfred Tay for all their support and guidance during my PhD. I also want to

convey my gratitude to the late Professor Geoffrey Shellam for his support and

encouragement during the early stages of my PhD.

I am grateful to all my lab members for their constant support and encouragement

during times of success and failure in experiments. Thank you all for making my

Australian study experience wonderful. I am truly thankful to Suzi McCarthy for

teaching me laboratory skills and being there to listen. I want to thank Timo for his

constant support in the lab, and his family for all those Christmas and Easter lunches

making me feel like home. I wish to thank Alice and Harapan for all their help in the

lab. My list for lab friends would not complete without the late Peter Dunstan. I cherish

moments we had together, from first moment of learning lab skills, thanks Pete, you

were one of the most wonderful souls I have met. And finally Kara, my Aussie-

American best friend, thank you for our friendship and unflinching support and

encouragement during my PhD.

I thank Australia Awards Endeavour Postgraduate Fellowship for sponsoring my PhD

studies. I am also grateful to scholarship case managers for their constant support. I

want to thank all the study volunteers of this study for their gracious support. I wish to

thank the Serology department of PathWest Laboratory Medicine WA for supporting

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laboratory procedures. I would also like to thank all the staff and PhD students of the

School of Biomedical Sciences for their support and little tips and tricks for

experiments.

Most importantly I am grateful to my hard-working mother for her undiminishing

support, love and prayers and for having faith in me. I still feel sorry I could not be

with you during the hardest times of the earthquake but I assure you all your sacrifice is

not wasted. I am also thankful to all my family members and friends back home in

Nepal and here in Perth. Words fail to express my gratitude to my husband Binit whose

constant encouragement, guidance and support throughout this journey has made it a lot

easier. Thank you Binit, for your faith in me, your constant support during all those ups

and downs during my PhD kept me going and thank you for making me smile even in

the worst situations.

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Table of contents

Abstract ______________________________________________________________ 3

Acknowledgements _____________________________________________________ 5

List of Tables ________________________________________________________ 12

List of Figures _______________________________________________________ 14

List of abbreviations ___________________________________________________ 17

List of conference/meeting abstracts ______________________________________ 18

Chapter 1:- Thesis Introduction _________________________________________ 19

1.1. Overview of the study ___________________________________________ 19

1.2. Aims of study __________________________________________________ 20

1.3. Outline of chapters ______________________________________________ 21

Chapter 2: - Literature review ___________________________________________ 23

2.1.Preamble ______________________________________________________ 23

2.2. Flavivirus ______________________________________________________ 23 2.2.1. Virus structure ________________________________________________________ 25 2.2.2. Replication ___________________________________________________________ 29

2.3. Dengue Virus __________________________________________________ 32 2.3.1. Origin and distribution __________________________________________________ 32 2.3.2. Classification _________________________________________________________ 37 2.3.3. DENV Vector and Transmission __________________________________________ 42 2.3.4. Clinical spectrum of dengue disease _______________________________________ 45 2.3.5. Dengue case classification _______________________________________________ 47

2.4. Zika Virus ___________________________________________________ 49 2.4.1. Classification _________________________________________________________ 52 2.4.2. Vector borne ZIKV transmission __________________________________________ 53 2.4.3. Non- vector transmission of Zika _________________________________________ 54 2.4.4. Infection and complications ______________________________________________ 55

A) Guillian-Barre syndrome (GBS) ______________________________________________ 55 B) Neonatal ZIKV-related complications _________________________________________ 56

2.5. Flavivirus vaccine strains ______________________________________ 58 2.5.1. Yellow fever _________________________________________________________ 58 2.5.2. Japanese encephalitis ___________________________________________________ 59

2.6. Flavivirus immunopathogenesis _________________________________ 62 2.6.1. Flavivirus-specific innate immune responses ____________________________________ 62

A) Interferons _______________________________________________________________ 62 B) Complement ______________________________________________________________ 63

2.6.2. Acquired immune responses _________________________________________________ 65 A) Cellular immunity _________________________________________________________ 65 B) Humoral immunity _________________________________________________________ 67

i) Neutralising or protective antibody-mediated immunity ______________________ 70 ii) Cross reactive and enhancing antibody mediated immune response _____________ 71

2.7. Laboratory diagnosis of Flavivirus infection _______________________ 76 2.7.1. Virus isolation ________________________________________________________ 78 2.7.2. Genome detection _____________________________________________________ 79

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2.7.3. Antigen detection ______________________________________________________ 80 2.7.4. Serology _____________________________________________________________ 81

2.8. Flavivirus infection in travellers _________________________________ 84 2.8.1. Dengue ______________________________________________________________ 84 2.8.2. Zika ________________________________________________________________ 85

Chapter 3:- Methods __________________________________________________ 87

3.1. Vero cells ______________________________________________________ 87

3.2. Virus amplification ______________________________________________ 87

3.3. Virus Titration _________________________________________________ 88 3.3.1. Determination of Tissue culture infectious dose __________________________________ 88 3.3.2. Determination of Focus forming units __________________________________________ 88

a. Preparation of monolayer ____________________________________________________ 88 b. Virus dilution _____________________________________________________________ 88 c. Test establishment __________________________________________________________ 88 d. Detection of FFU __________________________________________________________ 89

i. Optimisation of Fixative ________________________________________________ 89 ii. Optimisation of 4G2 monoclonal antibody concentration (primary antibody): _______ 89 iii. Optimising secondary antibody concentration ________________________________ 90 iv. Immunostaining _______________________________________________________ 90

3.3.3. Determination of Plaque forming units _________________________________________ 91

3.4. Methods for Chapters 5 and 6 _____________________________________ 92 3.4.1. Neutralising Antibody titre determination _______________________________________ 92

3.4.1.1. Preface ______________________________________________________________ 92 3.4.1.2. Establishment of neutralisation test ________________________________________ 94

a. Preparation of monolayer __________________________________________________ 94 b. Preparation of plasma sample ______________________________________________ 94 c. Focus reduction neutralisation test ___________________________________________ 94

i. Preparation of virus dilution ___________________________________________ 94 ii. Test establishment ___________________________________________________ 94 iii. Immunostaining and visualisation of FFU ________________________________ 95 iv. Plaque reduction neutralisation test ______________________________________ 95

d. Neutralisation titre determination ___________________________________________ 95 3.4.2. Hemagglutination inhibition antibody titre determination __________________________ 99

3.4.2.1 Principle _____________________________________________________________ 99 3.4.2.2. Methodology to establish HI test _________________________________________ 100

a. Preparation of viral antigen _____________________________________________ 100 b. Titration of inactivated virus ____________________________________________ 100 c. Preparation of plasma sample ___________________________________________ 100 d. Establishment of test __________________________________________________ 101

3.4.3. Mapping antigenic diversity using cartography _________________________________ 102 3.4.3.1. Preface _____________________________________________________________ 103 3.4.3.2. Cartography technique _________________________________________________ 103

3.4.4. Western blot ____________________________________________________________ 105 3.4.4.1. Preface _____________________________________________________________ 105 3.4.4.2. Methods ____________________________________________________________ 105

a. Optimisation of whole virus protein preparation _______________________________ 105 b. Quantification of Protein _________________________________________________ 107 c. Optimization of primary antibody concentration _______________________________ 107 d. SDS PAGE and Western blot ______________________________________________ 107 e. Detection of viral proteins ________________________________________________ 108

3.5. Method for chapter 7: - Antibody dependent enhancement ___________ 110 a. Cell lines ________________________________________________________________ 110 b. Virus stock and plasma samples ______________________________________________ 110

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c. Establishment of test _______________________________________________________ 111 d. Detection of infectious units and determination of enhancement of infection. ___________ 111

Chapter 4: Study population and viruses _________________________________ 114

4.1. Preamble _____________________________________________________ 114

4.2. The cohort ____________________________________________________ 114 a. DENV-1 sera panel _____________________________________________________ 116 b. DENV-2 sera panel _____________________________________________________ 117 c. DENV-3 sera panel _____________________________________________________ 117 d. DENV-4 sera panel _____________________________________________________ 117 e. ZIKV sera panel ________________________________________________________ 118

4.2. Viruses used in this study _______________________________________ 124 4.2.1 DENV Panel _____________________________________________________________ 124

a. DENV-1 ________________________________________________________________ 127 b. DENV-2 ________________________________________________________________ 130 c. DENV-3 ________________________________________________________________ 133 d. DENV-4 ________________________________________________________________ 136 e. Autologous DENV ________________________________________________________ 139

4.2.2. ZIKV Panel _____________________________________________________________ 139 4.2.3. Flavivirus vaccine strains __________________________________________________ 142

a. Japanese encephalitis vaccine ______________________________________________ 142 b. Yellow fever vaccine strain _________________________________________________ 142

Chapter 5:- DENV-specific antibody responses in monotypic infection _________ 144

5.1. Preamble _____________________________________________________ 144

5.2. Introduction __________________________________________________ 144

5.3. Aims _________________________________________________________ 146

5.4. Results _______________________________________________________ 146 5.4.1. Neutralising antibody _____________________________________________________ 146

a. Specificity and cross-reactivity post DENV-1 infection _________________________ 148 i. Anti-DENV-1 antisera differentially neutralize DENV-1 ______________________ 156 ii. Neutralization among homologous DENV-1 decreases over time _______________ 158 iii. Cross-neutralisation against heterologous DENV ____________________________ 161 iv. Longitudinal decrease in heterologous DENV cross-neutralization by anti-DENV-1

antisera _________________________________________________________________ 164 v. Persistent neutralisation against autologous virus ____________________________ 166 vi. Cross-neutralization of ZIKV, YF17D and IMOJEV by DENV-1 antisera ________ 168

b. Specificity and cross-reactivity of anti-DENV-2 antibody _______________________ 170 i. Anti-DENV-2 neutralisation specificity against homologous serotype ____________ 177 ii. DENV-2 homologous NT decreased with time post infection___________________ 180 iii. Cross-neutralisation against heterologous DENV ____________________________ 182 iv. Decreased cross-neutralisation over time___________________________________ 185 v. Neutralisation against autologous DENV-2 _________________________________ 187 vi. Neutralisation against ZIKV, YF17D and IMOJEV. __________________________ 189

c. Neutralising responses post DENV-3 infection ________________________________ 191 i. Neutralisation among homologous DENV-3 ________________________________ 197 ii. DENV-3 neutralisation over time ________________________________________ 199 iii. Neutralisation against autologous serotype _________________________________ 205 iv. Neutralisation against related serogroups __________________________________ 205

d. Specificity of neutralising antibody response post DENV-4 infection _______________ 207 i. Longitudinal DENV-4 antisera against homologous virus _____________________ 214 ii. Heterologous cross-neutralisation by anti-DENV-4 antisera ____________________ 218 iii. Neutralisation against autologous serotype _________________________________ 218 iv. Neutralisation against related serogroups __________________________________ 218

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5.4.2. Magnitude of neutralising antibody over time ________________________________ 221 5.4.3. Summary of neutralisation in DENV monotypic infection ______________________ 224 5.4.4. Total anti-DENV antibody: _______________________________________________ 225

a. Total antibody responses, DENV-1 infection _________________________________ 226 b. Total antibody responses, DENV-2 infection _________________________________ 230 c. Total antibody response post DENV-3 infection _______________________________ 235 d. Total antibody responses post DENV-4 infection ______________________________ 239

5.4.5. Summary of total antibody in DENV monotypic infection ______________________ 242 5.4.6. Longitudinal variation of neutralising and recognising anti-DENV antibody _______ 243

a. Cross-reactive anti-DENV responses within 2 months post infection _______________ 245 b. Anti-DENV antibody responses 2-12 months post infection ______________________ 247 c. Anti-DENV antibody responses 1-2 years post infection_________________________ 249 d. Anti-DENV antibody responses 2-4 years post infection_________________________ 251 e. Anti-DENV antibody responses 4 years post infection __________________________ 251

5.5. Summary discussion ____________________________________________ 254 5.5.1. Neutralisation endpoint stringency and increased specificity _______________________ 254 5.5.2. Anti-DENV antibody responses are virus- and time-dependent _____________________ 255

5.6. Research outcomes _____________________________________________ 256

Chapter 6: Antibody immune response post ZIKV infection __________________ 258

6.1 Introduction ___________________________________________________ 258

6.2. Aims of study _________________________________________________ 261

6.3. Results _______________________________________________________ 262 6.3.1. Neutralising antibody response ______________________________________________ 262 a. Within 1 month ___________________________________________________________ 267 b. 1 – 12 months post presentation ______________________________________________ 268 c. More than 12 months post presentation ________________________________________ 269

6.3.2. Total anti-ZIKV antibody _____________________________________ 274

6.3.3. Immunoblot analysis, ZIKV and DENV __________________________ 276 6.3.3.1. Positive and negative control ______________________________________________ 277 6.3.3.2. Anti-ZIKV antisera ______________________________________________________ 277 6.3.3.3. Anti-ZIKV/DENV antisera _______________________________________________ 278 6.3.3.4. Anti- ZIKV/YF17D _____________________________________________________ 278 6.3.3.5. Anti-DENV ___________________________________________________________ 278

6.4. Summary discussion ____________________________________________ 285 6.4.1. Anti-ZIKV specificity and cross-reactivity _____________________________________ 286 6.4.2. Anti-ZIKV antisera cross-neutralisation of DENV is time- and flavivirus background-

dependent ___________________________________________________________________ 288 6.4.3. Persistence of anti-ZIKV NAb in individuals with previous flavivirus infection ________ 289 6.4.4. Differential ZIKV MR766 and anti-ZIKV PRVABC59 neutralisation ________________ 289 6.4.5. Virus protein binding analysis by immunoblot __________________________________ 291


Chapter 7: - Enhancement of ZIKV infection by monotypic anti-DENV antibody 294

7.1. Preamble _____________________________________________________ 294

7.2 Introduction ___________________________________________________ 294 7.2.1. Mechanism of ADE _______________________________________________________ 295 7.2.2. ADE in DENV and ZIKV infection __________________________________________ 295

7.3. Results _______________________________________________________ 299 7.3.1. Acute phase plasma: DENV plasma sample within two months post infection _________ 302

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7.3.2. ZIKV enhancement by convalescent anti-DENV antisera collected 1-6 years post infection

____________________________________________________________________________ 303 7. 3.3. DENV-1 plasma samples __________________________________________________ 303 7.3.4. DENV-3 plasma samples __________________________________________________ 304 7.3.5. Pooled sera from DENV-1 and DENV-3 patients ________________________________ 304

a) At six months post infection _______________________________________________ 304 b) At 12 months post infection _______________________________________________ 304

7.4. Summary discussion ____________________________________________ 310 7.4.1. DENV and ZIKV in French Polynesia ________________________________________ 310 7.4.2. ZIKV enhancement by monotypic anti-DENV from travellers ______________________ 311


Chapter 8:- Summary and conclusion ___________________________________ 314

Chapter 9:- References _______________________________________________ 317

Appendixes _________________________________________________________ 338

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List of Tables

Table 2.1:- Assigning clades and clusters to flaviviruses reported by Kuno et al (8) .... 24

Table 2.2:- Flavivirus genes and their functions (12) ..................................................... 27

Table 2.3: Classification of DENV serotypes ................................................................. 40

Table 4. 1:- Demographic and laboratory findings of individuals with DENV-1

infection ........................................................................................................................ 119

Table 4. 2:- Demographic and laboratory findings of individuals with DENV -2

infection ........................................................................................................................ 120


infection ........................................................................................................................ 121


infection ........................................................................................................................ 122

Table 4. 5:- Demographic and Laboratory findings of individuals with ZIKV infection

....................................................................................................................................... 123

Table 4. 6:- Flaviviruses used in study ......................................................................... 126

Table 4. 7:- DENV-1 amino acid variation ................................................................... 129



Table 4. 10:- DENV-4 amino acid variation ................................................................. 138

Table 4. 11:- ZIKV amino acid variation ...................................................................... 141

Table 4. 1:- Demographic and laboratory findings of individuals with DENV-1

infection ........................................................................................................................ 150

Table 5. 1:- Anti-DENV-1 neutralising antibody titre at 50% reduction ..................... 152


Table 5. 3:-Anti-DENV-1 neutralising antibody titre at 90% ....................................... 154

Table 5. 4:- DENV-1-Sera panel statistical verification ............................................... 155

Table 4. 2:- Demographic and laboratory findings of individual with DENV -2

infection ........................................................................................................................ 171




Table 5. 8:- DENV-2-Sera panel statistical verification ............................................... 176


infection ........................................................................................................................ 192

Table 5. 9:- Anti-DENV-3 neutralising antibody titre at 50% reduction..................... 193

Table 5. 10:-Anti-DENV-3 neutralising antibody titre at 75% reduction..................... 194

Table 5. 11:- Anti-DENV-3 neutralising antibody titre at 90% reduction.................... 195

Table 5. 12:- DENV-3-Sera panel statistical verification ............................................. 196


infection ........................................................................................................................ 208

Table 5. 12:- Anti-DENV-4 neutralising antibody titre, 50% reduction ...................... 210


Table 5. 16:-DENV-4-Sera panel statistical verification .............................................. 212


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Table 5. 15:- Total antibody response in DENV-1 sera panel ...................................... 227

Table 5. 18:- Total antibody response DENV-2 sera panel .......................................... 232

Table 5. 19:- Total antibody response of DENV-3 sera panel ...................................... 236

Table 5. 20:- Total antibody response in DENV-4 sera panel ...................................... 240

Table 4. 5: - Demographic and laboratory findings of individuals with ZIKV infection

....................................................................................................................................... 264

Table 6. 1:- Neutralising antibody response in ZIKV ................................................... 265

Table 6. 2: - Total antibody titre against ZIKV ............................................................ 275

Table 7. 1:- Description of plasma samples used in ADE ............................................ 300

Table 7. 2:- Virus output from U937 post exposure to respective anti-DENV - ZIKV

immune complex ........................................................................................................... 301








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List of Figures

Figure 2.1:- Flavivirus Structure ..................................................................................... 28

Figure 2.2:- Flavivirus Replication ................................................................................. 31

Figure 2.3:-Dengue distribution in 2008 and 2016 ......................................................... 36

Figure 2.4:- DENV Phylogeny........................................................................................ 41

Figure 2.5:-DENV transmission cycle and mosquito-vectors (9) ................................... 42

Figure 2.6: Dengue clinical manifestations (World Health Organization 2009) ............ 46

Figure 2.8:- WHO Classification of dengue infection (2009) ........................................ 48

Figure 2.7:- WHO Classification of dengue infection (1997) ........................................ 48

Figure 2.9: -Zika virus spread from 1947-2016 ............................................................. 51

Figure 2.10:-The rise and fall of Zika ............................................................................. 51

Figure 2.11:- ZIKV Phylogeny ....................................................................................... 53

Figure 2.12- Immunological response during Flavivirus infection................................. 69

Figure 2.13:- Antibody threshold determining immune response in Flavivirus infection

......................................................................................................................................... 74

Figure 2. 14:- Adaptive immune responses in Flavivirus infection. ............................... 75

Figure 2.15:- Course of dengue infection and timing of diagnosis tests (7) .................. 77

Figure 2.16 :-Diagnosis of dengue. ................................................................................. 78

Figure 3.1: - Neutralisation ............................................................................................. 93

Figure 3. 2:- Establishment of neutralisation .................................................................. 96

Figure 3. 3:- Immunostaining and visualisation of FFU ................................................. 97

Figure 3. 4:- FRNT and PRNT ....................................................................................... 98

Figure 3. 5:- Hemagglutination of GRBC by flavivirus ................................................. 99

Figure 3. 6:- Hemagglutination inhibition by anti-flavivirus antibody ........................... 99

Figure 3. 7:- Measurement of total antibody by hemagglutination inhibition test ...... 102

Figure 3. 8:- Antigenic mapping strategy ..................................................................... 104

Figure 3. 9:- Comparison of protein lysate preparation methods ................................. 106

Figure 3. 10: Flow chart of Western blot analysis ........................................................ 109

Figure 3. 11:- Measurement of antibody-dependent enhancement of ZIKV infection by

anti-DENV antibody ..................................................................................................... 112

Figure 3. 12:- Viral load in the presence and absence of anti-DENV antibody ........... 113

Figure 4. 1:- Traveller cohort establishment and virus isolation. ................................. 115

Figure 4. 2:- Phylogenetic tree of DENV E-sequences including isolates from travellers

(11). ............................................................................................................................... 125

Figure 4. 3:- DENV-1 E- gene (1-394) alignment ........................................................ 128

Figure 4. 4:- DENV-2 E-gene (1-394) alignment ........................................................ 131

Figure 4. 5:- DENV-3 E-gene (1-394) alignment ......................................................... 134

Figure 4. 6:- DENV-4 E-gene (1-394) alignment ......................................................... 137

Figure 4. 7:- ZIKV E-gene alignment ........................................................................... 140

Figure 4. 8:- Phylogenetic tree showing the genetic relationship among the flaviviruses

strains used in this study. .............................................................................................. 143

Figure 5. 1:- Neutralising antibody response post DENV-1 infection, patient FLV-011

....................................................................................................................................... 151

Figure 5. 2:- Homologous strain neutralisation by DENV-1 antisera........................... 157

Figure 5. 3:- Differential neutralisation among DENV-1 homologous strains, patient

FLV-011 ........................................................................................................................ 159

Figure 5. 4:-Longitudinal variation of anti-DENV-1 neutralising antibody ................. 160













































































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Figure 5. 5:- Heterologous cross-neutralisation by anti-DENV-1 antibody ................. 163

Figure 5. 6:- Longitudinal heterologous cross-neutralisation by anti-DENV-1 antibody

....................................................................................................................................... 165

Figure 5. 7:- Autologous, homologous and heterologous neutralisation ...................... 167

Figure 5. 8:- Cross-neutralisation across flavivirus serogroup ..................................... 169

Figure 5. 9:- Neutralising antibody response by anti-DENV-2 antisera, patient FLV -

014 ................................................................................................................................. 172

Figure 5. 10:- Homologous strain neutralisation by anti-DENV-2 antibody ............... 179

Figure 5. 11:- DENV-2-specific neutralisation over time, patient FLV 014 ................ 181

Figure 5. 12:- Variation of homologous strain neutralisation by anti-DENV-2. .......... 183

Figure 5. 13:- Heterologous cross-neutralisation by DENV-2 antisera ........................ 184

Figure 5. 14:- Decrease in heterologous neutralisation time post infection .................. 186

Figure 5.15:- Neutralisation against DENV-2 autologous strains ................................ 188

Figure 5. 16:- Longitudinal cross-neutralisation of flavivirus serogroups by anti-DENV-

2 antibody ...................................................................................................................... 190

Figure 5.17:- Homologous strain neutralisation by anti-DENV-3 antisera. ................. 198

Figure 5. 18:- Strain specificity over time by DENV-3 sera panel, patient FLV-038. . 200

Figure 5.19:- Change in homologous neutralisation over time, anti-DENV-3 antibody

....................................................................................................................................... 201

Figure 5. 20:- Heterologous cross-neutralisation by DENV-3 antisera ........................ 203

Figure 5. 21:- Heterologous neutralisation over time by DENV-3 antisera. ................ 204

Figure 5.22:- Autologous strain neutralisation by DENV-3 antisera ........................... 206

Figure 5.23:- Homologous and heterologous DENV neutralisation by anti-DENV-4

antibody, patient FLV 049 ............................................................................................ 209

Figure 5. 24:- Homologous virus neutralisation by DENV-4 antisera ........................ 215

Figure 5. 25:- Homologous strain neutralisation by DENV-4, patient FLV049........... 216

Figure 5. 26:- Variation in homologous strain neutralisation by DENV-4 antisera over

time. .............................................................................................................................. 217

Figure 5. 27: - Heterologous strain neutralisation by anti-DENV-4 antisera ............... 219

Figure 5. 28: - Heterologous neutralisation over time by anti-DENV-4 antisera. ........ 220

Figure 5. 29:- Magnitude of anti-DENV within 1 year of infection ............................. 223

Figure 5. 30:- Magnitude anti-DENV 1-2 years post infection .................................... 223

Figure 5.31:- Magnitude of anti-DENV 3-6 years post of infection ............................ 223

Figure 5.32:- Summary of homologous and heterologous NAb responses .................. 224

Figure 5. 33:- Total antibody responses against homologous DENV-1 ....................... 228

Figure 5. 34: - Total antibody responses, anti-DENV-1 antisera against heterologous

DENV ........................................................................................................................... 229

Figure 5. 35:- Total antibody responses, Anti-DENV-2 antisera against homologous

DENV-2 ........................................................................................................................ 233

Figure 5. 36:- Total antibody responses, anti-DENV-2 antisera against heterologous

DENV. .......................................................................................................................... 234

Figure 5. 37: - Total DENV-3 antibody response against homologous DENV ............ 237

Figure 5. 38:- Total antibody responses, anti-DENV-3 antisera against heterologous

DENV ........................................................................................................................... 238

Figure 5.39: - Total DENV-4 antibody against homologous DENV ............................ 241

Figure 5.40: - Total antibody responses, anti-DENV-4 antisera against heterologous

DENV ........................................................................................................................... 242









































































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Figure 5. 41: - Summary of homologous and heterologous anti-DENV TAb .............. 243

Figure 5. 42:- Anti-DENV response and antigenic diversity at less than two months post

infection ........................................................................................................................ 246

Figure 5. 43:- Anti-DENV antibody response with a year of infection ........................ 248

Figure 5. 44:- Specificity of anti-DENV antibody 1-2-year post infection .................. 250

Figure 5. 45:- Anti-DENV-4 antibody at 2-4 years post infection ............................... 252

Figure 5. 46:- Anti-DENV-4 antibody 4 years post infection....................................... 253

Figure 6. 1:- Neutralising antibody response post ZIKV infection. ............................. 266

Figure 6. 2:- ZIKV neutralisation within 30 days post presentation of illness. ............ 270

Figure 6. 3:- ZIKV neutralisation 1-12 months post presentation of illness. ............... 271

Figure 6. 4:- ZIKV neutralisation more than 12 months post presentation of illness. .. 272

Figure 6. 5:- Variation in ZIKV neutralising antibody concentration over time based on

the flavivirus background ............................................................................................. 273

Figure 6. 6:- Coomassie stain. ....................................................................................... 280

Figure 6. 7:- Positive and Negative control .................................................................. 281

Figure 6. 8:- Monotypic anti-ZIKV antisera (FLV-064/FLV090) ................................ 282

Figure 6. 9:- Anti-ZIKV/DENV antisera (FLV-087).................................................... 283

Figure 6.10:- Anti-ZIKV/YFV antisera (FLV-032) ...................................................... 284

Figure 6. 11:- Anti-DENV antisera(FLV002/FLV030) ................................................ 285

Figure 7. 1:-ZIKV enhancement by monotypic anti-DENV pool at different stage post

infection. ....................................................................................................................... 306

Figure 7. 2:-ZIKV enhancement by anti-DENV-1 antisera .......................................... 307

Figure 7. 3:- ZIKV enhancement by anti-DENV-3 antisera ......................................... 308

Figure 7. 4 :-ZIKV enhancement based on French Polynesian co-circulation model. . 309











































17

List of abbreviations

% percent

/ per

µl Microliter

ADE Antibody dependent enhancement

CPE Cytopathic effect

DENV Dengue Virus

DMEM Dulbecco's Modified Eagle's Medium

E Envelope protein

ELISA Enzyme linked immunosorbent assay

FBS Fetal bovine serum

FFU Focus forming units

FRNT Focus reduction neutralization test

HA Hemagglutination assay

HI Hemagglutination inhibition

IgG Immunoglobulin G

IgM Immunoglobulin M

JE IMOJEV Japanese encephalitis vaccine strain

mAb Monoclonal antibody

NAb Neutralizing antibody

NT Neutralization titer

PBS Phosphate buffer solution

PCR Polymerase chain reaction

PFU Plaque forming units

prM Pre-membrane protein

PRNT Plaque reduction neutralization test

RPMI 1640 Roswell Park Memorial Institute 1640 medium

SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis

TAb Total antibody

WHO World Health Organization

YF17D Yellow fever 17D vaccine strain

ZIKV Zika Virus

18

List of conference/meeting abstracts

“Using cartography to define antigenic relationship among dengue viruses

(DENV) imported by travellers. American Society of Tropical Medicine and

Hygiene (ASTMH) 66th

Annual Meeting, Baltimore, United States of America,

2017.

“Using cartography to define antigenic relationship among dengue viruses

(DENV) imported by travellers”. Combined Biological Sciences Meeting

(CBSM), Perth, Australia, 2017.

“Antigenic mapping among imported Dengue virus in Western Australia”.

Centre for Research Excellence in Emerging Infectious Diseases (CREID)

Annual Colloquium, Sydney, Australia, 2017.

“Zika virus neutralizing antibodies in West Australian Travelers”. Australian

society of Microbiology (ASM) Annual Scientific Meeting, Perth, Australia,

2016.

“Illustrating antigen specific B-cell clonal expansion using RNA-seq”. West

Australian Society of Medical Research WA (ASMR-WA) symposium, Perth,

Australia, 2015.

“Illustrating antigen specific B-cell clonal expansion using RNA-seq” Science

on Swan Meeting, Perth, Australia, 2015.

19

Chapter 1:- Thesis Introduction

1.1. Overview of the study

This thesis is presented as a series of chapters that include the study of antibody

mediated immune responses elicited following dengue (DENV) and Zika (ZIKV) virus

infection.

Anti-DENV and anti-ZIKV humoral responses were studied in a group of travellers

who were infected during travel to endemic and epidemic countries. DENV and ZIKV

were strains isolated from viremic travellers; prototype strains; and representatives of

virulent and non-virulent lineages. Neutralising and total antibody responses were

studied. Cartographic maps were created to analyse antigenic relationships among

viruses and antisera. Furthermore cross-reactivity among flavivirus serogroups was also

studied.

Cross neutralisation among strains of ZIKV, cross-reactivity among DENV strains and

the flavivirus vaccine viruses YF17D and IMOJEV was studied. Identification of

antibody recognition site on viral protein was undertaken by Western immunoblot

analysis. Antibody-mediated enhancement of ZIKV in the presence of non-neutralising

concentrations of anti-DENV antibody was also studied.

Overall this thesis is an analysis of the different mechanism of antibody-mediated

immune response in monotypic and multitypic DENV and ZIKV infection among

travellers and how these responses change over time.

20

1.2. Aims of study

The main aim of the study is to understand of antibody mediated immune responses to

flavivirus infection in West Australian travellers, the great majority of whom present

with monotypic infection.

Specific Aims

1. To understand longitudinal variation of anti-DENV antibody responses against

contemporaneous DENV circulating in the Asia Pacific region, in a group of

travellers with monotypic infection

2. To determine specificity and cross-reactivity of anti-ZIKV antibody responses

against DENV and flavivirus vaccine viruses and to assess persistence of anti-ZIKV

immunity over time

3. To identify a marker that may be used to differentiate ZIKV and DENV infection

4. To determine ZIKV enhancement by anti-DENV antibody

21

1.3. Outline of chapters

Chapter 2 is a review of the literature on flaviviruses and immunopathogenesis of

flavivirus infection. The chapter begins with studies on flavivirus genus, structure and

replication and discusses dengue, Zika, and the yellow fever and Japanese encephalitis

vaccine viruses. The second section of the chapter reviews immunological responses

elicited by infection, with emphasis on the antibody-mediated immune response. The

final section reviews traveller-associated flavivirus infection and risk.

Chapter 3 details experimental methods used in the study. This chapter includes details

on optimization and establishment of tests used to derive DENV- and ZIKV-specific

antibody-mediated immune response.

The study population and viruses used in this study are described in Chapter 4. This

chapter includes demographic information and clinical history as well as genetic

characterisation of the viruses used in this study.

Chapter 5 defines the antibody-mediated immune response in monotypic dengue virus

infection. This chapter includes analysis of neutralising and total antibody responses

over time, against genetically diverse strains of DENV and other flaviviruses.

Furthermore, this chapter applies antigenic cartography to illustrate antigenic

relationships among cohort antisera and contemporaneous DENV.

In chapter 6, antibody-mediated immune responses among travellers with confirmed

and probable Zika virus infection was studied. This work identifies differential

neutralisation among patients with monotypic ZIKV infection, and patients with a

background of DENV infection and/or flavivirus vaccination. Differential neutralisation

22

among ZIKV strains and other flaviviruses is included in this chapter. Furthermore, this

chapter seeks to identify specific anti-ZIKV antibodies by westernblot.

Chapter 7 includes an assessment of enhancement of ZIKV in the presence of anti-

DENV antibody.

Chapter 8 summarize major findings of the study. In addition, this chapter outlines

implications of the work described in this thesis, and work that is being undertaken to

extend the findings of these studies.

23

Chapter 2: - Literature review

2.1. Preamble

This chapter provides an overview of the literature on flaviviruses and anti-flavivirus-

specific immune response. It provides background information on the Flavivirus genus

and focuses on dengue virus (DENV), Zika virus (ZIKV) and the vaccine strains of

yellow fever (YF17D) and Japanese encephalitis (IMOJEV). Available literature on

DENV and ZIKV with emphasis on viral genetic diversity and host immunological

responses was reviewed.

2.2. Flavivirus

The Flavivirus genus belongs to family Flaviviridae. It is a group that includes more

than 70 arthropod-borne viruses. Phylogenetically, these viruses are classified into

genotypes, clades, lineages and species; serological criteria divide the Flavivirus genus

into antigenic complexes (14).

Kuno et al. reported 14 clades based on conserved flavivirus genetic sequence and three

clusters based on vector associations (15). Gould et al described four

phylogenetic/ecological flavivirus groups: - two mosquito-borne, a tick-borne group,

and non-vectored viruses (16). In 1988, 68 then-recognised viruses were classified into

eight antigenic complexes: - tick-borne encephalitis (12 viruses), Rio Bravo (six),

Japanese encephalitis (10), Tyuleniy (three), Ntaya (five), Uganda S (four), dengue

(four) and Modoc (five) (15, 17). It is still uncertain which group is the oldest (18). The

most widely used classification of flaviviruses is clade and antigenic complex

classification by Kuno et al in 1998 as summarised in Table 2.1

24

Viruses in boldface belong to the corresponding antigenic complex to the right.

Viruses highlighted in grey are the flaviviruses used in this study

Refer Kuno et al for detail

Table 2.1:- Assigning clades and clusters to flaviviruses reported by Kuno et al (8)

25

2.2.1. Virus structure

Flaviviruses are ~500Å icosahedral asymmetric particles bearing ss-positive sense

RNA. Viral genomes code for single polypeptides that form structural and non-

structural proteins. Structure is formed by stoichiometric arrangement of three structural

proteins: core/capsid (C, 100AAs), membrane (M, 75AAs) and envelope (E, 495 AAs)

(2). The non-structural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 are

involved in immune invasion, pathogenesis and viral replication (19-22).

Capsid (C) protein forms the nucleocapsid which encloses the viral genome. The

approximately 100-amino acid C protein shares low homology among flaviviruses but

shares a similar distribution of basic amino acids and hydrophobicity profiles (23). It

plays an important role in encapsidation of the viral genome and may also be involved

in regulation of viral replication (24). C-protein has also been reported to inhibit stress

granule formation thus facilitating propagation of virus in host cells (25).

The envelope (E) protein contains a transmembrane anchor and 3 globular domains

linked to the anchor by a stem region. Flaviviruses share the common envelope protein

with ~40% amino acid homology in their envelope protein. Studies in TBEV show two

domain form an elongated shape consisting of a central domain (DI) that connects an

Ig-like domain (DIII) to a dimerization domain (DII), the whole structure lying flat

along the surface of the virus lipid bilayer (26). Similarly in DENV, DI is described as a

β-barrel at the core connecting DII finger-like domain with a fusion loop at one end and

DIII at other end with short polypeptide, DI/III linker (27). DII is a long finger-like

protrusion from DI and contains a second N-linked glycan that binds to dendritic cell

surface receptors (28). DII has been shown to be involved in virus attachment to Vero

cells in culture (29, 30). These binding characteristics have been confirmed using

expressed DIII (31). As a Class II fusion protein, the E glycoprotein can undergo an

26

acid-catalysed oligomeric reorganization to a fusogenic homotrimer (32-35). This event

occurs in the endosome, allowing the viral nucleocapsid to escape into the cytoplasm

and initiate RNA and protein synthesis (36).

Dynamic interaction between the E-protein heteromeric units determines the virion

surface texture (smooth-rough) and hence affects availably of virus epitopes.(37).

Alternation of viral envelope protein configuration triggered by temperature has been

reported as “virus breathing” motion of particles (38).

The prM protein functions as a chaperone protein for the E glycoprotein during viral

maturation. It helps to maintain the E glycoprotein structure until viral morphogenesis is

complete and the virion escapes the acidic exocytic vesicles (39, 40). Cleavage of prM

to form M protein defines the transition from immature non-infectious virus particle to

infectious forms (40, 41). Description of structure and function of flavivirus genes is

shown in Table 2.2. Structure of flavivirus and mature and immature virion is given in

Figure 2.1.

27

Table 2.2:- Flavivirus genes and their functions (12)

Structure Length of

amino acids

Function

C 113-114 Essential for genome encapsidation

prM 166 Functions as a cap-like structure that protects the fusion peptide E

from undergoing pre-mature fusion before virus release

E 493-495 Provides the first point of contact between the virus and the host cell

by mediating binding and fusion of the virus to the host cell

membrane

Confers protective immunity by triggering neutralising antibodies

that gives protective effect

NS1 352 Contributes to viral RNA replication complex and viral defence

through inhibition of complement activation

NS2A 218 Involved in the replication complex

Possibly interferon (IFN) antagonist

NS2B 130 Serves as a co-factor in the structural activation of the DENV serine

protease of NS3

NS3 618-619 Involved in the process of viral polyprotein synthesis and RNA

replication

NS4A 150 Contributes to virus replication by inducing membrane alterations

NS4B 245-249 Implicated in assisting viral RNA replication through its direct

interaction with NS3

Blocks IFN /

NS5 899-900 RNA cap-processing, RNA dependent RNA polymerase

Nuclear localisation

28

Figure 2.1:- Flavivirus Structure

A) Flavivirus genomic framework. Flavivirus RNA is ~11Kb, ss-RNA ORF genome that codes for

structural and non-structural proteins responsible for virion formation.

B) Mature and immature virion structure of dengue virus(2, 3)

Mature

virion Immature

virion

A

B

29

2.2.2. Replication

Virus enters host cells via inoculation by infected mosquito vectors across skin and is

introduced intradermally and intravenously, and dendritic cells enable virus migration

to lymph nodes (42, 43). Classic viral replication in all flavivirus includes attachment,

uncoating, RNA synthesis, assembly and release. Each step is illustrated in Figure 2.2.

Attachment of flavivirus to host is mediated by different cell surface receptors,

depending on virus and host cell type. C-lectin receptors DC-sign and mannose

receptors on dendritic cells and myeloid cells (36, 44), heat shock proteins (hsp),

heaparin sulphate (hs) (45-48) in mammalian and mosquito cell lines have been

identified as receptors. In other flaviviruses GAG glycosoaminoglycans have been

reported as the most common surface ligand. As the virion is attached receptor-

mediated endocytosis occurs, facilitating virus intake. The rate of virus intake by host

cells is host-dependent (40, 49). Antibody-dependent enhancement may also play a role

in enhancing virus uptake in the presence of sub-neutralising antibody induced in

previous homologous or heterologous flavivirus infection (50). Uncoating of the virion

is mediated by acidity in host cell compartments that triggers conformational change in

envelope protein, inducing fusion of viral and host membrane. Viral RNA is hence

released to mediate viral protein synthesis.

Synthesis of viral protein occurs in a series of process whereby structural and non-

structural proteins are synthesised using viral genome as template and host protein

machinery (36). Using host transcriptional systems viral RNA codes precursors of

structural and non-structural proteins. Structural protein form the virion while non-

structural proteins are involved in replication, assembly and modulation of host cell

responses (51). Non-structural proteins further play a role in viral particle movement

30

into cell organelles to complete the viral structure. Encapsidation of virion occurs in the

lumen of endoplasmic reticulum forming immature prM-C coated virion (52). Virus

utilises the host membrane as a scaffold for anchoring the viral replication complex

(53). Cleavage of prM to M to form mature infectious virion occurs in the trans-golgi

network mediated by furin proteases. This is followed by conformational change in E-

molecules forming mature smooth virion. Absence of transition of prM to M forms

immature non-infectious progeny (54). Release of infectious particles is pH mediated,

facilitating completion of the replication cycle and release of capsid coated RNA

particle in cytoplasm.

B

Figure 2.2:- Flavivirus Replication

Flavivirus replication utilizes host cellular mechanisms for formation of mature the virion, with cleavage of prM to M

(10).

32

2.3. Dengue Virus (DENV)

DENV is the causative agent of dengue fever (DF) and dengue haemorrhagic fever

(DHF). It emerged as human pathogen in the early 20th

century and is maintained in

circulation between human hosts and mosquito vectors. DENV comprises four

epidemiologically identical but genetically distinct serotypes. This section reviews

DENV classification, spectrum of disease and geographical distribution.

2.3.1. Origin and distribution

DENV may have originated in Africa or Asia. An African origin suggests DENV arose

in a sylvatic cycle in West Africa and disseminated Worldwide with the slave trade. An

Asian origin suggests evolution of DENV in the Malay Peninsula in a forest cycle

involving canopy-dwelling lower primates and mosquitoes (18, 55). Genetic analysis

suggests independent evolution of four serotypes of DENV from ancestral, sylvatic

viruses with the expansion of the sylvatic progenitors to new vectors and hosts. These

studies imply that sylvatic cycle in Asia and West Africa is the potential source of

endemic/epidemic strains of DENV (56). Phylogenetic analysis of sylvatic strains in

West Africa supports this hypothesis (57). Identification of all four sylvatic DENV

serotypes in canopy-dwelling non-human primates and Aedes mosquitos in Malaysia in

1978 supports a sylvatic lineage arising in the Asian-Oceanic region (58).

DENV was first isolated in 1943 during World War II by Hotta and Kimura (59). In

1944 Sabin and his colleagues isolated DENV (subsequently designated as the

prototype DENV-1Hawaii strain) from US soldiers stationed in India, New Guinea and

Hawaii (60). Significant spread of dengue occurred during this period of World War II

from 1941 to 1945. A dengue pandemic was reported among military personnel in

entire Pacific, from Australia to Hawaii and from New Guinea to Japan (61). However,

A

33

cases of haemorrhagic manifestations, shock and death associated with dengue were

reported as early as 1897 in Australia(62), 1915 in Taiwan (63) and 1928 in Greece(64).

Dengue has emerged as infectious disease that with widely distributed throughout the

tropic and subtropics. About 2.5 billion people, or 2/5th of the world’s population, live

in areas where there is a risk of dengue transmission. Dengue is endemic in more than

100 countries in Asia, the Pacific, the Americas, Africa, and the Caribbean. The World

Health Organization (WHO) estimates that 50 to 100 million infections occur yearly,

including 500,000 DHF cases and 22,000 deaths, mostly among children (65).

The history of dengue in Australia extends over more than 120 years. The earliest

reference described the import of eight cases by ship from Mauritius in 1873(66); the

first indigenous outbreaks likely occurred in Queensland at Townsville in 1879 and

Rockhampton in 1885. Several epidemics in northern Queensland were described in the

1890s and in the early part of the 20th century (67). The first cases in northern New

South Wales were reported in 1898, but epidemic activity did not extend southwards

until 1925-26 when cases were described as far south as Newcastle. Dengue was

reported in Western Australia in 1909-10 and was declared notifiable there in 1912.

Improvements in water storage systems and supply infrastructure lead to eradication of

Ae. aegypti and dengue by the 1960s. In 1914, dengue was reported from Darwin in the

Northern Territory (68). Recent Australian data from 2009 -2014 shows an average of

16.54 notifications annual crude rate per 100,000 population, with peak of 22.2 in 2010

and lowest 5.9 in 2009 (69).

Although dengue has not been present in WA since the 1940s (70), the state has

reported the highest number of annual notifications in the country (71). In recent years

new budget airlines began offering affordable package holidays and travel increased

300-fold between 2006 and 2010, coinciding with a sharp increase in dengue cases

notified to the Communicable Diseases Division of the WA Department of Heath (72).

34

Analysis of DENV derived from 6 febrile travellers entering WA after visiting seven

countries throughout Asia between 2010-2012 identified a diverse range of genotypes

and lineages within all four DENV serotypes (11). Most of the travellers had entered

from Bali, a popular holiday destination for residents of WA. Many of the imported

DENV were local regional variants with strong epidemic potential known to have

circulated in the region for some time. Other DENV were more recently introduced into

Bali from other countries. A new lineage of DENV-2 that appeared to be associated

with a major outbreak in Bali in 2012 was also identified and importation of this lineage

has continued up to 2017. Bali is a melting pot of substantial DENV diversity and

serves as a hub for DENV transmission and mixing. In October 2013 the first case of

locally acquired dengue fever occurred in a male with no history of travel outside WA

for many years and who was likely exposed in the Pilbara region in north-west WA

(73). The case may have been exposed to infected mosquitoes imported on international

cargo vessels docked at local iron ore export ports or via direct international flights into

Port Hedland or Perth airports. Between February 2014 and March 2016 Ae. aegypti

was frequently detected at Australian international airports (74, 75). The most regular

detections occurred at Perth International Airport but there is no evidence that Ae.

aegypti has become established. Dengue continues to be introduced into WA via

returning travellers (76).

In 2013, 390 million global DENV infections were estimated to have occurred of which

approximately one quarter - 96 million – were symptomatic, with varying severity

(77). Global incidence and prevalence continues to increase. In recent years there have

been outbreaks in countries where dengue has never been reported or has not occurred

for many years. In 2013, Japan had an outbreak in Tokyo after a lapse of 70 years (78).

Emergence of mosquito vectors in countries with no previous reports been identified

and movement of viruses, especially in travellers, is associated with increased disease

35

burden (79, 80). In 2015 India had its worst outbreak after 15 years. In 2016, large

outbreak of DENV was reported worldwide (78). Increased globalisation, travel and

trade are important determinants of dengue world-wide distribution (81, 82).

WHO dengue maps in Figure 2.3 show distribution of dengue and/or dengue

haemorrhagic fever, countries at risk of dengue in 2008 and the average number of

suspected or confirmed dengue cases reported to WHO from 2010-2016. These maps

illustrate the rapid geographic spread of DENV in an eight year period.

36

Figure 2.3:-Dengue distribution in 2008 and 2016

Widespread geographical distribution of DENV and increase in dengue notifications over a period of 8 years

2008-2016

37

2.3.2. Classification

DENV is classified as four distinct serotypes; DENV-1, DENV-2, DENV-3 and DENV-

4. DENV genetic diversity is attributed to interactions among virus, vector and host.

DENV has an error-prone RNA dependent RNA polymerase with no proof-reading

capacity, producing approximately one mutation per round of genome replication thus

leading to diversity among species and between serotypes (83, 84). Clonal evolution,

homologous recombination, intra-serotype recombination is also suggested due to co-

occurrence of different genotypes and serotypes in a single host or vector. However no

significant emergence due to recombination has been reported (85).Evolutionary forces

drive lineage replacement and clade turnover resulting in change in prevalent epidemic

strains. Vector migration, human movement and increased density of human living

conditions also impact generation of diversity (86, 87). Interaction between virus and

host immune mechanisms is also an important factor. Viruses associated with high

viremia and the capacity to escape immune responses are available to vectors for longer

periods and therefore are more likely to be transmitted during blood meals (88-90).

Phylogenetic analysis shows that emergence of modern serotypes of DENV occurred

after establishment of human populations large enough for urban transmission.

Emergence is believed to have been facilitated due to vectors switching from arboreal

Aedes to domestic and peri-domestic Aedes species. Diversity of serotypes is also

attributed to competition for susceptible hosts. Development of RNA sequencing

techniques allowed definition of genotypes within serotypes of DENV(57).

DENV serotypes, originally identified using serological approaches, are more precisely

classified phylogenetically into distinct genotypes representing clusters with nucleotide

sequence divergence of not more than 6%, and lineages within the genotypes may

represent strains with similar geographic origins. The definition of genotype and lineage

38

has varied in different studies; genotype distribution reviewed here is based on

definition by Chen et al (85).

Based on partial and whole genome sequences five distinct genotypes of DENV-1 have

been defined, named as Genotype I-V. The maximum nucleotide divergence is 6%

among genotypes (57, 91). The average nucleotide substitution rate is 6.56x10-4

substitution/site/year (92). Within genotypes there are distinct clades associated with

outbreaks sharing a specific spatiotemporal association. All the genotypes have

recorded human cycle transmission. Genotype III was reported to include sylvatic

isolates collected in Malaysia however recently E-gene analysis suggested possible

spill over from human to monkey, rather than true sylvatic DENV (56). Genotypes I

and IV are reported in recent epidemics in the Pacific while Genotype V has been

described in epidemics in Americas. Genotype II and Genotype III are not as widely

distributed as the other genotypes (24, 93). Spatiotemporal association of each genotype

is listed in table 2.3.

DENV-2 has six genotypes named after tentative topographical coverage: Asian

genotypes I and II, Cosmopolitan genotype, American genotype, Southeast Asia,

Asian/American genotype, in addition to Sylvatic genotype. Geographical distribution

of these genotypes is listed in Table 1. The sylvatic genotype of DENV-2 is the first

evidence of true sylvatic origin (94). Genotypic replacement of less viremic strains by

high viremic strains has been observed for DENV-2 (95, 96). Strain replacement is

attributed to greater fitness and longer viremia hence increased availability for the

vector. Distribution of DENV-2 genotypes is listed in table 2.3.

DENV-3 was delineated into four genotypes named Genotype I-IV by Lanciotti (97).

Genotype V was introduced subsequently (98). The prototype H87 strain used as a

WHO reference strain is included in this new genotype. DENV-3 genotypes evolved

39

and were maintained in circulation in particular regions and spilled over to other regions

with increased human and trade movement. However, in comparison to DENV-1 and

DENV-2, DENV-3 genotypes have shown more limited co-circulation. Topological

distribution of DENV-3 is described in table 2.3.

DENV-4 has four major genotypes, Genotype I-IV. Genotypes I and II show limited

topological distribution with the former circulating exclusively in Southeast Asia while

Genotype II circulates independently in Southeast Asia and Americas with limited

genetic exchange. Genotype I includes the prototype strain H241 isolated in the

Philippines, used as a WHO reference strain. Genotype III represents five distinct Thai

isolates circulating between 1997 and 2001 (99). Genotype IV is a sylvatic lineage and

is genetically distinct to the other genotypes (100) . The geographical topology of

DENV-4 is listed in table 2.3.

Figure 2.4. Shows phylogenetic relationships among DENV

40

Serotype Genotype Distribution

DENV-1

I

II

III

IV

V

Southeast Asia, China and East Africa

Isolated from Thailand between 1950s-1960s

Sylvatic strains originated from Malaysia with

possible spill over from human infection

Pacific Islands and Australia

Americas, West Africa, Asia, South East Asia

DENV-2

Asian genotype 1

Asian genotype 2

Cosmopolitan

American

Southeast Asia

Asian/American

Sylvatic genotype

Indonesia, Malaysia the Philippines and recent

isolates from South Pacific

Thailand Vietnam and Bangladesh

Sri Lanka, India, Africa and Samoa, two sub-clades

with global distribution

Indian subcontinent, Pacific Islands, Central and

South America.

Southeast Asia, South America likely introduced

from Vietnam, American strain.

Mixed strains from human and arboreal mosquitoes.

Genetically distinct from other strains representing

ancestral genotype.

DENV-3

I

II

III

IV

V

Strains from areas of Southeast Asia particularly

Indonesia.

Thailand

Sri Lanka, Taiwan, Singapore, Samoa, East Africa

Puerto Rico,Tahiti

Prototype H87-1956 Philippines isolates, China,

Japan and Brazil.

DENV-4

I

II

III

IV

Strains from Thailand, Philippines, Sri Lanka

Indonesia, Malaysia, Tahiti, Caribbean and

Americas

Thai strains that are distinct from other Thai strains

Sylvatic strains from Malaysia

Table 2.3: Classification of DENV serotypes

41

.

Figure 2.4:- DENV Phylogeny

Complete open reading frames of DENV sylvatic and endemic/epidemic strains ,

available in the GenBank library (Weaver and Vasilakis, 2009)(12)

42

2.3.3. DENV Vector and Transmission

Dengue is transmitted by the bite of an infected female mosquito. The predominant

mosquito vectors that maintain DENV in urban transmission cycles are from Aedes

genus of Culicidae family. Aedes aegypti, Ae albopictus and Ae polynesiensis are the

major human vectors. In 1906 Thomas Bancroft suggested Ae. aegypti as the carrier of

DENV, confirmed by John Burton in 1916 (101).

Aedes mosquitoes are day-biting vectors that breed mainly around urban environments

in clean fresh water and in manmade receptacles. Ae. aegypti is resilient to

environmental stress (e.g., droughts) or human interventions (e.g., control measures).

The ability of the eggs to withstand desiccation (drying) and to survive without water

for several months on the inner walls of containers allow greater adaptation to the

environment (102). The species of mosquito vector depends on the transmission cycle.

The widespread of DENV and its establishment in large human population was

Figure 2.5:-DENV transmission cycle and mosquito-vectors (9)

43

facilitated through vector switching from arboreal to peri-domestic mosquitoes (9).

DENV transmission between mosquitoes and humans does not need a sylvatic cycle for

maintenance. However, in forest settings sylvatic transmission exists between non-

human primates and Aedes mosquitoes. There are two distinct transmission cycles

described: sylvatic and human. The sylvatic cycle is hypothesised to be ancestral cycle

ecologically and is evolutionarily distinct from the human transmission cycle. This is

supported by phylogenetical studies showing sylvatic strains of West Africa are

genetically and evolutionarily distinct from all endemic and epidemic strains (103, 104).

The sylvatic transmission cycle is maintained between canopy-dwelling Aedes spp. and

non-human primates. The principle vectors responsible for this cycle are listed in Figure

2.5. West Africa and Southeast Asia are well-documented foci of sylvatic transmission

(9).

Establishment of human civilisation to a threshold that supported DENV transmission,

and adaptation of sylvatic DENV to both peri-domestic and urban mosquitoes resulted

in increased transmission via human cycles. DENV strains in human cycles are distinct

from sylvatic strains and circulate exclusively in domestic and peri-domestic

environments. Primary vector for transmission in human cycle is Ae. aegypti, Ae.

albopictus and Ae. polyenesiensis are secondary vectors (104-106).

Transovarial transmission (TOT) has been proposed as a mechanism of DENV

maintenance in both sylvatic and human cycles. Vertical transmission of DENV-1 and

DENV-4 was reported in Ae. albopictus in Brazil in 1990(107). Similarly, vertical

transmission of DENV-3 was reported in Ae. aegypti (108). A significant role for TOT

or vertical transmission in epidemiological persistence of DENV is yet to be confirmed.

A review of the literature of vertical transmission and its role in dengue epidemiology

by Grunnill and Boots concluded that a number of studies failed to establish evidence

for vertical transmission and maintenance of DENV circulation (109).

44

Other non-vector routes of DENV include needle stick injury, mother-to-child vertical

transmission, transfusion-related transmission and mucocutaneous exposure.

Transmission of dengue by accidental inoculation at a metropolitan hospital in France

was reported in 1990 (110). In 1996 a case of needle stick transmission of DENV-2

from an infected traveller to nurse was reported in France (111) and again in 2004, a

case of nosocomial transmission by needle stick injury was reported from an infected

traveller to a nurse in Germany (112). In 1994 vertical transmission of DENV-2 was

reported in a Thai woman; virus was isolated from her infant on the 6th

day of life(113).

Similarly, in a case of infant fatality with multi organ failure DENV-2 was isolated from

blood of the infant, borne to a mother with acute DENV infection (114). DENV-2 was

also detected in cord blood and infant’s blood in a case of vertical transmission from a

mother who was ill 5 days prior to birth, both mother and infant recovered and were

discharged 6 days after delivery (115). Boussemart et al confirmed two cases of

prenatal DENV transmission in 2001 (116). Intrapartum transmission of DENV-2 and

bone marrow transplant transmission of DENV-4 was reported during 1994-1995

DENV epidemic in Puerto Rico (117).

45

2.3.4. Clinical spectrum of dengue disease

Dengue clinical manifestations range from self-limiting to severe fatal disease. Dengue

fever (DF) is an acute febrile illness with two or more of the following symptoms and

signs: severe headache, severe eye pain, joint pain, muscle and/or bone pain, rash, mild

bleeding manifestation, low white cell count. Age, immune status, host genetic

background, infecting DENV strain are among the factors which contribute to infection

and disease severity (118). Severe dengue is characterised by fever that lasts from 2-7

days with general signs and symptoms consistent with dengue fever. Warning signs for

severe dengue develop with the decline in fever within 24-48 hrs. These include

capillary leakage leading to organ impairment, low platelet count, haemorrhagic

manifestations and possible fatality (30). The clinical spectrum of disease at different

day’s post-onset of acute illness is shown in Figure 2.6. There is no specific treatment

for dengue. Proper case characterisation and supportive treatment consisting of fluid and

electrolyte replacement therapy is the only available approach to management of severe

dengue. Successful management of dengue depends on early recognition of dengue

infection, recognition of warning signs for severe dengue, and access to medical care by

experienced health care workers (119).

46

Figure 2.6: Dengue clinical manifestations (World Health Organization 2009)

47

2.3.5. Dengue case classification

In 1997 WHO classified severe dengue cases as DF, DHF (Grades 1 and 2) and DSS

(DHF grades 3 and 4) (Figure 2.6) based on guidelines used in Thailand (120).

According to this classification, DF is characterised by high fever with two or more

clinical manifestation of headache, retroorbital pain, myalgia, arthralgia rash,

haemorrhagic manifestations. DHF fulfils four criteria: fever or history of fever lasting

2-7 days; a haemorrhage shown positive by tourniquet test or spontaneous bleeding,

thrombocytopenia and evidence of plasma leakage. DHF severity is classified based on

the presence or absence of spontaneous bleeding and severity of plasma leakage. (121,

122). However variations in clinical manifestations made application of this WHO

classification difficult and application of these criteria fails to detect severe dengue

(123). An assessment in Vietnam showed that the WHO classification resulted in a

considerable decrease in DHF cases and suggested that DF and DHF are part of a

continuous spectrum of dengue rather than being two separate clinical identities (124).

In 2009, WHO has introduced an algorithm to make clinical assessment of dengue

simple and intuitive. According to this new system dengue is now distinguished in three

categories: dengue without or with warning signs, and severe dengue. Dengue without

warning signs is an initial febrile phase with nonspecific symptoms but can be probable

dengue if there is history exposure to dengue in an epidemic/endemic area. Dengue with

warning signs occurs in the crucial phase soon after infection and may progress to

severe dengue. Dengue is classified as severe dengue if one or more of the following is

present: severe plasma leakage, severe haemorrhage and severe organ impairment.

Figure 2.7 and Figure 2.8 show the classical and recent classifications of dengue.

48

Figure 2.8:- WHO Classification of dengue (2009)

Figure 2.7:- WHO Classification of dengue

(1997)

49

2.4. Zika Virus

ZIKV is a flavivirus that belongs to the Spondweni serogroup, which includes Zika

(ZIKV) and Spondweni virus (SPONV). Both viruses show high level serological

cross-reactivity and have similar clinical presentations (125). ZIKV was first discovered

in 1947 in the Zika forest in Uganda following isolation from a sentinel monkey during

research on yellow fever virus. The first ZIKV isolate is the prototype strain MR766,

isolated from monkey no. 766 (126). In 1948, ZIKV was isolated from a pool of Aedes

africanus (ZIKV E/1 strain) (127). The first ZIKV isolate from humans was reported

during an outbreak of jaundice in Nigeria. Samples obtained from 3 cases were

investigated for yellow fever, ZIKV was isolated from one patient and two other

exhibited a rise in titre of serum antibodies against ZIKV(128).Subsequently, a strain of

ZIKV (P6-740) was isolated from Malaysia in 1966 (129) and first human case in

Asia was reported from Central Java, Indonesia in 1977-1978 (130). ZIKV re-emerged

in an outbreak mistakenly identified as dengue in 2007 in Yap state, Federated States of

Micronesia, and moved across the western and south Pacific to cause an outbreak in

French Polynesia in 2013 and subsequently, Brazil in 2015. This was the first known

introduction of ZIKV to the Americas.

More than 70% of the population in Yap were infected (131) during an epidemic where

the major symptoms were rash, arthralgia and conjunctivitis. Rapid diagnostic tests

suggested DENV as the causative agent. However samples from this outbreak when

analysed by CDC by RT-PCR demonstrated ~90% nucleotide identity with ZIKV,

confirming ZIKV as the causative agent of the ongoing epidemic. In 2013, 11.5% of the

population of French Polynesia was infected during an outbreak that lasted 21 weeks.

The first association between ZIKV infection and neurological complications (Guillian

Barre Syndrome) was also reported during this outbreak with a 20-fold increase in

incidence of GBS compared to the past four years. Phylogenetic analysis of ZIKV

50

isolated in French Polynesia showed homology with ZIKV isolated in Cambodia (132).

ZIKV outbreaks in the south Pacific followed, with transmission in New Caledonia,

Cook Islands and Easter Island in 2014 (126). In late 2014 and early 2015 cases of

exanthematic disease were reported in Brazil, with neurological syndromes, in regions

where ZIKV, DENV and CHIKV co-circulated (133). By the end of Sept 2015 cases of

Zika-associated neonatal microcephaly were reported and by the end of 2015, 440,000-

1,300,000 Zika cases in 18 states, with confirmed autochthonous transmission, were

estimated to have occurred in Brazil (134) and WHO declared a global emergency. In

October-November 2015 autochthonous ZIKV transmission was reported in Sincelejo,

Columbia. Virus isolated from this outbreak showed 99% sequence identity with FP

strain (135). ZIKV was then reported in 12 other countries and territories of the

Americas (126, 136). Systematic spread of ZIKV is illustrated in Figure 2.9 (137).With

global spread to more than 60 countries and the newly recognized association with

neurological complications Zika emerged as flavivirus with significant global disease

burden. However a steep fall in Zika cases has been noted (138). A single case was

confirmed locally transmitted in the United States to mid-August 2017. The decline in

incidence in countries with previous explosive transmission is proposed to result from

herd immunity and reduction in the number of susceptible people.

Figure 2.10 shows the rise and fall of Zika from late 2015 to 2017 in the Americas.

51

Figure 2.10:-The rise and fall of Zika

The rise and fall of Zika. Rapid increase in Zika cases in early 2016 and steep decline at the end of year

(Cohen, Science 2017)

Figure 2.9: Zika virus spread from 1947-2016

Zika virus spread 1947-2016. The chronological distribution of ZIKV from its earliest

discovery to neurological disorder-associated cases in 2016. (US Centres for Disease Control)

52

2.4.1. Classification

Phylogenetic analysis of flaviviruses has shown that ZIKV is placed in the mosquito-

borne cluster, based on the genomic sequence of 100-Kb genome segment at the 3′

terminus of the NS5 gene (8). As with other flaviviruses ZIKV is a positive sense ss-

RNA virus with a genome of approximately 11kb that codes for structural and non-

structural polyproteins. Among the flaviviruses of medical importance, ZIKV is closely

related to DENV with approximately 55.6 % amino acid homology across viral

polyprotein (139). ZIKV is reported to circulate as single serotype divided into strains

based on geographic origin as African and Asian strains. The African prototype MR766

1947 Uganda strain and the Asian prototype 2007 Yap strain share 96.5% amino acid

homology in the entire coding region (140).

Comparison of whole genome sequences shows 3 branches of ZIKV: East African

(Uganda cluster), West African (Senegal cluster) and Asian (131). The two African

lineages are believed to be due to independent introduction to these regions of prototype

MR766.

Phylogeny of ZIKV based on whole gene sequences is shown in Figure 2.11

53

Figure 2.11:- ZIKV Phylogeny

Phylogenetic tree of ZIKV . Strains used in the present study are

highlighted in red boxes.(13)

54

2.4.2. Vector borne ZIKV transmission

As for DENV Aedes genus is the principle vector for ZIKV. The ZIKV African strain

MR766 was isolated from Ae. africanus and the ZIKV Asian (Yap) strain was first

isolated from Ae. aegpti and Ae hensilli in Yap (2007) (141, 142). Ae. albopictus was

the vector in Africa (143). The earliest ZIKV isolations from Asia were from Ae. aeypti

in Malaysia in 1966 (129). In southeastern Senegal, a study of 1700 mosquito pools of

11,247 mosquitoes showed that Ae. furcifer and Ae vittatus were involved in ZIKV

transmission to humans(144). The role of mosquitoes as a ZIKV vector has been

confirmed in animal models. Boorman et al reported successful transmission of ZIKV

by Ae. aegypti to rhesus monkeys and infant mice (145). ZIKV shows wide divergence

of mosquito species as potential vectors for transmission; vector selection

predominantly depends on the availability of mosquito species. ZIKV seems to be

adapted to a large number of mosquito species including those not commonly known to

transmit other flaviviruses. Isolation of ZIKV from diverse species of Aedes, Culex and

Mansonia mosquitoes suggests a wide range of potential ZIKV vectors (126).

2.4.3. Non- vector transmission of Zika

Vector-borne transmission is the most common route for spread of ZIKV however

maternofetal and sexual transmission has been confirmed (146, 147).

In October 2013, perinatal transmission of ZIKV was confirmed in two mothers in

French Polynesia. ZIKV infection in a newborn child was confirmed by RT-PCR on

serum collected four days post-delivery. Possible transplacental infection of the infant

during delivery suspecte (148). Maternofetal transmission was confirmed during the

Brazil outbreak in 2015 with reports of neonatal malformations including microcephaly

linked with ZIKV infection. Virus was also isolated from amniotic fluid and tissue

samples from an infected infant during this outbreak (149, 150).

55

In 2008, clinical and serological evidence indicated that two American scientists who

recently returned after working in southeastern Senegal had contracted ZIKV infection.

One of the scientists transmitted ZIKV to his wife after his return home, most likely as a

sexually transmitted infection. Symptoms in the two scientists included maculopapular

rash, fatigue, arthralgia, headache and hematospermia in one of the patients. The wife

developed muscle aches and pains, headache, photophobia chills and malaise. Anti-

ZIKV hemagglutination inhibition antibody titres and neutralising titres were elevated.

(147). ZIKV was isolated from semen of a patient in Tahiti who presented with

hematospermia(151). Similarly, in 2016 ZIKV infection was diagnosed in a previously

healthy 24-year old woman with no travel history to Zika endemic areas after sexual

contact with man who stayed in Brazil from December 11, 2015 to February 9, 2016;

ZIKV was isolated from semen of a man 18 and 24 days post-onset of symptoms (152).

Other non-vector routes of transmission include via blood transfusion and laboratory

contamination. In the 2013 FP outbreak 3% of 1,505 blood donors, asymptomatic at the

time of donation were found to be ZIKV-positive positive by PCR (148). ZIKV

infection has also been reported as a result of laboratory work with arboviruses in 1973

(153).

2.4.4. Infection and complications

Acute symptomatic ZIKV infection is a self-limiting mild flu-like illness. Symptoms are

similar to other flavivirus infections and include fever, rash, arthritits, arthralgia or

myalagia, conjunctivitis and fatigue. However recent outbreaks have reported an

association between neurological complications and Zika. Most documented cases are

of Guillian-Barre syndrome and of microcephaly in neonates.

A) Guillian-Barre syndrome (GBS)

56

GBS is a neurological disorder related to autoimmune responses in which the immune

response targets the peripheral nervous system. GBS includes weakness of the arms and

legs and in severe cases paralysis of muscles that support breathing. During the 2013

French Polynesia outbreak a 20-fold increase in GBS following ZIKV infection was

reported (154). In the 2015 outbreak in Brazil 62% c of 121 Zika cases were reported to

precede symptoms of GBS. Similarly, 54% of 22 ZIKV infections in El Salvador were

also reported to proceed to GBS. In Columbia 2015-16, 67% of neurological cases were

reported to correspond to GBS (155). From April 2015-March 2016, 1474 cases of GBS

and 164,4237 suspected and confirmed Zika cases were reported from Brazil, Columbia,

the Dominican Republic, El Salvador, Honduras, Suriname and Venezuela. Compared

to baseline (pre-ZIKV) there were 172%, 211%, 150%, 100%, 144%, 400% and 877%

increases in GBS in the respective countries, strongly suggesting a connection between

GBS and ZIKV infection(156). However proof of causality is yet to be established

(157).

B) Neonatal ZIKV-related complications

Congenital Zika syndrome and microcephaly are neonatal defects in ZIKV infection.

Partial skull collapse due to subcortical calcification, damage to back of eye, macular

scarring, focal pigmentary and congenital contractures are among the indicators for

congenital Zika syndrome listed by CDC. Microcephaly is malformation resulting in

smaller head size related to birth defects and neurological conditions. Symptoms vary

depending on extent of brain disruption. During the 2015 Brazil outbreak 97.5%

increase in microcephaly was reported (158). Similarly, the 2013 French Polynesia

outbreak was followed by increase in microcephaly in the following years. In Hawaii,

USA, the presence of IgM Ab was reported in a mother who gave birth to full term baby

with microcephaly (159). In Columbia 50 cases of microcephaly reported in a period of

three months in 2016, a higher rate than the previous record of 140 cases per year (160).

57

Spatiotemporal occurrence suggests an association between microcephaly and ZIKV

transmission; however more data to establish this association is required and

investigations are ongoing.

58

2.5. Flavivirus vaccine strains

Yellow fever 17D and Japanese encephalitis IMOJEV vaccine are the most successful

flavivirus vaccines produced and are used in endemic areas as well as by travellers from

non-endemic countries. Antibody-mediated immune responses in flavivirus infection

are highly cross-reactive and pre-existing antibodies induced by previous natural

infection or vaccination means that interpretation of serological test results may be

difficult. Analysis of vaccine-mediated immune responses is therefore integral to any

analysis of anti-flavivirus immunity, especially in travellers. The background of the

flavivirus vaccines YF17D and IMOJEV is reviewed in this section.

2.5.1. Yellow Fever

Yellow fever (YF) is a well-known acute haemorrhagic flavivirus infection. Yellow

fever initially originated in Africa and was introduced to the western hemisphere with

the slave trade. Infection was often associated with ports, as outbreaks were reported

with arrival of ships from known epidemic foci (161). YF was endemic in tropical

regions of Africa and South America, and YF epidemics occurred upon introduction of

YFV to North America, Caribbean and Europe (162). It was the major health threat

during the 20th

century. Development of a live-attenuated vaccine in the 1930s led to

decline of the disease. However epizootics and human transmission still continues

(163). Outbreaks have been recently reported in Uganda (2010), Sudan and Ethiopia

(2012-2013) (162). In 2017- early 2018, 35 confirmed human cases of yellow fever

were reported in Brazil, from the states of São Paulo (20 cases, including 11 deaths),

Rio de Janeiro (three cases, including one death), and Minas Gerais (11 cases, including

seven deaths), and the Federal District (one fatal case) (164).

Infection with YFV may cause an acute infection with symptoms including fever,

arthralgia, nausea and photophobia. Multiple organ systems are affected including the

liver, kidneys, gastrointestinal tract and brain with fever, jaundice, severe haemorrhage

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and shock ensuing (165). Mortality is associated with jaundice and other severe liver

diseases. During the early 19th

century, yellow fever was mistaken as bacterial disease.

At the end of the 19th

century Ae. aegypti was shown to be the vector and that infection

was caused by a filterable agent (166). Max Thieler developed a vaccine for yellow

fever in 1937 by passaging the Asibi strain through laboratory animals including mice

and chickens; the attenuated, protective strain was named 17D (167).

YFV is classified into seven major genotypes: five African and two South American.

African genotypes have 10-23% nucleotide differences within genotypes while South

American genotypes have 16% differences in nucleotide sequences (168).

Wild type YFV is viscerotropic and affects a wide range of tissues and organs, with

liver being the predominant target .Virulence is highly dependent on virus strain and

host immune status (169).

YF17D was selected as the vaccine strain as it was believed to induce immune

responses similar to wild type infection. 17D escape variants are neutralised up to 10-

fold by monoclonal antibody derived from the sera of 17D vaccine recipients,

suggesting there is no risk of immune escape among mutant strains in the mouse model

(170). 17D has shown neurotrophic side effects in experimental lab animals but in

humans viremia induced by vaccination is very low and unlikely to invade the blood-

brain barrier however adverse effects have been reported in infants <- 6 months old.

17D-YFV is by far the most effective flavivirus vaccine ever produced.

2.5.2. Japanese encephalitis

Japanese encephalitis (JE) is an acute febrile illness caused by infection with Japanese

encephalitis virus (JEV).

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JEV first emerged in Japan during the 1870s and spread throughout Asia. Five

genotypes of JEV have been described so far, each genotype has a different

geographical distribution. The five genotypes are thought to have evolved from an

ancestral Indonesian strain (171).

JE is transmitted by Culex spp mosquito (Culex tritaeniorhychus) (172). Domestic pigs

are the major reservoir for mosquito-human transmission. High magnitude, long-lasting

viremia in pigs and close proximity to human hosts means pigs are a potent reservoir for

transmission(173). Birds are another major reservoir host and enable wide geographic

distribution. Humans and non-avian vertebrates are dead-end host as they do not

produce viremia to sufficiently high levels to infect new mosquitos. Virus circulation is

maintained via zoonotic transmission and vertical transmission in mosquitoes (174).

JEV infection may result in severe disease with febrile symptoms, aseptic meningitis,

acute flaccid paralysis or classic meningoencephalitis. In the first outbreak in Japan,

casualty rates reached 30% with 30-50% of survivors suffering neurologic disability. JE

the major public health problem in Southeast Asia and China (175). There are two broad

epidemiological patterns: in sub-tropic countries disease is mostly prevalent during wet

monsoon season while in tropical countries JE is seen throughout the year. The first

outbreak in Australia occurred in the Torres strait in 1995, and one case was reported in

northern Queensland (176). Relocation of pigs away from homes and major drainage

works markedly reduced transmission and diminished cases of human infection.

JEV vaccination is the only preventive measure available, apart from vector control.

There are four types of JEV vaccine available derived from different JEV strains in

different countries: mouse brain-derived killed inactivated; cell culture-derived live-

attenuated; cell-culture derived killed inactivated; cell culture-derived live-attenuated

chimeric. In Australia, JE-Vax and IMOJEV are approved vaccines however

inactivated JE-Vax is no longer manufactured. Live-attenuated vaccine IMOJEV is used

61

for vaccination. JEV vaccination in Australia is recommended for travellers to epidemic

areas and laboratory workers who are at risk of exposure.

62

2.6. Flavivirus immunopathogenesis

Flavivirus immunopathogenesis is dependent on virus-host interactions. Strain-specific

modulation of host immune system has been reported in DENV. Infection with

pathogenic stains and high viremia in the host are correlated with availability of virus to

vector during a blood meal. Similarly, susceptibility of the vector to particular strains

and immune escape has been associated with clade replacement in DENV. Flavivirus

pathogenesis is reviewed in this section.

2.6.1. Flavivirus-specific innate immune responses

Innate immune responses are the first line of host defence against infection. Following

inoculation by infected mosquitoes, the innate immune system is activated and in turn

stimulates acquired immune responses. Major elements of the innate response include

initiation of type I interferon production, and activation of the complement pathway.

A) Interferons

Type I interferons (IFN) confer an anti-viral environment that inhibits the replication of

viral nucleic acid. Germline-encoded pattern recognition receptors (PRRs) present on

innate immune cells detect viral components. Most reported PRR sensors in flavivirus

infection are Toll-like receptors (TLRs), and cytoplasmic receptors such as retionic

acid-inducible gene-I (RIGI) or RIGI like receptors (RLRS)(177, 178). Binding of

these receptors to conserved pathogen associated molecular patterns (PAMPs) on

viruses activates the cascade resulting in production of IFNα and IFNβ and other

cytokines(179-181). These cytokines trigger activation of IFN-stimulating genes (ISGs)

containing IFN-stimulated response elements (ISRE). ISGs inhibit DENV infection by

inhibition of viral entry/fusion and production of exonuclease (181). Several ISGs

including OAS1 and OAS3, Oas-1b, IFITM and STAT are reported to initiate antiviral

63

activity (182, 183). IFITM proteins restrict direct and ADE-mediated DENV infection

(184). Although the innate immune system has several antiviral mechanisms, human

epidemic flaviviruses have developed several mechanisms to evade host innate

responses. DENV may inhibit IFN activity by inhibiting production and signalling of

type 1 IFN (185). DENV NS2B3 protease can cleave STING involved in

phosphorylation that results in production of Type 1 IFN (186). STING is an important

restriction factor for infection of DENV in mice. In addition DENV inhibits production

of type 1 IFN in antigen presenting cells including dendritic cells (DC), among the first

cells to encounter virus post-entry. DENV proteins NS2A, NS4A and NS4B attenuate

IFN-mediated activation of ISRE(187) and NS4B prevents phosphorylation of STAT

rendering the complex inactive during viral replication and thus downregulating

production of pro-inflammatory cytokines. Mutations in NS4B have also been linked

with increase in virulence of DENV strains(188). Other flavivirus proteins, such as

NS5, have been shown inhibit IFN signalling in TBEV, WNV and JEV(189-191). NS5

in DENV is reported to degrade STAT (189, 192). Ashour et al showed NS5 is involved

directly or indirectly in proteolytic processing of STAT-2. Understanding the role of

innate immunity in host protection, and evolution of viral evasive mechanisms, is

necessary to better understand the role of innate immune responses in

immunopathogenesis of flavivirus infection.

B) Complement

The complement pathway is a co-ordinated sequential enzymatic cascade formed by

non-covalent association between nine activated protein components and fragments

(193). Complement components are named as C1, C4, C3, C5, C6, C7, C8 and C9 in the

order of activation. Activation of complement includes three well defined pathways:

Classical pathway triggered C1q component binding to antigen-antibody complex on

the surface of pathogens; the lectin pathway initiated by mannose binding protein

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(MBL) of carbohydrate structure on surface of microbes; and the alternative pathway

activated by hydrolysis of C3. These pathways activate the complement components

which assemble to form C5b-9 membrane attack complexes (MAC) that lyse infected

cells.

Complement is the heat labile component of plasma. Protective functions of

complement include priming of adaptive immune responses and complement-mediated

neutralisation (194). In flavivirus infection complement-mediated protective effects are

mainly by activation of B and T cells. All three complement activation pathways are

required for protection against WNV. Delay in priming adaptive immune response was

reported in mice lacking components of complement (195). Enhanced neutralising

effects of complement have been reported in DENV in the presence of complement

components (196). Another protective function of complement includes restriction of

antibody-dependent enhancement. Studies in WNV show that complement component

C1q restricts subclass-dependent antibody-dependent enhancement of flaviviruses in

cell culture and mice (197). However complement components C3 and C5 were

detected in DHF and DSS suggesting possible complement-mediated enhancement of

flavivirus infection (198, 199)

DENV, WNV and YFV can evade the complement activation pathway and thus protect

virus from complement-mediated neutralisation. WNV NS1 protein has been shown to

evade complement activation by recruiting complement regulatory protein H, which

results in attenuated deposition of MAC (200). Attenuation of the complement

activation pathway by DENV NS1 has also been reported. Avirutnan et al showed, by

binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 and thus

inhibits formation of convertase and activation of complement-dependent neutralisation.

Furthermore, binding of NS1 to C4b was shown to modulate complement activation by

enhancing C4b binding protein co-factor activity that attenuates the classical and lectin

65

pathways (201). NS1 binding to MBL has shown to protect virus from lectin-mediated

neutralisation by the lectin mediated pathway of complement (202).

Thus, host factors including genetic alleles that stimulate MBL concentration in

plasma, myeloid cells expressing complement receptors mediating antibody-mediated

enhancement, and viral interaction with complement proteins govern complement-

mediated immune responses in flavivirus infection.

2.6.2. Acquired immune responses

Flavivirus-specific acquired immune responses clears virus from the host and induces

immunological memory. Development of the acquired immune response in flavivirus

infection generally occurs after 4-7 days of primary infection. This includes genomic

rearrangement in B and T cells receptor genes forming a virus specific repertoire (203).

Acquired immune responses however may be protective or pathogenic. Sharing of

common viral epitopes among the flavivirus serocomplex confers immune responses

which may be protective or dysfunctional. Acquired immune responses elicited during

flavivirus infection are reviewed in this section.

A) Cellular immunity

DENV serotype-specific and cross-reactive T cell responses have been demonstrated

and include proliferative and cytotoxic responses. T-cell responses are activated by

recognition of viral peptide antigens bound to MHC class I or MHC class II molecules

on the surface of antigen presenting cells (APCs). These peptides are short fragment

derived from infecting virus. Peptides derived from processed intracellular virus are

restricted by MHC class I molecules and presented to CD8 T-cells, which differentiate

into cytotoxic T-cells. Peptide antigen from virus endocytosed by antigen presenting

cells are restricted by MHC class II molecules and presented to CD4 T-cells. Activated

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CD4 T-cells differentiate to effector TH1 and TH2 T-cells. TH1 cells activate

macrophages and induce B-cells to produce IgG while TH2 cells initiate naïve humoral

immune response to stimulate B-cells to produce IgM, and subsequently neutralising

and opsonizing subtypes of IgG.

The earliest in vitro studies showed that peripheral mononuclear cells from dengue-

immune individuals contain CD4+ T-cells which proliferate and produce interferon

gamma (IFN-gamma) when stimulated by dengue antigens (204). Analysis of JEV-

specific memory T-cells using a synthetic peptide library showed an association

between polyfunctional CD4+ T-cell memory response and complete recovery from JE

(205). Similarly, high magnitude YFV-specific T-cell responses have been shown to

contribute to success of yellow fever vaccination (206, 207).

DENV-specific T-cell responses are mostly directed towards non-structural proteins

(208). CD8+ T cells most frequently recognise NS3, NS4B and NS5 proteins while

CD4+ T cells mostly target E, in addition to non-structural proteins including NS3

(209-211). Predominant recognition of non-structural proteins has been postulated to

contribute to suboptimal performance of the chimeric dengue vaccine, which includes

DENV E protein in a YFV backbone. Balanced proliferation of polyfunctional CD8+

and CD4+ targeted to structural and non-structural proteins contributed to generation of

high titres neutralising antibodies (212, 213). In the murine model, protective T-cell

responses induced by a conserved peptide pool, activated in the absence of antibodies,

suggested that a dengue T-cell vaccine would reduce risk of antibody mediated

enhancement of infection (214).

An association between DENV-specific T-cell responses and development of severe

dengue in secondary infection has been shown (215). Peptide variation due to amino

acid diversity among DENV serotypes is thought to induce responses targeted to

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original infecting serotypes, resulting in biased cytokine production that leads to

exacerbation of infection. DENV-specific memory T-cell response in secondary

infection has been linked to original antigenic sin, resulting in low affinity cross-

reactive memory T-cells that are preferentially activated by infecting virus and

inducing dysfunctional responses (216-218). Furthermore, severe dengue is associated

with HLA type. In a large case control study of Vietnamese patients with DHF,

variation at the HLA-A locus was shown to influence CD8+ T-cell responses (219).

Comparison of cytokine profiles and T-cell degranulation in a cohort of children in

Southeast Asia with acute DENV infection showed HLA-A*24-restricted T cell

responses showed suboptimal degranulation but high cytokine production contributing

to vascular leakage in secondary dengue infection (220).

Advances in understanding T-cell mediated immune response have been made with

development of synthetic peptide antigen libraries, epitope peptide prediction

techniques and in vitro models to assess T-cell mediated anti-flavivirus responses.

However, gaps in our understanding of T-cell immunopathology in natural infection as

well as vaccine-mediated exposure to DENV and related flaviviruses still remain.

B) Humoral immunity

Humoral immunity is mediated by antigen-specific antibody-producing plasma and

memory B-cells. The high degree of homology among flaviviruses stimulates

production of highly cross-reactive but not necessarily cross-protective antibodies. Anti-

flavivirus-specific antibodies can be protective, cross-reacting or enhancing depending

on age, immune status, infecting virus strain and past exposure to other related

flaviviruses.

The human antibody response in dengue is complex as it involves polyclonal responses

to primary and secondary infections with four different DENV serotypes (221).The

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adaptive immune responses to DENV is driven by the presence or absence of virus

neutralising antibodies and T-cell responses involved in initiating antibody synthesis.

Upon infection, DENV elicits IgM and IgG directly against viral membrane protein. A

primary dengue infection is characterized by a slow and low titre antibody response.

IgM antibody is the first immunoglobulin isotype to appear. Anti-dengue IgG is

detectable at low titre at the end of the first week of illness, and slowly increases. In

contrast, during a secondary infection, antibody titres rise rapidly and antibody reacts

broadly with heterologous serotypes. In secondary infection, high levels of IgG are

detectable even in the acute phase and they rise dramatically over the two weeks. IgM

levels are significantly lower in secondary dengue infections and false negative anti-

dengue IgM reactions are observed during secondary infection. According to the Pan

American Health Organization (PAHO) guidelines 80% of all dengue cases have

detectable IgM antibody by day five of illness, and 93-99% of cases have detectable

IgM by day six to ten of illness, which may then remain detectable for over 90 days.

(222, 223).

Figure 2.12 shows production of antibody at different stages of infection (6).

69

Figure 2.12- Immunological response during Flavivirus infection

Immunological response during Flavivirus infection. Response is based on phases of DENV infection.

Virus is detected for approximately one week in serum. Detection of IgM followed by IgG indicates

primary infection while detection of IgG with the onset of illness indicates secondary infection (6)

Primary infection Secondary infection

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i) Neutralising or protective antibody-mediated immunity

Neutralising antibodies (NAb) prevent infection of cells by blocking attachment of virus

to the cell surface and preventing fusion to cell membrane; initiating complement or

antibody dependent cell cytotoxicity; and by opsonisation of virus. NAb produced

during primary dengue infection is thought to be life-long; anti-DENV-1 NAb has been

shown to persist for more than 60 years in individuals infected during the Second World

War in Hawaii, USA (224). Anti-DENV-2 NAb was reported to persist for 70 years

following an outbreak in Nagasaki, Japan, however DENV-1 was the epidemic virus in

original descriptions of the outbreak (225). NAb are used as marker protection and

indicator of vaccine efficacy. Mice lacking B cells are vulnerable to infection and

passive transfer of mAbs provides protection against viral infection (226-228).

Neutralization of infection by virus-specific antibodies can occur through several

mechanisms, including inhibition of binding to cell surface receptors or post-binding

inhibition of viral fusion. Neutralisation of infection is shown to be dependent on

availability of epitope and the number of antibodies binding to epitopes. There are two

proposed models of effective neutralisation; a single hit model proposed by Dulbecco et

al (1956) in which neutralisation requires binding of antibody to critical epitopes that

lock virus to a confirmation which prevents infection (229); and a multiple hit model

which proposes that neutralization requires a threshold number of neutralising

antibodies binding to viral epitopes to induce interference with attachment and fusion of

virus and hence prevent infection (230, 231). WNV monoclonal antibody (mAb-E16)

occupies ~25% of viral epitope sites per virion and gives effective neutralisation (218,

232). Pierson et al proposed a model of the stoichiometric requirements of for flavivirus

neutralisation based on analysis of WNV E-DIII specific mAbs Figure 2.13 shows that

if antibody can dock on to virion with stoichiometry sufficient to exceed threshold,

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neutralisation occurs, while engagement of virion with stoichiometry below this

threshold may support antibody mediated enhancement of infection (4, 44).

Flavivirus envelope protein is the major epitope for neutralising antibodies. It has been

shown in DENV-2 that all 3-domains of E-protein have antigenic characteristics and

conformation of these domains plays a significant role in eliciting immune response.

Antigenic properties of E protein are studied in detail by isolating mAbs against

different regions of protein and show anti-E-protein antibodies elicit virus neutralising,

hemagglutination-inhibiting and fusion-blocking antibodies (233-235). Domain III of E

protein is recognised by several anti-DENV mAbs which effectively block virus

absorption (236-239). However neutralising antibody response elicited by E-protein is

highly cross reactive and not all E-protein-directed antibodies are neutralising (240,

241). Low neutralising but enhancing antibody was reported against prM and M protein

(242, 243).

ii) Cross reactive and enhancing antibody mediated immune response

DENV-specific antibody mediated immune response triggered by a particular serotype

provides life-long immunity to the infecting serotype but not to the other three

serotypes. In secondary dengue, antibody directed towards original primary serotypes is

shown to cross-react with heterologous secondary infecting serotypes, which is termed

original antigen sin. It was first described in 1983 by Halstead el al, in the study of

severe dengue in Thai children. The highest neutralising antibody responses were

directed towards the initial infecting DENV type during secondary infection (244). A

large repertoire of DENV-reactive memory B cells persist for years after infection in

dengue-immune individuals, and human E-specific as well as prM-specific mAbs

extensively cross-react with the four DENV serotypes (245). The degree of relatedness

among infecting serotypes and preferential activation of cross-reactive memory cells is

proposed to promote original antigenic sin (18). This phenomenon of cross reactivity

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by past DENV is the proposed cause of the immunological cascade responsible for DHF

during secondary infection(246).

In secondary dengue infection with heterologous serotype, presence of non-neutralising

but cross-reacting antibody from past infection facilitates Fc - receptor mediated entry

of virus to mononuclear phagocytes. Fc receptor mediated entry triggers biased cytokine

responses thus switching off protective mechanism of macrophages and instead

enhances amplification of virus (247). This results in increase of viral load which then

drives an immunopathogenic cascade, exaggerating cytokine responses, that leads to a

transient increase in vascular permeability. This mechanism is termed antibody

dependent enhancement (ADE).The precise way in which vascular permeability is

altered is not clear but is more likely to be a functional change rather than structural

damage, as dengue shock is rapidly recoverable, and no inflammation is evident in the

leaking surfaces (246, 248, 249).

Wanning of neutralising antibodies to the concentration that is no longer capable to

neutralise infection is also shown to increase severity of dengue (221, 250). The most

compelling evidence for ADE has come from studies with infants, who have passively

acquired antibodies to DENV from their mothers (221). Studies in Thailand reported

DHF/DSS peaked in populations of first-time infected infants born to dengue-immune

mothers and children who had experienced a mild or asymptomatic dengue infection

and become secondarily infected by a different dengue serotype. This study showed

DHF/DSS is 15-80 times more frequent in secondary infections than in primary

infection, and that up to 99% of DHF cases had heterotypic antibodies to the DENV

serotype causing the DHF (249). Similarly, epidemiological studies in South-East Asia

clearly link DHF/DSS to individuals who have had a previous dengue infection or who

have acquired maternal dengue antibody (251). In Taiwanese patients dengue RNA

levels even after defervescence, correlated with disease severity. Cameron et al. studied

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Vietnamese infants and showed the majority experienced DHF when maternal anti-

DENV neutralising antibodies had declined to <1:20 (252). In vitro mechanism of ADE

with virus is studied in Fc-receptor bearing cells including U937, K562 and THP-1 cell

lines in the presence of varying antibody concentration. In the absence of antibody these

cell lines are selectively permeable to flaviviruses like DENV and ZIKV while in the

presence of non-neutralising antibody increased infection of these cell lines has been

reported (253-255).

With the recent outbreak of ZIKV the high degree of homology between DENV and

ZIKV has increased concern about enhancement of infection. Monoclonal antibody

studies show ZIKV cross-reacting and enhancing anti-DENV antibodies (256-261).

Possible outcomes of dengue and Zika immune primary and secondary infection is

shown in Figure 2.14 adapted from Culshaw et al. This figure shows that

immunopathogenesis in secondary infection is determined by interaction between

antibodies or T cells triggered during primary infection, and heterogenicity of secondary

infection virus.

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Figure 2.13:- Antibody threshold determining immune response in Flavivirus infection

Binding of Antibody with the virus requires a threshold in number of Ab and epitopes. Multiple binding of antibody to maximum accessible

epitopes results in neutralisation of viruses while the non-neutralising concentration of Ab triggers Fc- receptor mediated entry into

macrophages resulting in enhancement of virus production(4, 5).

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Figure 2. 14:- Adaptive immune responses in Flavivirus infection.

During primary immune response, virus-specific antibodies and T cells are activated. In secondary infection immune memory developed during primary

response can be protective or pathogenic depending on virus type involved in secondary infection. Cross-recognition within the serotypes of DENV and

flavivirus cross-reactivity (DENV-ZIKV) can be seen for both T-cells and antibodies however this elicits protective or pathogenic effects .

76

2.7. Laboratory diagnosis of Flavivirus infection

Accurate diagnosis is essential for clinical management, disease surveillance, studies of

pathogenesis, vaccine development and development of treatment strategies. Laboratory

diagnosis of flavivirus infection depends on availability of diagnostic markers. These

markers include virus and viral particles and immunological components produced in

response to infection and depend on timing of sample collection relative to time of

infection. In acute illness and during the viremic stage direct virus/viral particle

detection can be used while serological markers IgM, IgG are present at the end of the

acute phase and later in the post-infection period. Neutralising antibody has been

reported to persist for more than 60 years post infection in DENV-1 infection (224) and

70 years post DENV-1/2 infection (225).

Diagnostic markers and tests E-gene used at different times in dengue infection are

summarized in Figure 2.15.

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Diagnosis of dengue follows two methods:- direct detection of virus and virus particles

in the course of fever and viraemia and indirect detection of infection via measurement

pf anti-DENV antibodies in the post-febrile period (7). Figure 2.16 summarises

available direct and indirect methods for dengue diagnosis. Direct detection is highly

accurate but has limitations in terms of timing of sample collection and availability of

resources while indirect diagnosis is widely used due to long term persistence of

diagnostic markers and requiring less resources.

Figure 2.15:- Course of dengue infection and timing of diagnosis tests (7)

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2.7.1. Virus isolation

DENV was first isolated in 1943 by Ren Kimura and Susumu Hotta during a dengue

epidemic in Nagasaki, Japan(262) and in 1944 by Albert B. Sabin and Walter

Schlesinger in Hawaii, USA (263). ZIKV was isolated in April 1947 from serum of a

febrile rhesus monkey in the Ziika Forest, Uganda (127). Early virus isolation was done

by inoculation of mice and cell lines derived from mosquito and mammalian tissues.

Inoculation in newborn mice was widely applied (60) but this approach has limited

value as field strains DENV are not usually pathogenic for newborn mice and do not

consistently produce cytopathic effects (264). In 1964 recovery of DENV in a monkey

kidney cell line of grivet monkey Cercopithecus aethiope (Vero cells) was described in

attempts to use mammalian cell lines to isolate DENV from natural human infection

(265). Rosen and Gubler reported parenteral inoculation of Ae. albopictus mosquitoes in

1974 as a much more sensitive method to detect DENV present in serum from natural

infection or that had been adapted to cell lines or newborn mice (266).

Other cell lines used for DENV recovery include mosquito cell lines AP-61 (A.

pseudoscutellaris); Tra284 (Toxorynchites amboinensis;) C6/36 (Ae. albopictus); AP-64

Figure 2.16 :-Diagnosis of dengue.

Modified from Peeling, R. W. et al. Evaluation of diagnostic tests: dengue (1)

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(clone of an A. pseudoscutellaris cell line); CLA-1 (clone of an A. pseudoscutellaris);

and mammalian cell lines LLCMK2 (monkey kidney); Vero (monkey kidney); BHK21

(Baby hamster kidney) (267). ZIKV has also been isolated in these cell lines. However,

ZIKV is neurotropic and infects a wide range of cells including neural and placental

cells. Chan et al reported productive ZIKV replication in placental (JEG-3); neuronal

(SF268); muscle (RD); retinal (ARPE19); pulmonary (Hep-2 and HFL); colonic (Caco-

2); hepatic (Huh-7) cell lines; as well as nonhuman primate (Vero and LLC-MK2); pig

(PK-15); rabbit (RK-13); hamster (BHK21); and chicken (DF-1) cell lines (268).

Isolation of ZIKV has been successful in brains of suckling mice (269).

Isolated virus may be identified by immunofluoresecences assays (IFA) using virus

specific monoclonal antibodies or polyclonal antisera, and by molecular techniques

including RT-PCR. Virus isolation requires a viremic sample and is not possible in the

post-viremic phase of infection.

2.7.2. Genome detection

Molecular diagnosis of DENV and ZIKV is based on detection of viral genomic

material. These diagnostic tests result in rapid and precise identification of infecting

virus. The most widely used techniques are reverse transcriptase PCR (RT-PCR) and

real time RT-PCR. Viral RNA is extracted from serum, body fluid, tissues including

paraffin-imbedded tissues, or culture supernatant, purified, and reverse transcribed to

complementary DNA (cDNA), which serves as template for PCR. (18, 270). Detection

and typing of DENV was first shown by Lanciotti et al using a two-step nested PCR

(271), this approach was subsequently simplified to a single tube reaction with

equivalent sensitivity (272). Recent advances in DENV RT-PCR include enhanced

RNA extraction and reverse transcription and DNA amplification methods and primer

design. Primers specific for DENV serotypes, flavivirus consensus primers located in

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different genes including E, NS1, NS3, NS5, are widely used (267, 273-275). Real-time

RT-PCR is a more sensitive method compared to conventional PCR (276) and is

quantitative. Virus can be detected directly in blood by RT-PCR and real time RT-PCR

during the viremic phase of infection. Prolonged ZIKV detection up to 80 days in

vaginal secretions and whole blood has been reported in a case study of previously

healthy, non-pregnant travellers to United states from Honduras (277). ZIKV detection

in Urine samples has been reported for notably longer period of than for serum samples

(278). Study on travel-associated ZIKV infection imported to Italy, showed ZIKV RNA

detection in whole blood was comparable to urine with 100% detection rate till 10 days

while only 33.3% plasma samples were ZIKV RNA detection (279).

Nucleic acid slot-blot nucleic acid hybridization was also used to quantitatively detect

DENV-2 using radiolabelled cDNA probe, pVV17 (280). Reverse-transcription-loop

mediated isothermal amplification (RT-LAMP) assays target the 3’ noncoding region

for rapid detection of DENV (281). RT-LAMP has also been described for detection of

ZIKV (282). Mass spectrometry, microarray techniques, and luminescence based

techniques (Luminex) are increasingly used for detection of DENV genomes (283-285).

2.7.3. Antigen detection

The most frequently detected DENV antigen in the viremic phase is NS1 protein.

Serological detection of NS1 via EIA-based approaches is accessible in resource-poor

settings and is used worldwide for DENV surveillance whenever acute phase blood

samples are available. DENV NS1 has been detected in serum of infected person as

early as day one post infection and up to 18 days post infection. Several commercial

rapid diagnostic tests (RDTs) for NS1 antigen detection, and ELISA-based NS1 kits are

available for DENV identification. Pal et al showed that ELISA-based kits showed

higher sensitivity than antigen RDT kits (286). NS1 is a highly conserved glycoprotein

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used to detect DENV but does not differentiate among serotypes. Recent development

of anti-NS1 monoclonal antibodies has been successful in developing assays specific to

one or all serotypes. Anti-NS1 monoclonal antibodies that differentiate DENV and

ZIKV have been developed and can be used in an immunochromatography format to

identify DENV serotypes and ZIKV without cross-reaction (287). A retrospective study

in French Guiana using the Platelia Dengue NS1 Ag kit showed no false positive

dengue NS1 test results in acute-phase ZIKV infection (288). These data indicate a role

for NS1 antigen tests in differential diagnosis of DENV and ZIKV infection. Dot blot

assays directed to the envelope/membrane (E/M) antigen are also of value in detection

of viral antigen(289). Multiplex microsphere immunoassay (MIA) for the specific

diagnosis of differential virus non-structural proteins NS1 and NS5 has been reported in

ZIKV diagnosis (290).

2.7.4. Serology

Anti-DENV antibodies persist for many years after infection. Although serology is the

most accessible technique for viral diagnosis it is most challenging. The major difficulty

for serological diagnosis is the high degree of cross-reactivity among the DENV

serotypes and other flaviviruses (286). DENV-specific antibodies can be identified by

hemagglutination inhibition (HI) (291); MAC-ELISA / IgG ELISA assays (289, 292);

immunoblotting techniques (292, 293); and serum neutralisation test (294).

HI measure total anti-viral antibody titres based on the ability of virus binding antibody

to inhibit agglutination of red blood cells (RBC) by virus. For DENV diagnosis goose

RBC are generally used. HI is traditionally used to differentiate primary and secondary

infection due to its simplicity and sensitivity (289, 291). The WHO criteria for primary

infection includes HI titre <2560; in secondary infection HI titre > 2560. This test shows

cross-reactivity between DENV and related flaviviruses (295).

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IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is used for

detection of IgM if the sample is taken within appropriate time frame. This test aims to

identify recent infection but false positive results occur in patients with malaria or

previous dengue infections (296). IgG-ELISA is likely the most widely used sreening

assay for serodiagnosis of DENV infection in non-endemic areas. In primary infection

anti-DENV IgG appears shorty after IgM antibodies 7-10 days following onset of

symptoms; in secondary infection high levels of IgG are detectable during the acute

phase. The ratio of IgM/IgG hence can be used for differentiation of primary and

secondary DENV infection (297, 298).

Immunoblotting techniques detect anti-DENV IgM or IgG dependent on sampling time.

Commercial immunoblot kits for IgM show results comparable with IgM ELISA (299).

Comparative analysis of six DENV specific IgM and IgG kit compared sensitivity,

specificity and Kappa statistics of IgM and IgG and showed that immunoassays (EIA)

from MRL Laboratories and PanBio, a dot blot assay from Genelabs, and a dipstick EIA

from Integrated Diagnostics (INDX) are useful and reliable assays for dengue

immunoblotting (16).

Serum neutralisation tests determine anti-DENV neutralising antibody titre, which is

regarded as correlation of protection. It is also used as marker of vaccine induced

immunity (300). The gold standard test for determining neutralising antibody is the

serum dilution plaque reduction neutralisation test (PRNT). However, all field isolates

of DENV are not plaque-inducing and the traditional PRNT format has been modified

with different end-point determination techniques which includes focus reduction

neutralisation test (FRNT) (301, 302); microneutralisation test (MNT) (303, 304); and

flow cytometry (305, 306) These tests are based on measuring the capacity of anti-

DENV antibody present in patient serum or plasma to inhibit infection of a cell

monolayer. Briefly, consecutive serially diluted antiserum is incubated with a

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standardised amount of virus and this antibody-virus complex is exposed to the cell

monolayer; the highest dilution of anti-DENV that inhibits infection by 50% (or other

endpoint) is determined as the neutralisation titre. Commonly used cell lines include

Vero, C6/36, and U937-DCSIGN (6, 307, 308). All tests listed above vary in

determination of end point. In the classical PRNT this is determined by staining the

infected monolayer with dyes including methylene blue, crystal violet and neutral red,

which stain non-infected cells leaving plaques as clear areas in the cell monolayer

which are quantified. FRNT is useful for DENV strains which are not plaque-inducing

but do infect cells. FRNT measures infectious foci on the monolayer which are detected

by an immunostaining procedure using anti-DENV monoclonal antibodies or polyclonal

antisera. Liu et al reported high correlation between PRNT and FRNT in a 96 well

format (309). MNT is the cost-effective high-throughput version of neutralisation based

the technique of ELISA. Flow cytometric determination of neutralisation uses

fluorescein-conjugated anti-DENV monoclonal antibody to determine infection of cells,

to analyse 50% virus neutralisation. Kraus et al suggested flow cytometry-based

neutralisation assays offer significant advantages over PRNT, however flow cytometry

can only be performed in selected laboratories (308). Comparative evaluation of PRNT,

MNT and flow cytometry-based tests for measurement of anti-DENV neutralising

antibodies demonstrate high reproducibility and sensitivity (310).

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2.8. Flavivirus infection in travellers

The risk of travel-associated flavivirus infection depends on availability of the vector,

population density, immune status of the traveller. Highly viremic travellers pose a

greater risk for autochthonous transmission when the vector is present and can

contribute to the global spread of infectious disease and emergence of pathogens (311).

2.8.1. Dengue

Historically DENV has been reported to spread from Africa during travel for trade, and

troop movements in World War II. Spread of disease was due resilience of vectors

which could survive in water reservoir of sailing ships. In recent times vectors are

reported to effectively move within and between countries in automobile and truck tires.

Since the advent of frequent and cheap international airline travel travellers have

become the major cause of dengue notifications in non-epidemic areas where the

vectors may or may not be present. According to the US Geosentinel Surveillance

System, in 1997-2011 11% of travel-associated disease was dengue (311).

Travel related DENV infection has increased from 2% to 16% in 15 years from 1900s to

2000s. Within 60 years the number of countries reporting dengue increased from 9 to >

more than 100 (118). During 2003-2005, 219 imported dengue cases were reported in

Germany (312). Similarly, in 2004-2009, 16-46 travel-associated dengue cases were

diagnosed per year in Denmark (313). An incidence rate of 14.6 per 1000 person-

months was reported from the Netherlands in 2006-2007 (314). The majority of travel-

associated dengue is acquired at popular tourist destinations in the tropics and sub-

tropics.

Imported dengue is mostly acquired in Asia, particularly Southeast Asia, and the

Americas. Geosentinal Survey data from 2000-2010 reports 50% of cases were

imported from Southeast Asia (315). During 2010-2013 87-112 infective person-days

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per 100,000 travellers from Thailand was reported each year (316). Similarly, 70% of

imported dengue cases in Sweden in 2010 were reported to be acquired in Southeast

Asia (317).

In Australia, most dengue cases are travellers from endemic/epidemic areas. Local

autochthonous DENV transmission is only reported in northern Queensland where Ae.

aegypti is present. All other dengue cases in Australia are imported cases (318), with the

exception of an apparent locally-acquired DENV infection in northern Western

Australia in 2015 (73). Ae. aegypti has not been present in WA since the 1940s

however dengue represents 40% of all Indonesian-acquired infections notified to the

West Australian Department of Health. The number dengue notifications began to

increase when cheap flights to Southeast Asia were introduced in the first decade of the

21st century. Major outbreaks of dengue occurred in Southeast Asia including Bali,

Indonesia, a popular tourist destination for WA residents, in 2012, 2015 and 2016

(319).

Travellers are also sentinels for DENV endemic areas. Study of dengue in travellers

gives geospatial distribution of strains of DENV at a given time. This also explains the

pattern of spread of disease and emergence of strains of virus (312). Emergence of new

lineages has been reported in travellers. New lineage of DENV 3 and 4 was reported

from study in Germany in 2006-2015. New lineage of DENV-2 was reported in

travellers from WA was reported in 2014 (11).

2.8.2. Zika

ZIKV infection in travellers is one of the major factor for global spread of ZIKV. The

first large outbreak in the 21st century was in Yap State, Federated States of Micronesia,

in 2007 where more than 70% of the population was infected. The virus spread

throughout the Pacific and caused an outbreak in French Polynesia; the epidemic virus

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was of the Asian lineage and was closely related to Cambodian strain (320). A

subsequent outbreak in New Caledonia was reported in 2014; the first cases were

imported from French Polynesia (321).

The first travel-associated cases in the US were reported in two scientists returning from

working in Senegal. Possible sexual transmission was also reported in the same case

(147). In 2015, ZIKV infection in a traveller returning from Maldives in Finland was

reported (147). Cases of imported ZIKV have been reported in Europe since 2013. The

first case of ZIKV infection was reported in Germany in 2013, followed by Norway and

Italy in consecutive years. After the 2015 outbreak in Brazil the number of countries

with imported Zika has increased(320). In Australia the first travel-associated ZIKV

infection was reported in 2013 in a individual returning from Jakarta, Indonesia(322).

Increase in human mobilisation has increased spread of viruses and other pathogens.

Travellers are a medium for rapid global spread of flavivirus infection.

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Chapter 3:- Methods

3.1. Vero cells

The Vero cell line (Vero (ATCC® CCL81™)) was used throughout. This cell line is

derived from African green monkey (Cercopithecus aethiops) kidney fibroblasts. Cells

were grown in Dulbecco's minimum essential medium (DMEM) (Invitrogen) with 1%

Penicillin-Streptomycin (GIBCO, Invitrogen, 5,000 U/mL) and 1% L-Glutamine (200

GIBCO, Invitrogen mM), supplemented with 5% fetal bovine serum. All cell cultures

were maintained in 5% CO2 at 37°C in a humidified incubator.

3.2. Virus amplification

Viruses are detailed in Chapter 4. Vero cell monolayers at 80-90% confluency was

prepared. Half of the spent media was removed and 100ul of virus suspension was

inoculated onto the monolayer. The culture was incubated overnight spent media was

removed completely, followed by addition of maintenance media (DMEM

supplemented with 2% fetal bovine serum). Appearance of CPE was noted every day;

virus was harvested when ~100% CPE was obtained or before media colour changed

acidic whichever occurred first. Growth of virus was confirmed via hemagglutination

and NS1 antigen tests (Appendix 3.1). Harvested viruses were stored as 300ul aliquots

in -800C to preserve until further use.

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3.3. Virus Titration

3.3.1. Determination of Tissue culture infectious dose (TCID50)

Virus titration was performed using ELISA to determine 50% endpoint titre using Reed

and Muench method. This titration was done to ensure optimal growth of virus to

desired concentration. Virus with titres greater than 10-4

was used in assays. The

protocol for TCID50 determination is given in Appendix 3.2.

3.3.2. Determination of Focus forming units (FFU)

a. Preparation of monolayer

The first optimisation for establishment of the focus forming assay (FFA) was to

determine the number of cells per ml required for a 24-well plate format that would

allow the cell monolayer to remain adherent for at least 5 days to support optimal

growth of all DENV strains. A range of cell concentrations was tested; optimal input for

DENV FFA was found to be 6x104 cells/ ml.

b. Virus dilution

Virus stocks were diluted from 10-1

to 10-5

in maintenance media.

c. Test establishment

500ul of 6X104 cells/ml of Vero cell in 5% DMEM were seeded into 24 well plates to

obtain confluent monolayers in 16-24 hours. 200ul of 10-folddilutions of prepared virus

stocks was inoculated onto monolayers. After one hour a methylcellulose overlay was

added to stop virus progeny from spreading around the monolayer in a secondary

infection and allowing input virus to form infectious units at a single focus, reflecting

the number of infectious units at the time of inoculation. A plate template was prepared

to run duplicate dilutions of each virus and included no-virus cell-only controls well in

every plate. Plates were carefully monitored for formation of FFU every day under a

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phase-contrast microscope and immunostained when infectious units were apparent.

Each strain has different time-point of maturation and size of FFU. Average time for

optimal infectious unit formation for all viruses was 3-7 days. Allowing infection to

proceed past this optimal time increased the size but not the number of infectious units.

d. Detection of FFU

i. Optimisation of Fixative

To obtain optimal immunostaining without disturbing Vero monolayers three different

fixative agents were compared: 80% Methanol; 1X PBS/ACETONE; and 4%

Formaldehyde PBS. 80% methanol interfered in spot development as spots were faint

(compared to spots with other fixatives); during the drying step cells became

excessively dry and shred from the plate surface to be removed during washing.

PBS/Acetone drying also caused cells to become detached from the plates surface. Cells

were fixed properly using 4% formaldehyde and there was no cell shredding or faint

focus unit. Therefore 4% formaldehyde was used throughout to fix cells. All chemical

waste was collected in dedicated containers and disposed as per UWA chemical safety

guidelines.

ii. Optimisation of 4G2 monoclonal antibody concentration (primary antibody):

The monoclonal antibody 4G2 was used as primary antibody for detection of DENV

infectious units. Briefly, 4G2 was produced in large volumes from a hybridoma cell line

in 10% RPMI. The optimal 4G2 titre was determined by checkerboard ELISA

(Appendix 3.3). Supernatant from the hybridoma cell culture was then filtered with a

sterile vacuum filter and stored in aliquots at -80OC. The same batch of 4G2 was used

throughout.

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iii. Optimising secondary antibody concentration

Goat Anti-Mouse IgG (H+L)-HRP Conjugate (GAM) from Biorad (#1721011) was

used as secondary antibody for ELISA and FFA. Optimal GAM concentration was

determined along with 4G2 by checkerboard assay.

iv. Immunostaining

Visualisation of infectious units was done as previously described for mumps virus

(301). The overlay was removed, monolayers rinsed with phosphate buffered saline

(PBS, pH 7.4) and then fixed with 200 μL/well of 4% formaldehyde in PBS for 30-

45 min. 2% Casein with PBS/Tween-20 was used for blocking and permeabilization.

200ul of buffer was added and plates were gently placed on a plate rocker at the lowest

speed to ensure coverage by buffer for 30 minutes at room temperature (RT). Each well

was then incubated with 200ul of 4G2 in BB buffer (1/40 as determined by

checkerboard assay) for 1 hr at RT. Cells were then washed 2 times with PBS and

incubated with GAM-HRP secondary antibody (1/1500, determined by checkerboard

assay) for 1 hr at RT. Plates were again washed 2-times with 1X PBS followed by

incubation with 150ul of TrueBlue peroxidase substrate. Substrate was incubated for

10-20 mins maximum in the dark and the reaction was stopped with water. Blue-

coloured infectious units were observed.

FFU were counted and virus titre was determined from the mean number of infectious

foci obtained from duplicate runs. Virus dilutions giving 30–60 foci per well in a 24-

well plate were selected for ongoing experiments.

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3.3.3. Determination of Plaque forming units (PFU)

Selected viruses, including ZIKV, which form visible plaques were also used to cross-

validate FFA neutralisation data. Establishment of plaque assay was as described for

FFA. Plaques were detected by staining monolayers with 1% Methylene Blue in 10%

formaldehyde. Stained plates were stored in the dark overnight and washed with

running water.

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3.4. Methods for Chapters 5 and 6

3.4.1. Neutralising Antibody (NAb) titre determination

3.4.1.1. Preface

The neutralisation test is based on the principle that reaction between antibody and virus

renders the virus non-infectious and thus prevents entry of virus into host cells,

preventing or reducing infection. Known concentrations of virus are incubated with

varying dilutions of antibody as serum or plasma and added to susceptible target cells.

Virus that is not neutralised by antibody will induce infection. In the PRNT plaques

formed by infecting virus are visualised as clear areas on the cell monolayer; staining

with methylene blue dye stains only live Vero cells and enhance the appearance of

plaques. In the FFA, infection is detected by staining with virus specific monoclonal or

polyclonal antibody attached to an enzyme label; addition of the respective substrate

will visualise foci of infection.

Principals of neutralisation and methods for detection are shown in Figure 1.

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Figure 3.1: - Neutralisation

Neutralising antibody blocks entry of virus into cells hence preventing infection. In the absence of

antibody infection can be detected by formation of infectious units or distinct plaques

94

3.4.1.2. Establishment of neutralisation test

a. Preparation of monolayer

500ul of Vero cells at a concentration of 6X104 cells/ml in 5%DMEM ml were seeded

in 24-well plates to obtain confluent monolayers in 16-24 hours.

b. Preparation of plasma sample

Plasma samples used in the study are detailed in Chapter 4. 10-fold dilution of plasma

samples were prepared and heat-inactivated for 30min in a 56oC water bath. Serial 2-

fold dilutions starting from 20, to 320 were prepared for all DENV and control samples;

the dilution scheme for ZIKV samples varied based on available sample volume.

c. Focus reduction neutralisation test (FRNT)

i. Preparation of virus dilution

Virus dilutions (as determined in 3.3.2.) were prepared to obtain dilutions that produced

30-60 FFU; this was dependent on foci size in 24 well plates.

ii. Test establishment

Titrated virus with known FFU was mixed with respective plasma dilution at equal

volume and incubated for 1 hour at 37oC in 5%CO2. 200ul of plasma / virus mixture

was then added on to the Vero monolayer and incubated for 1 hour at 37oC with 5%

CO2. In each plate, two virus control wells (virus+media only; no antisera) and two cell

control wells (no virus; media only) were included. An overlay of 1% methylcellulose

in 5% DMEM with Amphotericin –B was added. Plates were incubated for 4-5 days at

37oC with 5% CO2. All test sets were performed in duplicate in two different

experimental runs.

95

iii. Immunostaining and visualisation of FFU

Immunostaining was done as optimised in section 3.3.2 (d). FFU in virus control well

and serum/plasma dilution was noted and percentage reduction at each dilution was

calculated to determine corresponding neutralising antibody titre that gave 50,75, and

90 percent reduction.

iv. Plaque reduction neutralisation test (PRNT)

Establishment of PRNT was same as for FRNT. Virus input was determined by PFU

titration in section 3.3.3. NAb titre was determined by comparison to virus control

wells.

d. Neutralisation titre (NAb) determination

For calculation of percent reduction an Excel spread sheet was created in which the

number of FFU or PFU in test wells was subtracted from the number of FFU or PFU in

the serum/plasma dilution, divided by FFU or PFU in virus control wells; the resulting

value was converted to a percentage. Neutralisation titre is expressed as the reciprocal

of serum/plasma dilution. Percent reduction value for corresponding reciprocal of

serum/plasma dilution was entered into Graphpad Prism 7.0. Nonlinear regression curve

fit was used to interpolate NAb titre at 50%, 75% and 90% reduction. A regression

analysis approach was used because of the censored nature of data (i.e. minimum of 20,

max of 320), tobit regression with random effects (the subjects) was used.

Figure 3.2 and 3.3 shows a flow chart of neutralisation establishment and detection by

FRNT. Sample FRNT and PRNT test plates are shown in Figure 3. 4

96

Flow chart for Neutralising antibody titre determination

Figure 3. 2:- Establishment of neutralisation

97

Figure 3. 3:- Immunostaining and visualisation of FFU

98

1/20

1/40

1/80

1/160

Plasma

dilution

1/320

Figure 3. 4:- FRNT and PRNT

FRNT and PRNT plates used for neutralisation assay in 24 well plate format. Virus control well

consists of virus loaded in absence of antibody representing total virus input. Each row represents

serial dilutions of plasma from 20-320-fold. Equal volume of virus was treated in each dilution. For

the virus control blank media was used as mock serum. Absence of infectious units at any dilution

represents neutralisation of virus. FRNT was used for all strains of DENV. PRNT was used in some

ZIKV cases to confirm PRNT results. Tests gave comparable results.

Virus

Control

Cell Control Virus

Control Cell Control

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3.4.2. Hemagglutination inhibition (HI) antibody titre determination

3.4.2.1 Principle

This test is based on the principle that in the presence of antibody, binding of virus to

goose red blood cells in (GRBC) is inhibited. Virus particles attach to GRBC surface

agglutinates and form a lattice thus preventing cells falling to the bottom of V- well

plates. This phenomenon is known as hemagglutination (Figure 3.5). In the presence of

virus-specific antibody, an Ab-virus complex is formed hence no virus is available to

agglutinate GRBC, this is called hemagglutination inhibition (Figure 3.6). The

maximum dilution of Ab that inhibits hemagglutination is the antibody titre.

Figure 3. 5:- Hemagglutination of GRBC by flavivirus

Figure 3. 6:- Hemagglutination inhibition by anti-flavivirus antibody

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3.4.2.2. Methodology to establish HI test

a. Preparation of viral antigen

Viruses for use in HI tests were amplified by cell culture. Inactivation of virus was done

with Binary Ethylenimine (BEI) as previously described (24). Virus stocks were first

centrifuged to clear dead Vero cells and treated with 0.1M Binary Ethylenimine (BEI) at

0.175N sodium hydroxide for 6-8 hours at 370C. Reaction was stopped by addition of

1M sodium thiosulphate and held overnight at 40C. After overnight incubation

inactivated virus was aliquoted and stored at -800C until further use. Preparation of all

reagents is given in Appendix 3.4.

b. Titration of inactivated virus

BEI-inactivated virus was titrated by hemagglutination assay (HA). HA was performed

in V-bottom 96 well plates. Virus was diluted in bovine albumin borate saline (BABS)

prepared from 4% bovine albumin (in saline) and borate saline at pH in 1:5 ratio. Each

virus was tested at pH 6.0, 6.2 and 6.4. Briefly, 50ul of virus dilution in BABS was

prepared in doubling dilution and 50ul of 10% Goose Red Blood cells (GRBC) was

added, followed by 1-hour incubation at room temperature. Positive reaction was

detected by a matrix of RBCs or the absence of pellet; negative reaction was indicated

by presence of a pellet.

c. Preparation of plasma sample

Non-specific inhibitors in patient serum or plasma were extracted using 20% Kaolin

suspension and 50% Goose Red Blood cells (GRBC). Briefly, 500ul of 20% Kaolin

was added to 100ul plasma samples in labelled micronic tubes. The mixture was

incubated at RT for 60 minutes with thorough mixing every 10 minutes to avoid false

positive results due to incomplete mixing. After 60 minutes tubes were centrifuged at

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3000rpm in 40c for 10 minutes. 50ul of 50% GRBC was added to the clear supernatant

after Kaolin purification and incubated at 40C for 60 minutes mixing by inversion every

10 minutes. Mixing at this step was done carefully to avoid lysis of GRBC that can

interfere with following testing. After 60 minutes tubes were centrifuged at

3000rpm/40c for 5 minutes. Clear plasma supernatant was then aliquoted in fresh

micronic tubes and stored at 40C and used within week of preparation.

d. Establishment of test

Plate plans were prepared beforehand for 96 well v-bottom plates. On test day plates

were labelled as according to plate plan. Plasma sample prepared after kaolin treatment

was further diluted two-fold in 25ul BABS from 1:10-1:640 convalescent plasma

samples (more than two months post infection) and 1:10-1:10240 for acute phase

samples (within two months post infection). Subsequently, 25ul of viral antigen at 4HA

unit, 50ul was added to each well containing 25ul of plasma dilution. Virus-plasma

mixture was then incubated overnight at 40C. Back-titration of virus dilution was

prepared as antigen control. HI inhibition was determined by adding 10% GRBC at

appropriate pH for that particular virus as determined in part (b). Positive HI test was

detected by presence of pellet at the bottom of plate. Figure 3. 7 shows systemic

diagram for HI test performance.

102

Figure 3. 7:- Measurement of total antibody by hemagglutination inhibition test

103

3.4.3. Mapping antigenic diversity using cartography

3.4.3.1. Preface

Neutralising antibody titres were used to create an antigenic map. Antigenic maps show

the variation in antibody responses across a range of viruses. Antigen maps were

initially used to derive antigenic distance between genetically related influenza strains

(323). In this project we expect to visualise systematic changes in neutralisation and

recognition patterns by anti-DENV antibody at different time points after infection and

to antigenically characterise DENV strains based these antibody responses.

3.4.3.2. Cartography technique

Antigenic maps were created with web based software https://acmacs-web.antigenic-

cartography.org/ designed by the Antigenic Cartography Group at the University of

Cambridge as previously described by Smith et al (323). Viruses and sera are given N-

dimensional locations on a grid in which each square side corresponds to two-fold

antiserum dilution or one antigenic unit (AU). A distance of one antigenic unit

translates to a two-fold drop in antibody titre; two square sides are four fold drop in

serum dilution, and so on (323). Briefly, the target distance from a serum to each virus

is derived by calculating the difference between the logarithm (log2) reciprocal NAb/HI

titre for that particular virus and the log2 reciprocal maximum titre achieved by that

serum (against any virus). The distance between viruses and sera in the map are

inversely proportional (323-325).

https://acmacs-web.antigenic-cartography.org/

https://acmacs-web.antigenic-cartography.org/

104

Figure 3. 8:- Antigenic mapping strategy

105

3.4.4. Western blot

3.4.4.1. Preface

Western blot analysis was used to investigate viral protein targets of anti-DENV and

anti-ZIKV. Vero cell lysates infected with DENV and ZIKV were gel electrophorized

through SDS-PAGE. Separated viral proteins were blotted onto nitrocellulose

membranes. Plasma from DENV- and ZIKV-infected individuals was used as primary

antibody. Viruses used in western blot analysis are ZIKV MR766 /ZIKV PRVABC59,

WHO reference strains of all four serotype of DENV (DENV-1 HW2001G4, D2-

NGCAII, D3-H87G5 and D4-H241GI) and YF17D vaccine isolate.

3.4.4.2. Methods

a. Optimisation of whole virus protein preparation

Viruses were inoculated onto Vero cell monolayers and cells were collected with a cell

scraper at 60-70% CPE. Two different techniques were used to prepare virus lysate:

radioimmunoprecipitation assay (RIPA) buffer lysis method and glutaraldehyde fixation

method, followed by sonication.

RIPA lysis buffer is widely used for preparation of protein lysates from cell culture and

to extract protein from cells and tissues for immunological assay. RIPA buffer was used

to prepare DENV lysate as previously reported (326). Scraped infected Vero cells were

lysed in RIPA buffer (ThermoFisher scientific) in the presence of 1% protease inhibitor

cocktail followed by sonication.

The glutaraldehyde fixation method was developed based on previously described

methods for DENV antigen preparation, followed by sonication to lyse fixed cells

(204). Infected cells were fixed for 20 minutes with glutaraldehyde (0.025%) at 4oC

with constant agitation in between. Cells were then sonicated at 60% pulse for 3 X 30

sec cycles or till cell suspension was clear. Comparative Coomassie stained protein

106

bands prepared by RIPA buffer lysis and glutaraldehyde fixation are shown in Figure

3.9. There are various factors that affect the stability of lysed protein. To maintain

proteins in the state at time of lysis it is critical to keep cells on ice and to use

appropriate the concentration of protease inhibitors. Based on the results obtained,

including and excluding RIPA buffer on the same batch of protein lysate,

glutaraldehyde fixation was selected for preparation of virus antigen.

Figure 3. 9:- Comparison of protein lysate preparation methods

For both methods protein samples were prepared from the same Vero cell lysates

107

b. Quantification of Protein

Protein was quantified using the bicinchoninic acid (BCA) colorimetric detection

method. Thermo Scientific™ Pierce™ BCA Protein kit was used for quantification. The

manufacturer’s protocol was followed to prepare samples. Serial dilution a of known

concentration of provided bovine serum albumin (BSA) was used as standard.

Absorbance was read using Polarstar Omega micro plate reader. MARS data analysis

software was used to construct standard curve and to quantify unknown samples.

c. Optimization of primary antibody concentration

Plasma samples from selected ZIKV and monotypic DENV subjects were used as

primary antibody. Dot blot assay was performed to optimize primary antibody

concentration for all plasma samples used. Three different concentrations of primary

antibody (1/50, 1/100 and 1/200) were tested against standardized secondary antibody

concentration (1/2000). The concentration of primary antibody that gave optimal

resolution was used in further western blot analysis. 2ul of sonicated cell lysate was

added onto nitrocellulose or PVDF membranes, allowed to dry, and the dot blot was

developed as described in the following section for immunoblot membranes.

d. SDS PAGE and Western blot

Cell lysate, prepared as described, was treated with SDS lysis buffer containing beta

mercaptoethanol (5X Laemmli sample buffer) at 95oC for 5mins. In-house prepared

12% SDS gel was used for initial optimisation of protocols. Once protein samples and

test protocol was established, a 4-15% gradient gel (Biorad TGX) was used to resolve

protein. Denatured proteins were electrophoresed along with the colour marker.

Electrophoresed protein was then blotted onto nitrocellulose membrane using trans-blot

turbo (Biorad). Semi-dry technique of transfer using homemade transfer packs was used

108

during optimisation. High background was seen for in-house transfer packs and

commercial transfer packs were used subsequently in attempts to reduce background, so

far without success. Optimisation is ongoing.

In-house transfer packs were prepared using semi-dry consumables as described in the

trans-blot turbo blotting system instruction manual. Transfer using commercial packs

was done as described by manufacturer. Transfer conditions were 30/10 mins at 25V

and 1.0A. Transfer completion was determined by complete transfer of marker.

e. Detection of viral proteins

Following transfer, membranes were dried for a few minutes to allow further protein

binding. Confirmation of transfer was done using Ponceau stain in some cases.

Membrane was then blocked for non-specific binding with 5% non-fat milk with 0.01%

Tween PBS as blocking buffer (BB) for one hour at RT on rocking platform.

Membranes were incubated with primary antibody prepared in BB (at the concentration

as determined by dot blot) overnight at 4oC on a rocking platform. The next day

membranes were washed 4X (10/5/5/5 minutes) with 0.01% tween PBS followed by

incubation with 1:2000 dilution of secondary antibody (Goat-Antihuman-HRP labelled)

for one hour at room temperature. After incubation with secondary antibody,

membranes were further washed 5X with 0.01% Tween-PBS. ECL clarity substrate

(BioRad) and Fujifilm LAS-3000 Imager was used for visualisation. The imager was set

at high precision and exposed for 30sec-1 min. Screen shots before and after exposure

was taken to determine positioning of protein bands.

109

Figure 3. 10: Flow chart of Western blot analysis

110

3.5. Method for chapter 7: - Antibody dependent enhancement

a. Cell lines

The Fc-γ receptor-bearing U937 (ATCC® CRL-1593.2™) cell line was used for

enhancement assay. The cell line was grown in RPMI (Invitrogen) supplemented with

10% fetal bovine serum, 1% Penicillin-Streptomycin (GIBCO, Invitrogen, 5,000 U/mL)

and 1% L-Glutamine (200 GIBCO, Invitrogen mM), designated as R-10. All cell

cultures were maintained in 5% CO2 at 37°C. Vero cells were used for virus titration in

plaque assays to determine enhancement of virus replication and output.

b. Virus stock and plasma samples

ADE of ZIKV PRVABC59 by anti-DENV antibody was determined by quantifying

enhanced infectious virus output from U937 cells due to the presence of antisera

obtained following DENV monotypic infection. Samples were selected to (1) assess

ADE by anti-DENV antibody at different stages of DENV infection and (2) to assess

sequential DENV and ZIKV circulation as occurred in French Polynesia in 2013.

ADE at longitudinal time points after infection was determined using a pool of

monotypic anti-DENV plasma collected within two months post infection and a pool of

plasma collected 1-6 years post-infection. Similarly, DENV-1 and DENV-3 antisera

samples collected 6 months and 1 year post-infection were selected to assess the French

Polynesia sequential DENV-1, DENV-3 and ZIKV outbreak model (327). Negative

control plasma was prepared individuals confirmed as seronegative by neutralisation

test, and the flavivirus-specific monoclonal antibody 4G2 was used as positive control.

111

c. Establishment of test

Plasma samples were serially diluted from 101 to 10

6. An equal volume of diluted

plasma mixed with ZIKV at MOI 1 was incubated for 1 hr at 37oC. The virus/plasma

mixture was then added to 2X104 U937 cells/100ul in 10% FBS RPMI in U bottom 96

well plates. Cells were infected for 18-24 hrs at 37oC. After infection cells were washed

5X with 1XPBS by centrifugation at 125g for 5 mins. Infected cells were further

incubated in the presence of lowest antibody dilution for 3 days in 10% FBS RPMI.

d. Detection of infectious units and determination of enhancement of infection.

Virus output was quantified by plaque assay. After 3 days supernatant from infected

U937 cells was harvested and inoculated onto a Vero monolayer in 24-well plate at 2-

fold and 10-1

to 10-3

dilution. Plates were incubated for 5 days until plaques were visible

in the positive control. After 5 days plates were stained overnight with 1% methylene

blue in 10% formaldehyde. PFU were counted at each sample dilution. Enhancement by

each plasma sample was tested at least twice and the average PFU/ml yield was

calculated for each dilution. A spline interpolating curve was constructed using

Graphpad prism 7.0 to graphically illustrate viral output at different plasma dilutions

and non-parametric Mann-Whitney t-test was used to determine statistical significance

of viral output in comparison with positive and negative control. Figure 3.11. shows the

flowchart to determine enhancement of ZIKV by anti-DENV antibody and Figure 3.12

shows example of PFU in the presence and absence of anti-DENV at different plasma

dilution.

112

Figure 3. 11:- Measurement of antibody-dependent enhancement of ZIKV

infection by anti-DENV antibody

113

Figure 3. 12:- Viral load in the presence and absence of anti-DENV antibody

An example of quantification of infectious ZIKV PRVABC59 output from U937 cells in the

presence or absence of anti-DENV antibody. Supernatants from infected U937 cell lines, in the

presence of diluted antisera was inoculated on Vero monolayers to quantify virus output. Column A

is a negative control which shows virus output in the presence of varying dilutions of flavivirus-

negative plasma. Similarly, column B and C illustrate virus output in the presence of anti-DENV

antisera from monotypic travellers. Column D shows virus output in the presence of positive control

anti-flavivirus monoclonal antibody 4G2.

114

Chapter 4: Study population and viruses

4.1. Preamble

This chapter describes the study population and viruses used in this project.

Demographic and clinical information is provided. West Australian residents who travel

overseas and infected with mosquito-borne viruses are enrolled according to a study

protocol approved by the Human Research Ethics Committee, UWA. DENV field

viruses used in the study were isolated from the travellers DENV and ZIKV reference

strains and YF and JEV vaccine viruses were included in the analysis.

4.2. The cohort

Study participants were travellers returning with DENV or ZIKV infection to WA from

dengue and Zika endemic/ epidemic countries. DENV infections were monotypic.

Samples were collected from DENV subjects up to 6 years post infection and allowed

analysis of longitudinal neutralising and total antibody responses. Presentation of febrile

illness was taken as starting time of infection and time post infection was calculated

henceforth. This population of well-defined DENV monotypic infections in a non-

endemic population allows careful characterisation of DENV-specific antibody

responses over time. ZIKV-specific antibody responses were assessed in returning

travellers with monotypic infection or travellers who had previously lived in DENV-

endemic areas and who were returning to their residence in Western Australia having

acquired ZIKV overseas. Neutralising and total antibody responses were studied among

35 monotypic dengue infections, 16 ZIKV infections to generate data on specificity and

cross-reactivity against homologous heterologous and autologous viruses and

persistence and variation of these responses over time.

Traveller study volunteers donated 150ml of blood at each visit. Serum and plasma

were separated, aliquoted and stored at -80OC until use. Peripheral blood mononuclear

115

cells were isolated and cryopreserved (Figure 4.1). Information sheet and Medical/travel

history forms used during enrolment of volunteers is included in appendix 4.1- 4.3.

Figure 4. 1:- Traveller cohort establishment and virus isolation.

116

At the time of writing 90 travellers with flavivirus infection returning to WA from

different parts of the world, mostly (73%) from Bali, Indonesia were enrolled, 38 of

whom were tested in this study.

Three sera panels were assessed: A) DENV monotypic sera panel B) ZIKV sera panel

4.2.1. DENV monotypic sera panel

DENV monotypic cohort is comprised of travellers returning to WA who were infected

with DENV overseas. All participants are PCR-confirmed dengue cases. This sera panel

has 35 individuals at different time points post-infection, ranging from five days to six

years. Sera were selected randomly based on availability.

Each study subject was assigned a code: FLV and a serial number; the number of

samples is denoted by number of visits. For Eg: FLV001 V1 denotes sample from

patient with serial number 001 at first visit.

a. DENV-1 sera panel

Total and neutralising antibody from nine monotypic DENV-1 infections was studied.

Samples represent infection acquired 2010-2016 and range from nine days to 72 months

post-infection. Six of nine DENV-1 plasma samples were used to determine neutralising

and total antibody responses and three were tested for total antibody only. Eight of nine

infections were acquired in Bali and one traveller became ill after visiting Thailand.

Longitudinal samples from five individuals were included. Two individuals had history

of yellow fever vaccination. Demographic and laboratory findings are listed in Table 4.

1. Diagnostics test results were generated at PathWest Laboratory Medicine WA.

117

b. DENV-2 sera panel

Ten plasma samples were randomly selected for this sera panel from cohort of DENV-2

participants, ranging from five days to 64 months post-infection. Six of ten samples

were tested for both neutralising and total antibody response, 4 were tested for total

antibody only. Infections were acquired in Bali (7), Thailand (2) and the Philippines (1).

Two had previous history of yellow fever vaccination. Infections were acquired from

2011-2016. Summary of this panel is listed in Table 4. 2

c. DENV-3 sera panel

Ten plasma samples were selected for this sera panel, ranging from 6 to 23 months post-

infection. Six of ten samples were tested for neutralising and total antibody and four

plasma samples were tested for total antibody only. Infections were acquired after

visiting Bali (9) and Mexico, Cuba, Bahamas and USA (Florida) (1) in 2015-2016. No

individual had received yellow fever vaccine. Demographic and laboratory findings are

summarised in Table 4. 3

d. DENV-4 sera panel

Six DENV-4 sera samples were available, ranging from 6 to 72 months post-infection.

All six samples were used to measure neutralising and total antibody. Samples were

obtained in 2010, 2015 and 2016. Infections were acquired after visiting Bali (5) and

Sri Lanka followed by Philippines (1). None individual had received yellow fever

vaccine. Two of the individuals in this sera panel were tested for multiple visits at

different time points after infection. Demographic and laboratory finding of DENV-4

cohort is given in Table 4. 4

118

e. ZIKV sera panel

ZIKV panel included sera from 16 individuals. This panel is divided into confirmed and

probable cases. Eight individuals enrolled in the Traveller study and assigned a code

(FLV-) and other eight individuals tested to diagnose infection collaboration with

PathWest Laboratory Medicine WA assigned study code (ZIKV-)

Five of 16 individuals are PCR-confirmed cases and 11 are probable ZIKV cases based

on serology and clinical symptoms. The three ZIKV confirmed individuals were

enrolled in study. These cases were acquired Bali (2014), El Salvador (2015) and

Mexico (2016). The traveller returning from Bali experienced a severe febrile illness on

their return to WA; despite extensive investigation the cause of their illness was not

identified. More than one year later, in October 2015, their samples were retrieved from

storage at -20OC and tested for ZIKV by PCR (urine) and IgM IFA (serum); positive

results indicated ZIKV was circulating in Bali in December 2014. The traveller to El

Salvador was in the country for more than three months. Onset of symptoms of acute

febrile illness occurred on the day they began their journey back to WA. Traveller to

Mexico felt illness in Canada and was diagnosed there with ZIKV. Probable ZIKV

cases of enrolled in study acquired their infection in Colombia, El Salvador, Tonga and

Mexico. The travellers to El Salvador and Tonga travel there frequently to visit family;

they lived in those countries for many years prior to 2015. Three of eight enrolled

individuals have received yellow fever vaccination.

8 individuals who developed acute febrile illness after returning from India, Southeast

Asia, the Pacific, South America and West Africa, whose sera were collected for

diagnostic testing. To differentiate among flaviviruses the sera tested for ZIKV, DENV,

YF17D and IMOJEV neutralising antibody. ZIKV infection was confirmed by PCR in 2

individuals. Table 4. 5 list the demographics and laboratory finding of ZIKV panel.

119

Sample ID Gender Origin of infection Flavivirus

Vaccination

Diagnostic tests

Months post onset of illness Location Year of

infection

Duration

of Stay

(Days) Flavivirus

HI NS1

Dengue

IgM

DENV

PCR^

FLV-001 M Kuta/Bali 2010 20 No <10 + - + 24 60 72

FLV-011 F N/A 2013 N/A No <10 + - + 0.3 (9days) 34

FLV-017 F Bali 2014 10 No <10 + - + 2 20

FLV-018 F Bali 2014 10 YF, 1978 <10 + - + 2 20 31

FLV-022 F Gili Island/Bali 2015 25 No <10 + - + 14 25

FLV-026 F Bali 2015 15 No <10 + - + 14

FLV-042* M Bali 2015 3 No <10 + - + 16

FLV-076* F Legian;Canghu/Bali 2016 7 YF, 1985 <10 + - + 6

FLV-085* M Ko Samui;

Krabi/Thailand

2015 21 No <10 + - + 18

“*”: - Samples included in HI test

N/A: - information not available

“^”: Serotyping PCR specific for DENV-1

“+”: - Positive

“- “: - Negative

Table 4. 1:- Demographic and laboratory findings of individuals with DENV-1 infection

120

Sample ID Gender Origin of infection Flavi

Vaccination

Diagnostic tests Months post onset of

illness Location Year of visit Duration

of Stay

(Days) Flavi HI NS1 result Dengue

IgM

DENV

PCR^

FLV-004 M Bali 2011/2012 14 No <10 + - + 16 51 64

FLV-005 F Bali 2011 10 No <10 + + + 16 51

FLV-009 F Bali 2012 10 No 40 + + + 17 36

FLV-010 M Thailand 2012 10 No <10 + - + 19 36 48

FLV-012 F Java, Bali 2013 N/A No <10 + - + 0.1 33

FLV-014 F Bali 2013 N/A YF 2006 + - + 7 36

FLV-070* M Bali 2016 5 YF <10 + + + 7

FLV-072* M Bali 2015 10 No <10 + - + 15

FLV-073* M Thailand 2015 10 No <10 + - + 15

FLV-074* M Philippines 2015 10 No <10 + + + 13

“*”:- Samples included only in HI test



Table 4. 2:- Demographic and laboratory findings of individuals with DENV -2 infection

“+”: - Positive


121


Vaccination

Diagnostic tests Months post onset

of illness Location Year of

visit

Duration of Stay

(Days)

Flavi

HI

NS1

result

Dengue

IgM

DFENV

PCR^

FLV-029 M Indonesia 2015 21 No <10 + + + 12

FLV-030 F Bali 2015 8 No <10 + + + 12

FLV-033 F Bali 2015 14 No <10 + + + 14

FLV-035 F Bali 2015 >14 No <10 + + + 8

FLV-037 F Semiyak 2015 6 No <10 + - + 14

FLV-038 M Semiyak 2015 10 No <10 + - + 14 23

FLV-044* F Mexico; Cuba; Bahamas;

USA (Florida)

2015 28 No 160 + + + 8

FLV-045* M Gili Islands/ Bali 2016 11 No <10 + + + 8

FLV-046* M Manado 2015 28 No <10 + - + 18

FLV-047* F Bali 2015 14 No <10 + + + 12





“+”: - Positive


122

Sample

ID

Gender Origin of infection Flavi

Vaccination


illness Location Year of

visit

Duration of Stay

(Days)

Flavi

HI

NS1

result

Dengue

IgM

DENV

PCR^

FLV-002 M Bali 2010 8 No 20 Equivocal + + 24 60 72

FLV-049 F Amed; Ubud; Legian/

Bali

2015/2016 10 No <10 + + + 8 14

FLV-068 M Tuban/ Bali 2016 5 No <10 + + + 6

FLV-080 M Pak Thong Chai/

Thailand

2015 13 No <10 + - + 16

FLV-086 F Sri Lanka; Philippines 2015 10 No <10 + - + 21

FLV-099 M Kuta; Seminyak;

Ahmed

2016 14 No <10 + + + 9


“+”: - Positive


MPI: - Months post infection


123

ZIKV case

classification

Sample

ID Gender

Origin of infection

Flavivirus

Vaccination

Diagnostic tests Samples available at

Location Year of

visit

Duration

of Stay

(Days)

Flavi HI ZIKV

IgM

ZIKV

PCR

Presentation

of illness

Days post onset

of illness

Confirmed

FLV-064 M Indonesia 2014 10 No <10 + + no 605

FLV-087 F El Salvador 2015 105 No 640 -/weakly

+ + yes 12/39/425

FLV-093 M Mexico 2016 60 YF-2015 N/A N/A + no 330

ZIKV 01 M South Pacific 2015 N/A Yes (date N/A) 20-640 + + yes 210

ZIKV 02 F Fiji 2016 N/A N/A 20 + + yes 11

Probable

FLV-032 F Colombia 2015 N/A YF-2015 160 + N/A yes 7/37/90/240/390

FLV-088 M El Salvador 2015 60 Not sure >640-320 - N/A yes 23/425

FLV-089 F El Salvador 2015 60 Not sure 320 equivocal ND yes 23/425

FLV-090 F Tonga End

2015 14 No

<10

+ ND

yes 425

FLV-091 M Mexico 2016 30 YF-2015 160 + ND yes 3/240

ZIKV 03 F Colombia 2016 N/A N/A 1280 - N/A yes 38

ZIKV 04 M Philippines 2016 N/A N/A <10 + N/A yes N/A

ZIKV 06 F Tonga End

2015 N/A N/A

<10

+ N/A

yes N/A

ZIKV 07 F Tonga End

2015 N/A N/A

<10

+ N/A

yes N/A

ZIKV 09 F India 2016 N/A N/A 10 equivocal N/A yes N/A

ZIKV011 M Thailand 2017 N/A N/A <10 + N/A yes N/A

Table 4. 5:- Demographic and Laboratory findings of individuals with ZIKV infection



ND: Not done

Flavi HI : MVE 151 antigen

ZIKV PCR: Urine

“+”: - Positive


124

4.2. Viruses used in this study

A collection of 32 DENV isolates, two ZIKV strains, and two vaccine viruses YF17D

and IMOJEV was used to determine cross-neutralisation and cross-recognition among

flaviviruses. DENV were contemporaneous circulating viruses isolated from travellers,

and reference viruses. ZIKV strains are prototype African and Asian lineages; vaccine

viruses were included to allow assessment cross-reactivity among flaviviruses. Virus

origin and year of isolation is listed in Table 4. 6.

4.2.1 DENV Panel

DENV isolates were selected to represent genetic diversity (serotypes and genotypes),

geographic diversity (lineages) and temporal diversity (year of infection). Different

genotypes of DENV included in study are shown in Figure 4.2.

Comparison of full length E gene deduced amino acid sequences showed 98%

similarity among homologous serotypes and 60% diversity between heterologous

serotypes. Residues 1-394 in E include determinants of host range tropism, and sites

that affect binding of neutralising monoclonal antibodies (33).

125

2289 DENV-1 including 28

from WA travellers

2263 DENV-2 including 46

from WA travellers

1256 DENV-3 including 3 from

WA travellers 640 DENV-4 including 9 from

WA travellers

Figure 4. 2:- Phylogenetic tree of DENV E-sequences including isolates from travellers (11).

Virus sampled from travellers from WA is coloured in red and red boxed area represents the genotypes used in this study

126

Flavivirus

type Types

Virus definition

(Year/Location/genotype/lineage)

Code used in

study

Used for tests Genbank

accession

number FRNT HI

DENV

DENV1

2001/Hawaii/ Genotype-4 D1-HW2001G4 ▄ ● DQ672563

2010/Bali/Genotype-1/Lineage 14 D1-10BG1L14 ▄ ●

2015/Laos/Genotype-1 D1-15LG1 ▄ ●

2010/Bali/Genotype-2/Lineage 4 D1-10BG2L4 ▄ ●

2010/India/Genotype-3/Lineage 4 D1-10INDG3L4 ▄ ●

2014/Philippines/Genotype-4 D1-14PHLG4 ▄ ●

**2010/Bali/Genotype-2/Lineage 2(FLV001) D1-10BG2L2 ▄ ●

** 2014/Bali/Genotype-1/Lineage 14

(FLV018)

D1-14BG1L14 ▄ ●

**2015/Bali/ Genotype-1/Lineage H

(FLV022)

D1-15BG1LH ▄ ●

DENV2

1944/New Guinea C/ Asian II D2-NGCAII ▄ ● M29095

2010/Laos/Asian1/Lineage 9 D2-10LGA1L9 ▄ ● KM216697

2011/Bali/Cosmopolitan/Lineage 4 D2-11BGCL4 ▄ ●

*2011/Bali/Cosmopolitan/Lineage 4 D2-11BGCL4 ● KM216709

2013/Malaysia/Cosmopolitan/Lineage N D2-13MGCLN ▄ ●

2015/Bali/Cosmopolitan/Lineage 4 D2-15BGCL4 ▄ ●

2015/Bali/Cosmopolitan/Lineage N D2-15BGCLN ▄ ●

*2015/Timor

Leste/Indonesia/Cosmopolitan/Lineage G

D2-

15TLS/IDNGCLG ●

1994/ Puerto Rico/ (non-virulent strain) D2-PR1940-NV ▄ ● GQ398308

1994 /Puerto Rico/ (virulent strain) D2-PR 6913-V ▄ ● GQ398279

**2012/ Bali/Cosmopolitan/Lineage 4

(FLV004) D2-12BGCL4 ▄

**2013/ Bali/Cosmopolitan/Lineage 4

(FLV014) D2-13BGCL4 ▄

DENV3

1956/Philippines/Genotype 5 D3-H87G5 ▄ ● M93130

**2015/Bali/Genotype1 (FLV037) D3-15BG1 ▄ ●

**2015/Bali/Genotype1 (FLV038) D3-15BG1 ●

1983/Sri Lanka/(Virulent) D3-UNC3002-V ▄ ● AF547226

1989/Sri Lanka (non-virulent) D3-UNC3008-NV ▄ ● AF547232

DENV4

1956/Philippines/Genotype1 D4-H241GI ▄ ● AY947539

2010/Bali/Genotype:3/Lineage:1 D4-10BG3L1 ▄

2010/Bali/Genotype:3/Lineage:2 D4-10BG3L2 ▄ ●

*2014/Bali/Genotype:3/Lineage:2 D4-14BG3L2 ●

ZIKV ZIKV

1947/African/ Prototype ZIKV MR766 ▄ ● LC002520

2015/Puerto Rico/ Asian strain ZIKV

PRVABC59 ▄ ● KU501215

Flavi ref

strains

Yellow fever Yellow fever 17D YF-Vax YF 17D ▄ X03700

Japanese

encephalitis IMOJEV IMOJEV ▄

Kunjin Virus HI reference KUNV ●

Table 4. 6:- Flaviviruses used in study

“*”:- Tested only for total anti-DENV

antibody content

“**”:- Autologous viruses

127

a. DENV-1

Nine DENV-1 strains were used in this study. These strains represent four different

genotypes. DENV-1HW2001 used as a reference strain while all other isolates were

derived from febrile WA travellers in 2010, 2014 and 2015. Two strains represent

DENV-1 (Genotype 1) isolated in 2010 and 2015 from patients infected in Bali and

Laos respectively. Both viruses induced CPE in Vero cells although not at the same

rate: DENV-1-2015-Laos induced CPE more rapidly than DENV-1-2010-Bali. DENV-

1 (Genotype 2) acquired in Bali, 2010, was isolated from one individual. Two strains

representing DENV-1 (Genotypes 3) and DENV-1 (Genotype 4) originated in India and

Philippines in 2010 and 2014 respectively. Both viruses showed similar pattern in

inducing CPE. Three autologous viruses were isolated from individuals enrolled in the

Traveller study at the time of their acute illness; antisera were obtained from these

individuals (FLV-001; FLV-018, FLV-022) at 2 or more visits and allowed detailed

analysis of long-term immunity. Genetic diversity of DENV-1 genotypes included in

this study is shown in Figure 4.2. Comparative alignment of E-gene neutralising domain

(1-394) is shown in Figure 4.3. E gene diversity is summarised in Table 4.7.

128

Figure 4. 3:- DENV-1 E- gene (1-394) alignment

Alignment of E-gene functional domain (1-394) for DENV-1 including reference strains and patient isolates. Each virus name represents serotype year of isolation, origin,

genotype and lineage. ** :- are autologous viruses. GenBank accession: DQ672563 is strain D1-HW2001G4.

129

Amino acid position

D1-HW2001G4

D1-10BG1L14

D1-15LG1

D1-10BG2L2

D1-10INDG3L4

D1-14PHLG4

**D1-14BG1L14

**D1-15BG1LH

**D1-10BG2L4

8 N S S . . . S S .

37 D . . . N . . . .

88 T A A . A . A A .

96 F . . . . L . . .

114 I . . . L . . . .

139 V . . . . I . . .

145 N T T T T T T T T

155 T S S . . . S S .

161 I T T T . T T T .

171 S T T . . . T T .

227 S . . . . . . T .

297 I L M V V V M M T

305 S . . . . . . . P

311 E . . . D . . . .

324 V I I . . . I I .

337 F . . . I . . . .

339 S T T . T . T T .

345 V . . . . . . L .

352 V I I I I I I I .

359 T . . . . I . . .

362 E . . . . . . G .

369 A . . . T . . . .

378 I . . L . L . . .

379 V . . . . . M . .

380 V I I . I . I I .

388 K R . . . . . . .

436 V . . I . I . . .

439 I . . . V . . . .

461 I V V . V . V A .

480 I V . . . . . L .

484 M L L . . . L L .

**:- Autologous virus

Table 4. 7:- DENV-1 amino acid variation

130

b. DENV-2

This panel consists of ten strains representing two genotypes of DENV-2: Asian

1 and Cosmopolitan. DENV-2-New Guinea C was included as reference strain.

WA traveller isolates from 2010, 2011, 2013 and 2015 were used to test

antibody mediated immune response against these strains. DENV-2 (Asian 1

genotype) 2010 was acquired in Laos. DENV-2 (Cosmopolitan genotype)

strains were acquired in Bali and Malaysia 2011 and 2015. Lineage 4,

Cosmopolitan genotype is a newly identified strain that emerged in Bali,

Indonesia during a DENV-2 outbreak and was identified in WA travellers (11)

and has continued to be imported. DENV-2 isolated in Puerto Rico, PR1940 and

PR6913, represent virulent and non-virulent epidemic activity, respectively.

Non-virulent strain PR1940 induced CPE more slowly than all other DENV-2 in

this analysis. Traveller DENV-2 induced CPE at similar rates. Genetic diversity

of DENV-2 genotypes included in this study is shown in Figure 4.2. Comparison

of full length E gene sequences of viruses used this analysis is shown in Figure

4. 8. Comparative alignment of E-gene neutralising domain (1-394) is shown in

Figure 4.4. E gene diversity is summarised in Table 4.8.

131

Figure 4. 4:- DENV-2 E-gene (1-394) alignment

Functional E-domain (1-394) in all the DENV-2 isolates and reference strains. Each virus name represents serotype year of isolation, origin, genotype and

lineage. ** :- represents autologous viruses. GenBank accession: M29095.1 was used as reference sequence (D2-NGCAII)

132

Amin

o acid

Positi

on

DENV-2 sequences used in study

D2-

NGCGAII

D2-

10GAIL9

D2-

11BGCL4

D2-

11BGCL4-2

D2-

13MGCLN

**D2-

12BGCL4

**D2-

13BGCL4

D2-

15BGCL4

D2-

15BGCLN

15-

TLS/IND/GC

PR1940-

NV

PR6913

-V

47 E K K K K K K K K K K K

52 Q . H H H H H H H H . .

71 D E A A A A A A A A E E

83 N K . . . . . . . S . .

91 V . . . . . . . . . . I

126 K E E E E E E E E E E E

129 V I I I I I I I I . . .

131 Q . . . . . . . . . L L

141 I V . . . . . . . . . .

149 H . N N N N N N N N . .

164 I V V V V V V V V V . .

203 N . . . . . . . . D D D

226 T K . . . . . . . . . .

228 G E . . . . . . . . . .

308 V . . . . . . . . . I I

346 H Y . . . . . . . . . .

359 T . . . . . . . . . A .

379 I . . . . . . . . V . .

390 N . S S S S S S S S . .

402 I F F F F F F F F F F F

454 I T T T T T T T T T T T

462 I . V V V V V V V V . .

484 V I . . . . . . . . . .

491 V . . . . . . . . . A .


**:- Autologous virus

133

c. DENV-3

DENV-3 virus panel includes three traveller isolates obtained 2010-2015, and 3

reference strains. All DENV-3 isolated from WA travellers to date belong to Genotype-

1. The reference strain DENV3 H87 and virulent and non-virulent strains (UNC-3002

and UNC-3008) which belong Genotype 3 were also included in the analysis. Details of

virus and GenBank accession number wherever available is given in Table 4. 6.

Genetic position of DENV-3 genotype included in this study is shown in Figure 4.2.

Phylogenetic position of DENV-3 isolates shown in Figure 4. 8. Comparative alignment

of E-gene neutralising domain (1-394) is shown in Figure 4.5. E gene diversity is

summarised in Table 4.9.

134


Comparison of functional E-domain (1-394) in DENV-3 traveller isolates and reference strains. Each virus name represents serotype year of isolation, origin, genotype and

lineage. **:- represents autologous viruses used in the study. Reference strain D3-H87: GenBank Accession No. M93130.

135


Amino acid position

DENV-3 strains used in study

D3-H87G5

**D3-15BG1

**D3-15BG1

UNC3002-V

UNC3008-NV

6 V . . I .

68 I V V . .

81 I . . V V

124 S . L P P

132 H . . Y Y

140 I . S . .

164 S P P P P

169 A V V T T

225 K E E E E

231 R K K . .

270 T . . N N

291 K E E E E

301 L S S T T

303 T A A . .

320 I . . V .

340 G . E . .

344 A . . . .

345 H Y . . .

377 V I I . .

383 K . . N N

391 R K K K K

452 I . . V V

459 V . . . .

475 F . L . .

479 A V V . .

489 V A A A A

136

d. DENV-4

Three isolates derived from travellers infected in 2010 or 2014, and one reference strain

were included. Traveller viruses belonged to Genotype 3, with 2 distinct lineages (L1

and L2). Reference strain H241 belongs to Genotype 1. Details of virus and GenBank

accession number wherever available is given in Table 4. 6. Genetic relations of DENV-

4 strains included in this study are shown in Figure 4.2. Phylogenetic distribution is

shown in Figure 4. 8. Comparative alignment of E-gene neutralising domain (1-394) is

shown in Figure 4.6. E gene diversity is summarised in Table 4.10.

137


Alignment of E-gene functional domain (1-394) DENV-4 patient isolates and reference strains. Each virus name represents serotype year of isolation,

origin, genotype and lineage. An incomplete E-gene sequence for DENV-4 isolate (genotype 2 lineage 2) was not included in this alignment.

Reference sequence H241 GenBank accession: AY947539.1

138


Amino acid position

DENV-4 strains used in study

D4-H241G1

D4-10BG3L1

D4-10BG3L2

D4-14BG3L2

46 I T T T

120 S L L L

132 I . . V

155 I T T T

156 P S S S

157 N . . S

221 A T T T

233 Y N . .

265 T A A A

360 Y N N N

363 S G . .

385 S G . .

402 L F F F

429 L F F F

455 V I I I

461 F . . L

463 V . A .

494 H Q Q Q

139

e. Autologous DENV:

DENV was isolated from 7 individuals who were enrolled in the Traveller study at the

time of their acute illness. Serum samples collected at subsequent visits were assessed

for cross-reactivity and antigenicity against the same panel of homologous and

heterologous DENV serotypes, ZIKV, vaccine strains YF17D and IMOJEV, as well as

autologous DENV. Three DENV-1, two DENV-2 and two DENV-3 autologous isolates

from 2010,2012,2013,2014 and 2015 were available for study. Details of autologous

viruses are summarised in Table 4. 6.

4.2.2. ZIKV Panel

Two strains of ZIKV were included in the analysis: prototype African strain ZIKV

MR766 isolated from sentinel RHESUS monkey April 20, 1947 from Zika forest,

Entebbe, Uganda; and Asian strain ZIKV PRVABC59 isolated from a human in Puerto

Rico, 2015. NS5 sequencing and GenBank BLAST analysis confirmed that ZIKV

MR766 showed 100% homology with Uganda LC002520 and ZIKV PRVABC59 was

100% homologous with Puerto Rico KU501215. E gene diversity is summarised in

Table 4. 11. Comparative alignment of E-gene neutralising domain (1-394) is shown in

Figure 4.7.

140

Figure 4. 7:- ZIKV E-gene alignment

NS5 sequencing of isolates used in study showed 100% match with Uganda LC002520 (ZIKV MR766) and Puerto Rico KU501215(ZIKVPRVABC59), used to map E-gene

141

Amino acid

position

ZIKV strains

ZIKV MR766 ZIKV PRVABC59

120 T A

152 T I

156 I T

158 Y H

169 V I

283 K R

285 F S

317 V I

341 I V

343 V A

393 D E

437 V A

438 F L

473 V M

487 T M

495 M L

Table 4. 11:- ZIKV amino acid variation

142

4.2.3. Flavivirus vaccine strains

a. Japanese encephalitis vaccine

JEV vaccine strain was isolated from live attenuated JE vaccine “IMOJEV” (Sanofi-

Aventis Australia Pty Ltd) by inoculating Vero cells with vaccine preparation, available

lyophilised. IMOJEV is a monovalent chimeric that includes JEV prM and E coding

sequence of SA14-14-2 in a backbone of YF17D-204. JEV isolation was confirmed by

real time RT-PCR. Passage 2 of the isolate was used to determine neutralising antibody

responses to DENV- and ZIKV-infected travellers and vaccine recipients.

b. Yellow fever vaccine strain

YFV17D vaccine virus was isolated from YF-VAX, originally prepared from strain

17D-204 in living avian leucosis virus free (ALV-free) chicken embryos. The vaccine

was obtained lyophilised in sorbitol and gelatin stabilizers and was reconstituted and

inoculated onto Vero monolayer cell culture. CPE was noted every day and virus was

harvested at 5 days for first passage. In the second passage virus growth was rapid and

100 % CPE was obtained within 3 days of inoculation. YF17D isolation was confirmed

with Real time PCR. Passage 2 and Passage 3 were used for further assays.

143

Figure 4. 8:- Phylogenetic tree showing the genetic relationship among the flaviviruses strains used in this

study.

Tree was constructed using neighbour joining method with 100 bootstrap replications Different colour

represents different virus panels used in the study. Reference and vaccine strain sequences were downloaded

from NCBI GenBank. GenBank Accession numbers are listed in Table 4.1.

144

Chapter 5:- DENV-specific antibody responses in monotypic infection

5.1. Preamble

Longitudinal data on anti-DENV-specific antibody responses from the time of acute

infection to 72 months post-infection is described. Viruses and antisera were obtained

from febrile travellers returning to Western Australia and who were diagnosed with

well-characterized monotypic dengue infection upon their return. Phylogenetic analysis

of traveller DENV, originating in the Asia Pacific region (most often in Bali, Indonesia)

allowed identification of serotype, genotype and lineage; viruses represent genetic

diversity (genotypes), geographic diversity (lineages) and temporal diversity (year of

infection) within serotype. Autologous, homologous and heterologous antisera were

analysed for neutralizing and total antibody responses over time. Cross-neutralisation of

ZIKV, YF17D and IMOJEV was also assessed. Antigenic relationships among

monotypic antisera and contemporaneous circulating DENV were visualised and

quantified using antigenic cartography.

5.2. Introduction

Dengue is an acute viral infection and immunity against the infecting serotype is

thought to be lifelong (328-330). DENV-specific antibody-mediated responses post

infection include neutralising antibodies, assumed to be protective, (331) and enhancing

antibodies, assumed to be pathogenic (38, 332, 333). The high degree of genetic

homology among the four DENV serotypes means that understanding the nature of

antibody-mediated immunity is not straightforward. Although good animal models that

mirror human anti-DENV responses are lacking, IFN-knockout mice are currently one

of the best models available. Analysis in humans is complicated by the high degree of

cross-recognition among serotypes. This is particularly true for study populations in

145

dengue endemic areas, where multitypic anti-DENV responses are common once

children reach adolescence.

DENV-specific neutralising antibody (NAb) is a marker of infection and vaccine-

mediated protection. NAb epitopes are situated on the dengue envelope protein (E) on

the virion surface. Anti-E NAb block the interaction between host and virus by binding

to epitopes that facilitate virus entry into target cells (231). Flavivirus envelope proteins

consist of three non-overlapping antigenic domains DI, DII and DIII, each domain has

multiple epitopes. DIII is believed to be the specific NAb -binding epitope while cross-

reactive antibodies are also reported to bind to DII (236, 334). Conserved residues and E

protein hydrophilicity facilitates shared E epitopes and cross-neutralisation of closely

related flaviviruses. Anti-DENV neutralisation capacity in vitro is correlated with

development of E-specific humoral immune responses in vivo (335). Variation in viral

antigenic properties is due to amino acid variation that alters antibody binding site, thus

affecting binding of antibody to viral epitopes (325). Minor changes in AA orientation

can cause noticeable immunological variation, however major AA changes may not

result in immunological change (203). Seven AAs differences in E domain I and II was

proposed to result in displacement of the dominant circulating DENV-2 lineage in

Brazil, in 2007/2008 (336). Single AA residue change in DENV-1 E-gene at virus

breathing site was reported to alter neutralisation susceptibility (337). In secondary

dengue infection antibody responses directed against infecting serotype may be

predominantly derived from repertoires induced during previous flavivirus infection.

This phenomenon is termed original antigenic sin (244) and is a consequence of the

high degree of homology among flaviviruses (246).

The Study. This study, in a cohort of travellers with well-defined monotypic DENV

infection, aimed to characterize persistence of autologous, homologous and

146

heterologous DENV-specific neutralizing and total antibody responses. Antigenic

relationships among 35 monotypic antisera and 32 contemporaneous circulating DENV

were visualised and quantified using antigenic cartography.

Antigenic cartography was utilized to define antibody and antigenic diversity over time.

Antigenic cartography is derived from geographic cartography to determine relative

distance between places and derives the distance between antigenically variable

pathogens and host response based on antibody binding assay. This was first

implemented to describe emergence trends in Influenza (323). Antigens and anti-sera

are projected on a grid where corresponding distances are derived from antibody titre

obtained by total antibody (hemagglutination inhibition) or neutralising antibody. This

antigenic mapping creates a unified framework to analyse results obtained from

antibody-dependent tests, despite random error due to assay conditions and variability

among patients’ immune history (338, 339). This allows quantitative interpretation and

easy visualisation of antibody response data (340).

5.3. Aims

To characterize persistence of autologous, homologous and heterologous

DENV-specific neutralizing and total antibody responses

To determine antigenic relationships among monotypic antisera and

contemporaneous circulating DENV and to visualise and quantify antigenicity

using antigenic cartography

5.4. Results

5.4.1. Neutralising antibody

Serum neutralisation data are reported here in sections according to the infecting virus,

hereafter referred as sera panel. Each sera panel details neutralisation against (i)

infecting DENV serotype i.e. homologous serotype, (ii) other DENV serotype i.e.

147

heterologous serotype and (iii) DENV autologous virus i.e. virus isolated from patient’s

own acute sera (iv) other flavivirus serogroups i.e. ZIKV, YF17D and IMOJEV.

Data for each sera panel is presented in order of increasing months past infection.

Columns are colour coded for the same patient at different time points. Mean

neutralisation reduction for all individuals is shown in Appendix 5.1. Increase in

stringency increases serotype-specificity; however this also leads to missing results

from individual with low magnitude immune responses. Only 50% reduction did not

exclude homologous serotype NAb responses, across all serotypes (DENV-1 – DENV-

4, as shown in Tables 5.1-5.4; 5.5-5.8; 5.9-5.12; 5.13-5.16) and thus 50% reduction was

used throughout.

Neutralisation data was statistically analysed considering each FRNT 50 measurement

as a function of the person, the panel, the virus, and time since infection. Because the

measurements are restricted at both the lower and upper level, tobit regression (341)was

chosen for initial analyses. This method was derived for use with these kinds of data

where measurements cannot be taken below or above certain values. The method

assumes that there is some latent (or unobservable) variable that is being measured

imperfectly, such that below one level or above another, only the values of the levels are

known, but that between those levels the observed value equals the value of the latent

variable. The regression method then estimates the effect of various predictors (such as

time, strain similarity and their interactions) on this latent variable. In all analyses the

response variable in the regression was the natural log of the FRNT 50, with the

censoring cut-offs set to 2.99 and 5.77 as data points were between 20-320. An

alternative fairly robust method that can be (and is often) used with data restricted in

this way, is to simply replace those below the lower limit with half that value and those

above the upper limit with double that value (342).This method was also used and the

148

results compared with those obtained using tobit regression (343).Estimate of variance

on the same person at different times after infection was done with robust variance

estimation of confidence intervals for the regression coefficients. The regression

coefficients together with their robust standard errors then provide measures of the

differences in log(FRNT50) (and their statistical significance) according to: panels,

whether serotypes are homologous or heterologous, time since infection (both linear and

non-linear), whether a reference strain or not, and the individual viruses.

a. Specificity and cross-reactivity post DENV-1 infection

DENV-1 antisera showed differential neutralisation among 6 homologous and 15

heterologous strains over time. Neutralisation against homologous serotype was ~85 %

greater than against heterologous serotype. Figure 5. 1 shows an example of acute and

convalescent anti-DENV-1 NAb in a DENV-1-infected individual. Patient FLV-011

acute phase antisera neutralized all homologous DENV-1 and heterologous DENV

strains at high magnitude, whereas convalescent sera, 2 years after infection,

differentially neutralized homologous and heterologous DENV. Of note, one

homologous virus, strain D1-10INDGL4, was not neutralized by convalescent anti-

DENV-1 antiserum, whereas two heterologous DENV strains, both serotype 3, were

neutralized.

The DENV-1 sera panel consists of 6 individuals with monotypic DENV-1 infection,

sampled less than 1 week to 6 years post infection. Sera from 3 individuals were

collected within 2 months of infection and after 1 year of infection; from 2 individuals

after 1 and 2 years of infection; and from 1 individual 2, 5, and 6 years after infection

(Table 4.1 also shown below). Neutralisation titres at 50%, 75% and 90% reduction

were compared (Table 5. 1- Table 5. 3). Example of mean reduction of neutralisation is

shown in Figure 5.1.

149

Statistical verification for each FRNT-50 measurement was done using tobit regression

analysis and because there were repeated measures on the same person at different times

after infection, robust variance estimation was used to estimate confidence intervals for

regression coefficients. The regression coefficient together with robust standard errors

measures the difference in log (FRNT50) and statistical significant according to

serotypes, time post infection and individual viruses.

150

Sample ID Gender Origin of infection Flavivirus

Vaccination

Diagnostic tests

Months post onset of illness Location Year of

infection

Duration

of Stay

(Days) Flavivirus

HI NS1

Dengue

IgM

DENV

PCR^

FLV-001 M Kuta/Bali 2010 20 No <10 + - + 24 60 72

FLV-011 F N/A 2013 N/A No <10 + - + 0.3 (9days) 34

FLV-017 F Bali 2014 10 No <10 + - + 2 20

FLV-018 F Bali 2014 10 YF, 1978 <10 + - + 2 20 31

FLV-022 F Gili Island/Bali 2015 25 No <10 + - + 14 25

FLV-026 F Bali 2015 15 No <10 + - + 14

FLV-042* M Bali 2015 3 No <10 + - + 16

FLV-076* F Legian;Canghu/Bali 2016 7 YF, 1985 <10 + - + 6

FLV-085* M Ko Samui;

Krabi/Thailand

2015 21 No <10 + - + 18

“*”: - Samples included in HI test



“+”: - Positive


Table 4. 1:- Demographic and laboratory findings of individuals with DENV-1 infection

151

Figure 5. 1:- Neutralising antibody response post DENV-1 infection, patient FLV-011

Specificity and cross-reactivity of antibody response post DENV-1 infection.

152

Note:

Value 1=<20

Value 320=>320

Table 5. 1:- Anti-DENV-1 neutralising antibody titre at 50% reduction

Note:

Value 1=<20

Value 320=>320

*MPI: Months Past Infection

*Acute: <=2 months past infection

*Convalescent: > 2 months past infection

FLV011/ 9

days

FLV018/

2

FLV017/

2

FLV022/

14

FLV026/

14

FLV018 /

20

FLV017/

20

FLV001 /

24

FLV022/

25

FLV018/

31

FLV011/

34

FLV001/

60

FLV001 /

72

D1-HW2001G4 320 320 242 320 204 263 54 320 320 278 81 320 248 227

D1-10BG1L14 320 320 320 320 320 320 193 320 320 185 320 320 320 295

D1-15LG1 320 320 65 320 223 246 52 320 108 314 256 320 320 212

D1-10BG2L4 320 135 129 320 154 122 244 320 320 89 320 320 320 218

D1-10INDG3L4 320 320 189 320 35 136 145 320 320 82 67 320 242 178

D1-14PHLG4 320 320 320 320 198 320 98 320 320 276 247 320 320 273

GMT-D1 320 277 184 320 158 219 111 320 267 179 181 320 293 230

D2-NGCGAII 320 320 240 61 1 94 39 79 161 23 1 252 70 53

D2-10LGA1L9 320 122 79 189 41 77 1 56 87 81 34 229 294 73

D2-11BGCL4 108 1 1 22 22 27 1 1 46 1 24 22 45 9

D2-13MGCLN 187 1 23 41 1 46 1 1 51 46 20 24 40 13

D2-15BGCL4 320 209 193 20 1 1 39 1 1 1 1 27 28 10

D2-15BGCLN 186 1 1 50 1 37 1 1 51 51 1 70 44 9

D2-PR6913-V 171 44 44 22 1 1 21 1 40 1 1 106 60 11

D2-PR1940-NV 320 320 122 50 1 22 21 106 139 49 47 130 89 59

GMT-D2 226 24 29 43 2 17 5 5 42 11 5 73 63 20

D3-H87G5 320 37 73 37 1 26 21 47 114 1 1 52 26 21

D3-15BG1 320 42 77 320 1 42 60 116 130 55 106 320 320 82

D3-UNC3002-V 123 21 1 41 23 35 1 70 42 1 1 40 35 13

D3-UNC3008-NV 304 66 184 282 57 134 1 40 75 30 135 54 36 62

GMT-D3 249 38 32 108 6 48 6 63 83 6 11 78 57 34

D4-H241G1 320 245 246 137 57 82 21 320 93 47 46 137 111 107

D4-10BG3L1 320 58 1 80 25 45 1 113 1 1 1 77 37 14

D4-10BG3L2 320 85 82 93 22 33 36 102 67 1 31 201 114 53

GMT-D4 320 107 27 100 31 49 9 155 18 4 11 128 78 43

ZIKV MR766 320 320 88 43 28 199 1 71 21 152 1 58 58 43

ZIKV PRVABC59 1 1 1 1 1 1 1 1 1 1 1 1 1 1

YF 17D 64 320 28 1 1 269 1 1 1 217 1 1 1 6

IMOJEV 223 1 1 1 1 1 1 1 1 1 1 1 1 2

GMT 46 18 7 3 2 15 1 3 2 13 1 3 3 5

Other

Flaviviruses

GMT-flavi

acutes: 18

(<20-320+)

GMT-flavi

convalescent: 3

(<20-320+)

GMT-flavi:5 (<20-320+)

Heterologous

serotype

GMT-

heterologous

serotype= 31

(<20-320+)

GMT-D2 strains

convalescent: 15

(<20-294)

GMT-D2 all

strains:28

(<20-320+)

GMT-D3

strains

acutes: 67

(<20-320+)

GMT-D3 strains

convalescent: 28

(<20-320+)

GMT-D4

strains

acutes: 97

(<20-320+)

GMT-D4 strains

convalescent: 33

(<20-320+)

GMT-D3 all

strains:34

(<20-320+)

GMT-D4 all

strains:43

(<20-320+)

GMT-D2

strains

acutes: 54

(<20-320+)

Study No/MPI

Virus<1 year

Homologous

serotype

GMT-D1

strains

acutes: 254

(65-320+)

1- 2 year 2- 6 yearsGMT

GMT-D1 strains

convalescent:224

(35-320+)

GMT-homologous

serotype=230 (35-320+)

Geometric Mean Titer (GMT)

153





Note:

Value 1=<20

Value 320=>320

FLV011/

9 days

FLV018

/ 2

FLV017/

2

FLV022/

14

FLV026/

14

FLV018 /

20

FLV017

/ 20

FLV001/

24

FLV022/

25

FLV018/3

1

FLV011/

34

FLV001/

60

FLV001 /

72

D1-HW2001G4 306 312 121 320 75 112 28 320 320 74 38 240 98 137

D1-10BG1L14 320 320 320 320 320 320 76 198 288 98 232 150 320 230

D1-15LG1 214 103 35 320 87 130 26 245 46 161 128 320 320 125

D1-10BG2L4 320 44 43 206 42 54 82 145 211 37 112 320 320 109

D1-10INDG3L4 320 320 37 320 25 77 55 320 320 158 39 320 120 130

D1-14PHLG4 320 218 86 320 116 320 45 320 187 64 91 320 320 172

GMT-D1 297 178 76 297 79 135 47 248 194 87 88 269 223 146

D2-NGCGAII 320 274 124 28 1 1 1 42 28 1 1 143 37 15

D2-10LGA1L9 320 57 34 74 1 28 1 29 74 29 1 54 82 22

D2-11BGCL4 22 1 1 1 1 1 1 1 1 1 1 1 1 1

D2-13MGCLN 68 1 1 1 1 1 1 1 1 1 1 1 1 1

D2-15BGCL4 320 53 57 1 1 1 1 1 1 1 1 1 1 3

D2-15BGCLN 63 1 1 24 1 1 1 1 24 1 1 1 24 3

D2-PR6913-V 56 1 1 1 1 1 1 1 1 1 1 30 28 2

D2-PR1940-NV 320 72 45 23 1 1 1 47 23 1 1 46 37 11

GMT-D2 124 9 8 6 1 2 1 4 6 2 1 8 10 4

D3-H87G5 139 1 32 1 1 1 1 24 47 1 1 22 1 4

D3-15BG1 320 24 29 54 1 25 31 56 50 1 54 320 320 35

D3-UNC3002-V 37 1 1 1 1 1 1 1 1 1 1 1 1 1

D3-UNC3008-NV 115 1 1 66 1 25 1 1 37 1 1 25 21 5

GMT-D3 117 2 6 8 1 5 2 6 17 1 3 20 9 6

D4-H241G1 320 119 114 58 1 34 1 137 44 24 13 62 47 33

D4-10BG3L1 161 1 1 1 1 23 1 1 1 1 1 1 1 2

D4-10BG3L2 320 26 21 35 1 1 1 34 32 1 1 33 1 7

GMT-D4 255 15 13 13 1 9 1 17 11 3 2 13 4 8

ZIKV MR766 288 157 1 1 1 85 1 32 1 47 1 45 29 10

ZIKV PRVABC59 1 1 1 1 1 1 1 1 1 1 1 1 1 1

YF 17D 21 285 1 1 1 76 1 1 1 80 1 1 1 4

IMOJEV 35 1 1 1 1 1 1 1 1 1 1 1 1 1

GMT 22 15 1 1 1 9 1 2 1 8 1 3 2 3

Study No/MPI

Heterologous

serotype

Homologous

serotype

GMT-D4 all

strains:8

(<20-320+)

Other

Flaviviruses

<1 year 1- 2 year

GMT-flavi

acutes: 7 (<20-

288)

GMT-flavi

convalescent: 2

(<20-45)

GMT-flavi:2 (<20-288)


GMT-D1

strains

acutes:159

(37-320+)

GMT-D1 strains

convalescent:

142(25-320+)

GMT-homologous

serotype=146 (25-320+)

2-6 yearsVirus

GMT

GMT-D2

strains

acutes: 21

(<20-320+)

GMT-D2 strains

convalescent: 3

(<20-143)

GMT-D2 all

strains:5

(<20-320+)

GMT-

heterologous

serotype= 6

(<20-320+)GMT-D3

strains

acutes: 11

(<20-320+)

GMT-D3 strains

convalescent: 5

(<20-320+)

GMT-D3 all

strains:6

(<20-320+)

GMT-D4

strains

acutes: 37

(<20-320+)

GMT-D4 strains

convalescent: 5

(<20-137)

154




Note:

Value 1=<20

Value 320=>320

Table 5. 3:-Anti-DENV-1 neutralising antibody titre at 90%

FLV011/

9 days

FLV018/

2

FLV017

/ 2

FLV022/

14

FLV026/

14

FLV018 /

20FLV017

/ 20

FLV001 /

24

FLV022/

25

FLV018/

31

FLV011/

34

FLV001/

60

FLV001 /

72

D1-HW2001G4 147 159 60 171 41 53 19 313 194 37 26 134 51 78

D1-10BG1L14 320 320 80 320 320 320 39 87 141 52 98 70 320 147

D1-15LG1 64 47 25 320 44 70 20 120 26 82 69 152 161 68

D1-10BG2L4 320 27 23 89 1 33 22 68 112 18 45 237 166 45

D1-10INDG3L4 320 140 21 169 21 47 32 187 266 88 29 190 66 82

D1-14PHLG4 167 106 39 320 68 320 29 172 63 40 46 173 256 102

GMT-D1 193 99 36 210 31 91 26 139 105 46 47 149 139 81

D2-NGCGAII 320 130 66 1 1 1 1 29 1 1 77 25 9

D2-10LGA1L9 244 29 1 35 1 1 1 20 35 1 1 27 38 7

D2-11BGCL4 1 1 1 1 1 1 1 1 1 1 1 1 1 1

D2-13MGCLN 38 1 1 1 1 1 1 1 1 1 1 1 1 1

D2-15BGCL4 166 1 1 1 1 1 1 1 1 1 1 1 1 1

D2-15BGCLN 31 1 1 1 1 1 1 1 1 1 1 1 1 1

D2-PR6913-V 30 1 1 1 1 1 1 1 1 1 1 1 1 1

D2-PR1940-NV 209 1 22 1 1 1 1 22 1 1 1 15 1 3

GMT-D2 56 3 2 2 1 1 1 3 2 1 1 4 2 2

D3-H87G5 59 1 1 1 1 1 1 1 22 1 1 1 1 2

D3-15BG1 320 1 1 25 1 1 21 31 25 1 34 169 150 12

D3-UNC3002-V 21 1 1 1 1 1 1 1 1 1 1 1 1 1

D3-UNC3008-NV 52 1 1 32 1 1 1 1 24 1 1 1 1 2

GMT-D3 67 1 1 5 1 1 2 2 11 1 2 4 4 3

D4-H241G1 320 61 56 25 1 1 1 51 27 1 1 29 26 11

D4-10BG3L1 59 1 1 1 1 1 1 1 1 1 1 1 1 1

D4-10BG3L2 289 1 1 21 1 1 1 23 1 1 1 1 1 2

GMT-D4 176 4 4 8 1 1 1 11 3 1 1 3 3 3

ZIKV MR766 95 38 1 1 1 35 1 22 1 20 1 1 1 4

ZIKV PRVABC59 1 1 1 1 1 1 1 1 1 1 1 1 1 1

YF 17D 1 113 1 1 1 25 1 1 1 17 1 1 1 2

IMOJEV 1 1 1 1 1 1 1 1 1 1 1 1 1 1

GMT 3 8 1 1 1 5 1 2 1 4 1 1 1 2

Other

Flaviviruses

GMT-homologous serotype=81

(<20-320+)

GMT-D2 strains

acutes: 7 (<20-

320+)

GMT-D2 strains

convalescent: 2

(<20-77)

GMT-D2 all

strains:2.4 (<20-

320+)

GMT-

heterologous

serotype= 3

(<20-320+)GMT-D3 strains

acutes: 4

(<20-320+)

GMT-D3 strains

convalescent: 2

(<20-169)

GMT-D3 all

strains:2.6 (<20-

320+)

GMT-D4 strains

acutes: 15 (<20-

320+)

GMT-D4 strains

convalescent: 2

(<20-51)

GMT-D4 all

strains:3.4(<20-

320+)

GMT-flavi

acutes: 3 (<20-

113)

GMT-flavi

convalescent: 1

(<20-35)

GMT-flavi:1.6 (<20-320+)

Heterologous

serotype


GMT-D1 strains

acutes: 88 (21-

320+)

GMT-D1 strains

convalescent: 79

(35-320+)

Virus<1 year 1- 2 year

Study No/MPI

2- 6 years

Homologous

serotype

GMT

155

Table 5. 4:- DENV-1-Sera panel statistical verification

Variable Effect (difference in

log(FRNT50)) Std. Err. Tobit p-value

(95%

Conf.interval)

Change per month -0.006 0.035 -0.18 0.868 -0.095 0.083

Homologous serotype 2.786 1.063 2.62 0.047 0.053 5.519

D1-HW2001G4 0.000

D1-10BG1L14 1.888 0.638 2.96 0.032 0.248 3.529

D1-15LG1 -0.027 0.750 -0.04 0.973 -1.954 1.901

D1-10BG2L4 0.063 1.185 0.05 0.960 -2.984 3.109

D1-10INDG3L4 -0.603 0.459 -1.31 0.246 -1.783 0.576

D1-14PHLG4 1.203 0.350 3.44 0.018 0.303 2.102

D2-NGCGAII 0.000

D2-10LGA1L9 0.045 0.682 0.07 0.949 -1.707 1.797

D2-11BGCL4 -3.270 0.600 -5.45 0.003 -4.813 -1.726

D2-13MGCLN -2.718 0.466 -5.83 0.002 -3.916 -1.519

D2-15BGCL4 -2.292 0.789 -2.91 0.034 -4.319 -0.265

D2-15BGCLN -2.615 0.520 -5.03 0.004 -3.952 -1.279

D2-PR6913-V -2.716 0.516 -5.26 0.003 -4.043 -1.388

D2-PR1940-NV -0.268 0.424 -0.63 0.556 -1.359 0.823

D3-H87G5 0.000

D3-15BG1 2.951 0.979 3.01 0.030 0.435 5.467

D3-UNC3002-V -0.831 0.583 -1.42 0.213 -2.329 0.668

D3-UNC3008-NV 1.362 0.436 3.12 0.026 0.242 2.482

D4-H241G1 0.000

D4-10BG3L1 -2.532 0.281 -9.02 <0.001 -3.253 -1.810

D4-10BG3L2 -1.339 0.427 -3.13 0.026 -2.438 -0.241

Note: Virus values are average differences from reference strain in each individual panel

Variable: Time post infection and strains of DENV tested

Effect:- Difference in log FRNT50 with censoring cut-offs set to 2.99-5.77

p-value:- Significance of effect of neutralisation compared with reference strains in each serotype

95%CI:- robust variance estimation of CI for regression coefficients.

156

i. Anti-DENV-1 antisera differentially neutralize DENV-1

Variation in neutralisation of homologous DENV-1 was observed. DENV-1 strains

were isolated from travellers infected in 2010-2015. Each strain is coded with serotype,

year of isolation, location of isolation genotype and lineage, as described in (11) as

shown in (Table 4.6). The reference virus D1-HW2001G4 isolated in 2001 DENV-1

Hawaii outbreak was included. Neutralisation against all the homologous strains was

significantly greater (p=0.047) than against heterologous viruses.

When geometric mean titres (GMT) were compared, NAb response against strain D1-

10BG1L14 was 32% greater than NAb against reference strain D1-HW2001G4, as well

as 41% greater than another isolate D1-15LG1 belonging to the same genotype and

circulating in 2015. Genotype 1 strains were more strongly neutralized than Genotype 2

or Genotype 3. Using tobit regression analysis, the difference in neutralization

(FRNT50) for 2010 Genotype 1 was statistically significant compared to reference

strain if we take p<0.05 as evidence to reject null hypothesis (there is no difference)

while difference in neutralisation for D1-15LG1, a virus belonging to the same

genotype and originating in Laos in 2015 was not statistically significant (p>0.05).

Statistical values for each dataset are given in Table 5.4. Thus, within DENV-1

serogroup the order of NAb responses against different strains of DENV-1, from highest

to lowest was D1-10BG1L14>D1-14PHLG4>D1-10BG2L4>D1-2001HWG4>D1-

15LG1>D1-10INDG3L4. Figure 5. 2 show comparative NAb response against

homologous strains.

157

Study no. MPI Color code

FLV 011 0.3/34

FLV018 2/20/31

FLV017 2/20

FLV022 14/25

FLV026 14

FLV001 24/60/72

Figure 5. 2:- Homologous strain neutralisation by DENV-1 antisera

This scatter plot shows NAb titre at 50% reduction of 11 plasma samples from 6 individuals at

different time points against various homologous strains. Bold line represents geometric mean titre

for each strain at 95% CI. Each study cohort is color coded as indicated. Neutralisation potency was

variable depending on individual. At acute phase (0.3 MPI) neutralisation titre is above threshold of

320 while a same serum (FLV011) at 34MPI has neutralisation below GMT. While for sera FLV001

neutralisation was above GMT at all times (24/60/72) post infection.

158

ii. Neutralization among homologous DENV-1 decreases over time

Anti-DENV-1 acute phase sera neutralized all DENV-1 strains. Each patient for whom

there were follow-up samples demonstrated a decline in magnitude of responses,

measured as GMT, with time after infection, for example FLV011/9 days: 320,

FLV011/34 months: 181; FLV017/2 months:184, FLV/017/20 months:111. Variation

among antisera was apparent – for example patient FLV001 antisera 24 months post

infection neutralized all DENV-1 at high magnitude (>320 for all strains) whereas

patient FLV017 antisera 20 months GMT was significantly lower (111) (Table 5.1).

This pattern of differential homologous NAb responses was patient-specific: FLV017

also showed lower magnitude responses at 2 months post infection with GMT 184

whereas patient FLV018 antisera, also sampled 2 months after infection, demonstrated

GMT 277.

DENV-1 strain-specific neutralization was observed. FRNT50 titres were greatest for

strain DI-10BG1L14, with titres of 320 for 11 of 13 antisera and highest GMT; lowest

magnitude responses were seen for strain D1-10INDG3L4 (Figures 5.2 and 5.4).

Homologous neutralization persisted for all patents’ antisera however decline in

neutralization capacity that was strain-specific was apparent: Figure 5.3 shows relative

decline in neutralization against 2 strains, compared to the other 4. Despite this pattern

of patient (antisera) and strain- specific differential neutralization, overall variation

within homologous DENV-1 was not significant (p>0.05) (Table 5.4).

159

9 days after infection

2 years 10 months after infection

Figure 5. 3:- Differential neutralisation among DENV-1 homologous strains,

patient FLV-011

Acute phase sera neutralize all DENV-1 strains at high magnitude. At 2 years 10

months after infection strain-specific decline in neutralization capacity was

apparent.

1 .0 1 .5 2 .0 2 .5 3 .0

0

1 0 0

1 5 0

L o g 1 0 P la s m a D ilu tio n

Re

du

cti

on

%

50

75

90

1 .0 1 .5 2 .0 2 .5 3 .0

0

1 0 0

1 5 0


Re

du

cti

on

%

50

75

90

D 1 -H W 2 0 0 1 G 4

D 1 -1 0 B G 1 L 1 4

D 1 -1 5 L G 1

D 1 -1 0 B G 2 L 4

D 1 -1 0 IN D G 3 L 4

D 1 -1 4 P H L G 4

160

Figure 5. 4:-Longitudinal variation of anti-DENV-1 neutralising antibody

Heat map illustrates the change in NAb titre and time post infection. If NAb

decline consistently after infection there would be a clear pattern of red falling to

green and this pattern in seen in some samples. However, the overall pattern is

variable among individuals.

Strains of DENV-1

Stu

dy n

o (

MP

I)

161

iii. Cross-neutralisation against heterologous DENV

Cross-neutralisation against all heterologous DENV strains was seen within 2 months of

infection. Convalescent antisera more than one year after infection showed significantly

less cross-neutralization, with some exceptions that were patient- and virus-specific.

Neutralisation capacity of anti-DENV-1 antisera against 15 heterologous DENV: 8

DENV-2, 4 DENV-3 and 3 DENV-4 strains were tested (Table 5.1). Each strain

representing patient isolates are coded with serotype-date of isolation-genotype-lineage,

reference strains include DENV-2 NGC (D2-NGCAII), DENV-3 (D3-H87G5) and

DENV-4 (D4-H241G1) was used. In addition, viruses with distinct epidemiological

traits – ‘virulent’ and ‘non-virulent’ DENV-2 (PR-1940-NV and PR-6913-V) and

DENV-3 (UNC-3002-V and UNC-3008-NV) were included as heterologous strains.

DENV-4 was the most highly neutralised serotype (GMT 97) compared to DENV-2

(54) and DENV-3 (67). Differential strain-specific neutralisation was observed. Among

DENV-2 strains, strain D2-10LGA1L6, originating in Laos 2010 and belonging to the

Asian 1 Genotype, was most strongly neutralised compared to others. Similarly, within

DENV-2 Cosmopolitan genotype, strain D2-15BGCL4 originating in Bali Indonesia in

2015 is neutralized at higher magnitude than strains originating in Malaysia in 2013 and

Bali in 2015 (Figure 5.5). Non-virulent strain PR1940 was highly neutralised compared

to virulent strain PR6913, suggesting that strains with higher epidemic potential are less

susceptible to neutralisation with heterologous DENV antisera. A similar pattern was

observed for DENV-3 virulent and non-virulent strains, non-virulent UNC3002 highly

neutralised compared to virulent UNC3008. The DENV-3 strain D3-15BG1, originating

in Bali in 2015 and a representative of all DENV-3 isolated from travellers, was

strongly cross-neutralized by anti-DENV-1 antisera. DENV-4 reference strain D4-

162

H241G1 was neutralized at greatest magnitude highest neutralisation, titre compare to

DENV-4 patient isolates. Figure 5. 5 shows differential strain -specific neutralisation

by anti-DENV-1 antibody.

Variable neutralisation among heterologous strains was compared using regression

analysis. Each strain was compared with the respective reference strain.

163


FLV 011 0.3/34

FLV018 2/20/31

FLV017 2/20

FLV022 14/25

FLV026 14

FLV001 24/60/72

Figure 5. 5:-Heterologous cross-neutralisation by anti-DENV-1 antibody

Line represents geometric mean titre at 95% CI. NT-50 values were different among

different patient ranging from <10->320 for some strains within serotypes. No strains

within a serotype were neutralized equally by any of plasma sample. Variation of strain

selection was seen depending on patient’s immune response and strain of virus. Each

colored dot represents individual patients as described in legend. Acute samples (<2MPI)

had high degree of cross reactivity across the heterologous strains. And sample from

individual with frequent travel history had NT above GMT against heterologous strains.

164

iv. Longitudinal decrease in heterologous DENV cross-neutralization by anti-

DENV-1 antisera

An acute phase sample collected 9 days after infection (patient FLV011) cross-

neutralized all heterologous DENV at high magnitude. Convalescent antisera from the

same patient 34 and 72 months after neutralized homologous DENV at higher

magnitude than heterologous viruses (Table 5.1). A similar pattern was observed for

patients FLV018 and FLV017, for acute phase sera (2 months after infection) and

convalescent sera (31 and 20 months, respectively). However, patient FLV001 showed

an increase in heterologous cross-neutralization with development of anti-DENV-2,

DENV-3 and DENV-4 cross-neutralization at 60 months that was not present at 20

months. This individual travelled in between his 20 and 60-month visits, but not to any

countries where dengue is endemic (although it is possible that he was exposed to

DENV in Northern Territory, which neighbours dengue endemic countries and where

DENV is frequently introduced by travellers).

Serotype cross-neutralization was strain-dependent. Cross-neutralization of strain D3-

15BG1 was maintained at significant levels for 5 of 6 patients, whereas responses

against D4-10BG3L1 had declined to <20 in 3 of 4 patients with antisera available 2-6

years after infection.

Change in cross-neutralisation with the progression of time post infection is illustrated

in heat map (Figure 5. 6).

165

Figure 5. 6:- Longitudinal heterologous cross-neutralisation by anti-DENV-1 antibody

Decrease in cross-neutralization with time was observed across heterologous DENV. With some exceptions

cross-neutralization largely declines 14 months after infection.

Stu

dy

no

(M

PI)

166

v. Persistent neutralisation against autologous virus

Autologous virus isolated from acute phase sera for 3 patients from whom serum

samples were available up to 60 months (5 years) post infection were neutralised at high

magnitude. Homologous virus was also neutralized at high magnitude however in 5 of 8

cases the magnitude of the response was less than for autologous strains, within the

limits of the neutralization test endpoint dilution. Heterologous serotype cross-

neutralization was always lower than for autologous and homologous virus. In 2 of 3

patients a decline in homologous neutralization, compared to autologous neutralization,

was apparent at 25 months post infection; in the third patient this differential decline

was not apparent until 72 months. Data are shown in Figure 5.7.

All 3 autologous isolates originated in Bali, Indonesia, in 2010, 2014 and 2015 (Table

4.6).

Mo

nth

s

pos

t

inf

ecti

on

Heterologous DENV

strains

167

Figure 5. 7:- Autologous, homologous and heterologous neutralisation

Preferential neutralisation against autologous virus was observed.

FLV001 antisera were collected 24, 60 and 72 months post infection;

FLV081 2, 20 and 31 MPI, and FLV022 14 and 25 MPI.

168

vi. Cross-neutralization of ZIKV, YF17D and IMOJEV by DENV-1 antisera

No patients were known or suspected to be previously infected with ZIKV. However,

the ZIKV prototype strain MR766 was neutralized by 10 of 11 anti-DENV-1 antisera.

One of 6 patients (FLV017) showed a decline in anti-MR766 neutralization by 20

months, to undetectable levels. Despite this strong response, no antisera cross-

neutralized the 2015 epidemic strain ZIKV PRVABC59.

Cross-neutralization of the vaccine viruses YF17D and IMOJEV was mostly observed

for acute phase sera. Anti-YF17D responses were maintained in one patient, FLV018, at

20 and 31 months after infection; this patient had been vaccinated in 1978.

Change in NAb responses with time post infection is summarized in Table 5.1

169

Figure 5. 8:- Cross-neutralisation across flavivirus serogroup

Cross neutralizing responses against ZIKV MR766 but not ZIKV

PRVABC59 were observed. Cross neutralization of YF17D was a feature of

acute phase antisera.

Stu

dy

no

(M

PI)

170

b. Specificity and cross-reactivity of anti-DENV-2 antibody

DENV-2 antisera showed differential neutralisation among 8 homologous and 14

heterologous strains over time. Responses were greatest for homologous DENV-2

strains and were consistently high for acute phase antisera collected 5 days after

infection. Heterologous cross-neutralization declined with time, however persisting

responses were observed up to 64 months that were patient- and virus-specific. An

example of a DENV-2-infected 7 months and 3 years after infection is shown in Figure

5. 9 . One of 6 heterologous DENV-1 strains; 1 of 4 DENV-3 and 2 of 3 DENV-4 were

neutralized 7 months after infection. By 3 years only 1 DENV-4 strain was neutralized

at low magnitude, however 1 DENV-2 strain, D2-PR6193V, was also no longer

neutralized. D2-PR6193V is a reference strain that represents epidemic virulence, and it

is noteworthy that this virus was not neutralized whereas the non-virulent reference

strain D2-PR1940NV was neutralized. List of samples included in this sera-panel is

given in Table 4.2 (also shown below).

Non-linear regression curve was used to determine neutralisation potency at different

reduction cut-off. Neutralisation against homologous and heterologous strain (as shown

in Figure 5. 9) for all anti-DENV-2 plasma samples and non-linear regression curve at

different time points post infection are given in appendix 5.2. Antibody titre at different

cut-off is given in Table 5. 5- Table 5. 7. Statistical variables derived using tobit

regression using FRNT-50 as functional measurement and robust variance estimation is

listed in Table 5.8 and described in each section below.

171


Vaccination

Diagnostic tests

Months post onset of

illness Location Year of visit Duration

of Stay

(Days) Flavi HI NS1 result Dengue

IgM

DENV

PCR^

FLV-004 M Bali 2011/2012 14 No <10 + - + 16 51 64

FLV-005 F Bali 2011 10 No <10 + + + 16 51

FLV-009 F Bali 2012 10 No 40 + + + 17 36

FLV-010 M Thailand 2012 10 No <10 + - + 19 36 48

FLV-012 F Java, Bali 2013 N/A No <10 + - + 0.1 33

FLV-014 F Bali 2013 N/A YF 2006 + - + 7 36

FLV-070* M Bali 2016 5 YF <10 + + + 7

FLV-072* M Bali 2015 10 No <10 + - + 15

FLV-073* M Thailand 2015 10 No <10 + - + 15

FLV-074* M Philippines 2015 10 No <10 + + + 13





“+”: - Positive


172

Figure 5. 9:- Neutralising antibody response by anti-DENV-2 antisera, patient FLV -014

DENV-2 antisera acute and convalescent antisera differentially neutralize homologous and

heterologous DENV.

173




Note:

Value 1=<20

Value 320=>320


>5 years

FLV012

/ 5 days

FLV014

/ 7

FLV004

/ 16

FLV005

/ 16

FLV009

/ 17

FLV010

/ 19

FLV012

/ 33

FLV009

/36

FLV010

/36

FLV014

/36

FLV010

/48

FLV004

/51

FLV005

/51

FLV004/

64

D2-NGCGAII 320 232 320 320 320 320 221 40 46 138 61 176 289 51 159

D2-10LGA1L9 320 192 320 218 128 129 148 139 250 225 225 272 201 155 199

D2-11BGCL4 320 132 217 113 230 64 123 154 66 320 68 186 70 251 142

D2-13MGCLN 320 320 320 243 320 320 261 159 149 320 96 170 84 128 208

D2-15BGCL4 320 320 320 320 320 235 320 73 34 247 31 81 48 83 144

D2-15BGCLN 320 302 320 88 68 47 265 71 48 320 46 111 97 98 120

D2-PR6913 V 121 320 131 241 50 64 241 29 29 53 29 49 59 64 76

D2-PR1940 NV 320 320 224 155 163 320 320 121 74 96 88 75 96 79 149

GMT-D2 283 256 261 194 164 145 226 84 67 183 65 122 99 101 144

D1-HW2001G4 258 1 20 1 65 50 1 1 1 1 1 1 1 1 3

D1-10BG1L14 232 1 127 233 52 1 103 48 1 1 1 51 63 1 13

D1-15LG1 129 1 32 1 1 1 1 1 1 1 1 32 1 23 3

D1-10BG2L4 320 102 320 262 157 45 66 78 53 24 38 73 93 1 66

D1-10INDG3L4 320 320 66 118 43 35 1 1 1 23 1 1 1 1 9

D1-14PHLG4 210 1 1 1 1 1 1 1 1 1 1 1 1 1 1

GMT-D1 234 6 35 14 17 7 4 4 2 3 2 7 4 2 7

D3-H87G5 320 320 58 21 119 1 1 1 1 1 1 44 1 27 9

D3-15BG1 320 34 73 27 30 1 42 30 1 1 23 1 1 1 9

D3-UNC3002-V 123 34 129 76 45 1 1 1 1 1 1 1 1 24 6

D3-UNC3008-NV 320 69 320 50 46 43 30 1 1 1 30 162 29 175 30

GMT-D3 252 71 115 38 52 3 6 2 1 1 5 9 2 18 11

D4-H241G1 320 168 60 74 170 184 31 104 1 79 1 56 29 55 46

D4-10BG3L1 149 39 30 51 66 33 58 1 1 41 1 1 21 1 12

D4-10BG3L2 320 100 159 74 87 69 47 31 23 27 1 36 38 21 42

GMT-D4 248 87 66 66 99 75 44 15 3 45 1 13 29 10 28

ZIKV MR766 320 111 59 29 30 34 1 1 1 1 1 1 1 1 6

ZIKV PRVABC59 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

YF 17D 320 245 1 1 320 1 1 100 1 191 1 1 1 1 7

IMOJEV 47 1 1 1 1 1 1 1 1 1 1 1 1 1 1

GMT 47 13 3 2 10 2 1 3 1 4 1 1 1 1 3

GMT-D4

s tra ins for

convalescent

sera : 24 (<20-

184)

GMT-D1

s tra ins for

convalescent

sera : 5 (<20-

320)

GMT-D1

stra ins for

acute sera :

234(129-320+)

GMT-D3

stra ins for

acute sera :

252(123-320+)

GMT-D3

stra ins for

convalescent

sera :8 (<20-

320)

GMT-D4

stra ins for

acute sera :

248(149-320+)

GMT-flavi

convalescent:

2 (<20-191)

Virus Geometric Mean Titre (GMT)

GMT- total sera homologous

serotype=144 (29-320+)

GMT-D1

strains

total : 7

(<20-320+)

Homologous

serotype

GMT-D2

stra ins for

acute sera:

283(121-320+)

GMT-D2

convalescent

sera: 136 (29-

320+)

Heterologous

serotype GMT-D3

strains

total : 11

(<20-320+)

GMT-D4

strains

total :28

(<20-320+)

GMT- total sera

heterologous

serotype= 10 (<20-

320+)

Other

Flaviviruses

GMT-flavi

acutes: 47

(<20-320+)

GMT-flavi:3 (<20-320+)

< 1 year 1-2 year 2-3 years

GMT

Study No./ MPI

4-5 years

174

Note:

Value 1=<20

Value 320=>320





>5 years

FLV012 /

5 days

FLV014 /

7

FLV004 /

16

FLV005 /

16

FLV009 /

17

FLV010 /

19

FLV012 /

33

FLV009

/36

FLV010

/36

FLV014

/36

FLV010

/48

FLV004

/51

FLV005

/51

FLV004/6

4

D2-NGCGAII 320 139 87 320 193 320 112 27 27 72 34 75 121 32 94

D2-10LGA1L9 320 66 97 119 48 59 75 51 105 100 125 91 96 52 88

D2-11BGCL4 320 44 94 54 126 24 40 82 34 207 37 94 39 96 70

D2-13MGCLN 320 320 193 122 238 320 57 72 50 320 46 90 47 26 114

D2-15BGCL4 320 320 265 140 140 90 81 39 23 128 1 44 30 40 65

D2-15BGCLN 79 155 126 41 36 28 85 40 29 174 28 59 51 52 58

D2-PR6913 V 47 132 43 79 27 32 57 20 1 32 1 28 29 29 23

D2-PR1940 NV 320 124 90 62 78 150 33 34 1 46 38 35 49 32 47

GMT-D2 211 134 109 95 86 80 63 41 15 106 17 59 51 41 64

D1-HW2001G4 100 1 1 1 1 24 1 1 1 1 1 1 1 1 2

D1-10BG1L14 79 1 33 90 26 1 1 1 1 1 1 1 1 1 3

D1-15LG1 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1

D1-10BG2L4 320 1 60 69 68 25 1 31 23 1 1 21 42 1 12

D1-10INDG3L4 320 174 36 46 27 23 1 1 1 1 1 1 1 1 6

D1-14PHLG4 50 1 1 1 1 1 1 1 1 1 1 1 1 1 1

GMT-D1 103 2 6 8 6 5 1 2 2 1 1 2 2 1 3

D3-H87G5 82 79 1 1 1 1 1 1 1 1 1 1 1 1 2

D3-15BG1 100 1 29 1 1 1 1 1 1 1 1 1 1 1 2

D3-UNC3002-V 1 1 37 21 1 1 1 1 1 1 1 1 1 1 2

D3-UNC3008-NV 65 1 94 22 23 1 20 1 1 1 1 1 1 27 5

GMT-D3 27 3 18 5 2 1 2 1 1 1 1 1 1 2 2

D4-H241G1 320 57 24 32 62 50 21 39 1 39 1 32 20 26 24

D4-10BG3L1 23 1 1 1 32 1 1 1 1 1 1 1 1 1 2

D4-10BG3L2 320 32 47 1 37 22 28 21 1 1 1 22 1 1 8

GMT-D4 132 12 11 3 42 10 8 9 1 3 1 9 3 3 7

ZIKV MR766 288 1 1 1 1 1 1 1 1 1 1 1 1 1 1

ZIKV PRVABC59 1 1 1 1 1 1 1 1 1 1 1 1 1 1

YF 17D 174 117 28 21 187 1 39 49 1 87 1 1 1 1 11

IMOJEV 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

GMT 15 3 2 2 6 1 2 3 1 3 1 1 1 1 2

Other

Flaviviruses


Homologous

serotype

Geometric Mean Titre (GMT)

GMT-D2

strains for

acute sera:

211(47-320+)

GMT-D2

convalescent

sera: 58 (<20-

320+)


serotype=64 (<20-320+)

Heterologous

serotype

GMT

GMT-flavi

acutes: 15

(<20-288)

GMT-flavi

convalescent: 2

(<20-117)

GMT-flavi:2 (<20-288)

Virus

GMT-D1

strains for

acute sera :

103(29-320+)

GMT-D1

strains for

convalescent

sera : 2 (<20-

174)

GMT-D1

strains

total : 3

(<20-320+)

GMT- total sera

heterologous

serotype= 3(<20-

320+)

GMT-D3

strains for

acute sera :

27(<20-100)

GMT-D3 strains

for

convalescent

sera :2 (<20-

94)

GMT-D3

strains

total : 2

(<20-100)

GMT-D4

strains for

acute sera :

132(23-320+)

GMT-D4

strains for

convalescent

sera : 5 (<20-

62)

GMT-D4

strains

total

:7(<20-

320+)

4-5 years

Study No./ MPI

175




Note:

Value 1=<20

Value 320=>320


>5 years

FLV012 / 5

days FLV014 / 7

FLV004 /

16

FLV005 /

16

FLV009 /

17

FLV010 /

19

FLV012 /

33

FLV009

/36

FLV010

/36

FLV014

/36

FLV010

/48

FLV004

/51

FLV005

/51

FLV004/

64

D2-NGCGAII 268 83 38 124 85 223 49 22 1 42 24 39 59 24 44

D2-10LGA1L9 320 34 47 62 27 35 39 31 48 46 64 47 52 28 48

D2-11BGCL4 320 1 44 29 64 1 23 48 23 93 25 51 27 43 27

D2-13MGCLN 147 320 109 53 62 320 29 42 21 144 28 51 32 1 51

D2-15BGCL4 115 320 120 64 62 44 27 27 1 67 1 29 23 26 31

D2-15BGCLN 33 79 42 26 27 21 37 29 22 72 22 37 33 31 33

D2-PR6913 V 21 52 27 41 1 1 1 1 1 24 1 21 21 16 6

D2-PR1940 NV 242 48 47 37 37 51 21 1 1 30 22 24 31 18 22

GMT-D2 129 52 52 48 29 24 20 13 5 56 12 35 33 17 28

D1-HW2001G4 46 1 1 1 1 15 1 1 1 1 1 1 1 1 2

D1-10BG1L14 42 1 1 43 1 1 1 1 1 1 1 1 1 1 2

D1-15LG1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

D1-10BG2L4 84 1 33 31 21 1 1 20 1 1 1 1 25 1 4

D1-10INDG3L4 320 84 25 23 21 1 1 1 1 1 1 1 1 1 4

D1-14PHLG4 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1

GMT-D1 33 2 3 6 3 2 1 2 1 1 1 1 2 1 2

D3-H87G5 23 36 1 1 1 1 1 1 1 1 1 1 1 1 2

D3-15BG1 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1

D3-UNC3002-V 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

D3-UNC3008-NV 27 1 1 1 1 1 1 1 1 1 1 1 1 1

GMT-D3 12 2 1 1 1 1 1 1 1 1 1 1 1 1 1

D4-H241G1 293 30 1 1 32 27 1 20 1 25 1 23 1 1 6

D4-10BG3L1 320 1 1 1 18 1 1 1 1 1 1 1 1 2

D4-10BG3L2 56 20 1 1 21 1 20 1 1 1 1 1 1 3

GMT-D4 174 9 1 1 23 3 3 20 1 3 1 3 1 1 4

ZIKV MR766 95 1 1 1 1 1 1 1 1 1 1 1 1 1 1

ZIKV PRVABC59 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

YF 17D 79 58 1 1 82 1 20 28 1 38 1 1 1 1 5

IMOJEV 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

GMT 9 3 1 1 3 1 2 2 1 2 1 1 1 1 2

GMT-flavi

acutes: 9 (<20-

95)

GMT-flavi

convalescent: 1

(<20-82)

GMT-flavi:2 (<20-95)

Study No./ MPI

Geometric Mean Titre (GMT/NT range)

GMT-D2

strains for

acute sera:

129 (21-

320+)

GMT-D2

convalescent

sera: 25 (<20-

320+)

GMT- total sera

homologous serotype=28

(<20-320+)

GMT-D1

strains for

acute sera :

33(<20-320+)

GMT-D1

strains for

convalescent

sera : 2 (<20-

320)

GMT-D1

strains

total : 2

(<20-320+)

GMT- total

sera

heterologous

serotype= 2

(<20-320+)

GMT-D3

strains for

acute sera :

12(<20-29)

GMT-D3 strains

for

convalescent

sera :1 (<20-

36)

GMT-D3

strains

total : 1

(<20-36)

GMT-D4

strains for

acute sera :

174(56-320+)

GMT-D4

strains for

convalescent

sera : 3 (<20-

30)

GMT-D4

strains

total :4

(<20-293)

Heterologous

serotype

Other

Flaviviruses

1-2 year 2-3 years

GMT

< 1 yearVirus

4-5 years

Homologous

serotype

176


Variable

Effect

(difference in

log(FRNT50))

Std. Err. tobit p-

value (95% Conf.interval)

Change per month -0.085 0.023 -3.71 0.014 -0.144 -0.026


Homologous

serotype

D2-NGCGAII 0.000

D2-10LGA1L9 -0.482 0.661 -0.73 0.498 -2.181 1.216

D2-11BGCL4 -1.167 0.868 -1.34 0.237 -3.399 1.065

D2-13MGCLN 0.706 1.032 0.68 0.524 -1.946 3.358

D2-15BGCL4 0.029 0.880 0.03 0.975 -2.232 2.290

D2-15BGCLN -1.079 1.170 -0.92 0.399 -4.086 1.928

D2-PR6913-V -2.627 0.755 -3.48 0.018 -4.567 -0.686

D2-PR1940-NV -0.552 0.972 -0.57 0.594 -3.049 1.945

Heterologous

serotype

D1-HW2001G4 0.000

D1-10BG1L14 2.097 0.598 3.5 0.017 0.559 3.636

D1-15LG1 -0.217 0.615 -0.35 0.739 -1.798 1.365

D1-10BG2L4 3.544 0.617 5.75 0.002 1.959 5.129

D1-10INDG3L4 1.604 0.898 1.79 0.134 -0.703 3.911

D1-14PHLG4 -0.541 0.298 -1.81 0.129 -1.307 0.226

D3-H87G5 0.000

D3-15BG1 -0.553 0.758 -0.73 0.499 -2.501 1.396

D3-UNC3002-

V -1.031 0.786 -1.31 0.246 -3.051 0.989

D3-UNC3008-

NV 1.166 1.150 1.01 0.357 -1.790 4.122

D4-H241G1 0.000

D4-10BG3L1 -1.963 0.362 -5.43 0.003 -2.893 -1.034

D4-10BG3L2 -0.551 0.336 -1.64 0.162 -1.414 0.312




p-value:- Significance of effect of neutralisation compared with reference strains in each

serotype


177

i. Anti-DENV-2 neutralisation specificity against homologous serotype

Anti-DENV-2 antisera neutralized all DENV-2 strains. Acute phase antisera collected 5

days after infection neutralized all homologous DENV at high magnitude as did antisera

collected 7 months after infection. Convalescent antisera (16 – 64 months post

infection) neutralized homologous DENV-2 at lower magnitude GMT 136) than acute

phase sera (GMT 283).

Most travellers returning to Western Australia with DENV-2 infection have been

infected with the same lineage (DENV-2 Cosmopolitan Genotype, Lineage 4). This

novel lineage emerged in 2010-2012 as the dominant epidemic virus in Bali, Indonesia

and has continued to be imported up to 2017. One other virus was imported, belonging

to the Asian 1 Lineage and originating in Laos (D2-10LGA1L9). Neutralization was

assessed against traveller isolates, mostly Lineage 4 or the closely related Nepal lineage

(designated N: D2-13MGCLN; D2-15BGCLN); D2-10LGA1L9; the epidemic strains

D2-PR6193V and D2-PR6193V, and the reference strain D2-NGCAII.

Field isolates imported by travellers therefore represent 2 DENV-2 genotypes, Asian I

and Cosmopolitan. Each field isolate is coded with serotype, year of isolation, genotype

and lineage.

Neutralisation against homologous DENV-1 was approximately 10-fold greater than

that against heterologous serotypes. Difference in homologous vs heterologous

neutralisation was statistically significant (p=0.004).

Within-serotype neutralization varied depending on the virus strain. Asian 1 Genotype

strain D2-10LGA1L9 was neutralized at high magnitude, as was strain D2-13MGCLN;

interestingly the other 3 closely related Lineage 4/Nepal viruses were neutralized to a

178

lesser degree. (Figure 5. 10). Among the patient isolates GMT for Asian 1 D2-

10LGA1L9 was 25% higher than GMT of Cosmopolitan isolates, as a group. Within the

4 Cosmopolitan viruses there was ~2-fold difference in GMT. D2-13MGCLN

originating in Malaysia in 2013 was neutralized with highest GMT compared to other

DENV-2. Neutralisation against the epidemic virulent PR6913-V strain was

significantly lower (p=0.018) compared to other DENV-2 strains. Reference strain

DENV-2NGC was neutralised efficiently by all the test samples.

Thus, the same pattern of patient- and virus-specific neutralization was seen for DENV-

2 as was noted for DENV-1.

179

Study no. MPI Color code FLV 012 0.1/33

FLV014 7/36

FLV004 16/51/64

FLV005 16/51

FLV009 17/36

FLV010 19/36/48

Figure 5. 10:-Homologous strain neutralisation by anti-DENV-2 antibody

Scatter plot includes result from 15 different plasma samples from 6 individuals collected at

different time points. Bar represents geometric mean at 95%CI. GMT mean titre varies with

in serotype. Each study individuals are color coded as represented in legend. Neutralisation

titre varied in each individual at different time points post infection. Antisera at acute phase

(0.1 MPI) had neutralisation above threshold, same antisera at 33 (MPI) had neutralisation

below GMT for some of the homologous strains. Neutralisation titre for FLV010 was

below GMT for 3/8 homologous strains at all time post infection. Hence the pattern of

neutralisation was variable in each individual.

180

ii. DENV-2 homologous NT decreased with time post infection

Anti-DENV-2 antisera neutralized all homologous DENV. Responses were of greatest

magnitude within the first year of infection and decreased with time. Acute phase sera

showed highest NT (283); convalescent NT were of lesser magnitude; lowest NT (65)

was 48 months post infection in one patient (Figure 5. 12) Consistently high-magnitude

NT were observed for Cosmopolitan Genotype viruses, although D2-15BGCL4 NT

were of lower magnitude, > 1 year post infection, compared to other Cosmopolitan

isolates. D2-10LGA1L9 (Asian 1 Genotype) NT were of greater magnitude than

Cosmopolitan isolates. Lowest NT were directed against the virulent epidemic reference

virus D2-PR6913V. Thus, homologous NT over time post infection is patient- and

virus-specific.

181

Figure 5. 11:- DENV-2-specific neutralisation over time, patient FLV 014

DENV-2 field isolates, epidemic strains and reference strains were

neutralized by anti-DENV-2 antisera. Magnitude of responses declined

between 7 months and 3 years in a strain-specific manner. The greatest

decline was against the virulent epidemic reference strain D2-PR6193V.

Responses against the autologous virus were maintained at the highest

magnitude over time.

3 years after infection

7 months after infection

182

iii. Cross-neutralisation against heterologous DENV

Anti-DENV-2 antisera differentially neutralized heterologous DENV. Cross-

neutralisation was assessed against 13 heterologous DENV strains. Neutralisation

against DENV-4 was approximately 3-fold higher than DENV-1 and DENV-3.

Acute phase sera cross-neutralised all heterologous strains. GMT for acute phase

sera was 320 (DENV-2); 234 (DENV-1); 252 (DENV-3); 248 (DENV-4). The

magnitude of responses declined with time, convalescent sera GMT were lower:

136 (DENV-2); 5 (DENV-1); 8 (DENV-3); 28 (DENV-4). DENV-1 D1-10BGL4

genotype-2-lineage-4 strain was neutralized to the highest magnitude among

DENV-1; non-virulent DENV-3 strain (UNC-3008) was neutralised to the highest

magnitude among DENV-3; and DENV-4 reference strain (D4-H241G1) and patient

isolate DENV-4-genotype 3-lineage 2 were neutralised to the highest magnitude

among DENV-4.

183

Figure 5. 12:- Variation of homologous strain neutralisation by anti-DENV-2.

Change in colour from red to green indicates decrease in neutralisation. The number of strains with

>200 NT decreases with time and the overall response becomes more strain-specific. Responses are

patient- and strain-dependent.

Stu

dy

no

(M

PI)

184

Study no. MPI Color code FLV 012 0.1/33

FLV014 7/36

FLV004 16/51/64

FLV005 16/51

FLV009 17/36

FLV010 19/36/48

Figure 5. 13:- Heterologous cross-neutralisation by DENV-2 antisera

Scatter plot includes result from 15 different plasma samples from 6 individuals

collected at different time points. Black bar represents geometric mean at 95%CI.

Different position of GMT bar represents variation of neutralisation across heterologous

strains of DENV. Each coloured dot represents study individuals. Heterologous NT was

seen up to 16 months post infection.

185

iv. Decreased cross-neutralisation over time

Heterologous cross-neutralization declined over time post infection. Acute phase serum

from patient FLV012 collected 5 days after infection was highly cross-reactive against

all DENV; this cross-neutralization had declined significantly by 33 months post

infection. However, even at 33 months 2 DENV-1, 1 DENV-3 and 3 DENV-4 were still

neutralized by anti-DENV-2 antisera. Persistence of cross-serotype neutralization was

seen in all 6 patients, at low magnitude. Responses were virus-specific: D1-10BG2L4

was neutralized by all DENV-2 antisera with the exception of one serum sample

collected 64 months after infection (patient FLV004); an earlier sample from this patient

collected at 51 months neutralized this strain FRNT50 titre 73 (Table 5.5) Cross

neutralisation against other heterologous strains declined completely: anti-

D1HW2001G4 was completely absent after 19 months.

Heterologous cross-neutralization was strain- and patient-specific. Responses may be

maintained up to 51 months post infection. Longitudinal variation in cross-serotype

neutralization by anti-DENV-2 antisera is shown in Figure 5.14

186

Figure 5. 14:- Decrease in heterologous neutralisation time post infection

Neutralizing capacity of anti-DENV-2 antisera against heterologous DENV strains.

Change in colour of heat map from red to green with increasing time indicates

decrease in cross reactivity. Cross-neutralization of heterologous DENV-1 isolate

D1-10BG2L4 is maintained up to 51 months post infection, whereas neutralization

of D114PHLG4 is lost by 7 months. Differential neutralization of D1-10BG1L14 is

patient antiserum dependent.

Stu

dy

no

(M

PI)

187

v. Neutralisation against autologous DENV-2

Autologous virus and antisera were available for 2 patients, FLV004 and FLV014.

Antisera were collected 14, 51 and 64 months post infection for FLV004, and 7 and 36

months for FLV014. Autologous virus was neutralized by autologous antisera at high

magnitude and this was consistent for both patients at all timepoints. Neutralization by

homologous antisera was of lesser magnitude than for autologous antisera and declined

with time; FLV004 homologous responses had declined significantly at 51 and 64

months compared to autologous responses, and also declined for FLV014. Heterologous

responses declined across all visits for both patients (86% and 94%).

Both autologous viruses belonged to the DENV-2 Cosmopolitan Genotype, Lineage 4,

as did the homologous DENV-2 strains, indicating that variation in neutralization

capacity was patient-dependent.

188

Figure 5.15:- Neutralisation against DENV-2 autologous strains

Neutralisation against autologous virus was compared with neutralisation against heterologous and

homologous virus. Neutralisation of autologous strain was maintained to the highest titre at 14,51 and 64

months post infection for patient FLV004, whereas there was a considerable decrease in homologous and

heterologous over time. Similarly, high magnitude neutralization against FLV014 autologous virus was

maintained at 7 and 36 months post infection, while heterologous and homologous neutralisation

declined.

189

vi. Neutralisation against ZIKV, YF17D and IMOJEV

Fifteen antisera from six individuals were tested against the Flavivirus serogroup.

DENV-2 antisera collected between 0.1 – 19 months post infection neutralized

prototype ZIKV MR 766 only; none neutralised the 2015 epidemic ZIKV PRVABC59,

even among the earliest acute-phase sera. The anti-MR 766 was no longer present at 33

months.

Neutralisation against YF17D vaccine virus was measured only in vaccinated

individuals, in addition to acute phase antisera sampled 1-week post infection. Two

individuals showed neutralisation against YF17D at all time points. One of them was

vaccinated 7 years before, the other individual did not recall being vaccinated. IMOJEV

was neutralised by a single, acute phase antiserum. Change in NAb with time post

infection is shown in Figure 5.16.

190

Figure 5. 16:- Longitudinal cross-neutralisation of flavivirus serogroups by anti-DENV-2 antibody

Cross-neutralisation against ZIKV, YF17D and IMOJEV was patient- and virus-specific.

Stu

dy

no

(M

PI)

191

c. Neutralising responses post DENV-3 infection

Anti-DENV-3 antisera collected 8-23 months post-infection neutralized all homologous

DENV-3. Heterologous cross-neutralization was observed across most heterologous

viruses within the first year of infection and diminished significantly after 12 months.

Antisera from two of 3 patients sampled in the first year after infection neutralised

DENV-1, DENV-2, or DENV-3 strains at low magnitude; no strains were neutralised by

both antisera. No antisera collected more than 1 year after infection neutralised

heterologous DENV. Compared to DENV-1 and DENV-2, DENV-3 neutralization was

more restricted to homologous viruses. The field strain D3-15GB1, representing

Genotype 1 imported by all DENV-3-infected travellers, and the epidemic non-virulent

reference virus D3-UNC3008-NV were neutralised at greatest magnitude.

Demographic details are summarised in Table 4. 3 (also shown below).

Homologous strain neutralisation by anti-DENV-3 antisera was 27-fold higher than

neutralisation against heterologous serotypes. Tobit regression and robust variance

estimation shows effect of neutralisation (FRNT-50) against homologous serotype was

significantly greater than against heterologous serotype (Table 5.9) The difference

between mean neutralisation between homologous and heterologous strain was ~96%.

No cross-neutralization of ZIKV, YF17D or IMOJEV was detected.

Neutralisation data are listed in Table 5.9-5.12.

192


Vaccination

Diagnostic tests Months post onset

of illness Location Year of

visit

Duration of Stay

(Days)

Flavi

HI

NS1

result

Dengue

IgM

DFENV

PCR^

FLV-029 M Indonesia 2015 21 No <10 + + + 12

FLV-030 F Bali 2015 8 No <10 + + + 12

FLV-033 F Bali 2015 14 No <10 + + + 14

FLV-035 F Bali 2015 >14 No <10 + + + 8

FLV-037 F Semiyak 2015 6 No <10 + - + 14

FLV-038 M Semiyak 2015 10 No <10 + - + 14 23

FLV-044* F Mexico; Cuba; Bahamas;

USA (Florida)

2015 28 No 160 + + + 8

FLV-045* M Gili Islands/ Bali 2016 11 No <10 + + + 8

FLV-046* M Manado 2015 28 No <10 + - + 18

FLV-047* F Bali 2015 14 No <10 + + + 12

Table 4. 3:- Demographic and laboratory findings in individuals with DENV -3 infection




“+”: - Positive


193





FLV035

/ 8

FLV029

/12

FLV030

/ 12

FLV033

/14

FLV037

/14

FLV038

/14

FLV038

/ 23

D3-H87G5 320 216 164 51 320 117 108 157

D3-15BG1 320 320 320 209 320 320 113 259

D3-UNC3002-V 320 167 33 48 320 78 55 102

D3-UNC3008-NV 320 227 320 107 320 159 134 208

GMT-D3 320 226 154 86 320 147 97 172

D1-HW2001G4 1 1 1 1 1 1 1 1

D1-10BG1L14 320 31 1 1 51 70 1 12

D1-15LG1 38 40 1 1 27 1 1 5

D1-10BG2L4 27 178 20 1 27 1 1 8

D1-10INDG3L4 22 48 21 1 25 1 1 7

D1-14PHLG4 39 100 1 1 1 1 1 3

GMT-D1 26 32 3 1 10 2 1 5

D2-NGCGAII 1 40 1 1 1 1 1 2

D2-10LGA1L9 184 79 25 1 36 1 1 10

D2-11BGCL4 1 1 1 1 1 1 1 1

D2-13MGCLN 29 27 1 1 83 51 1 9

D2-15BGCL4 1 29 1 1 1 1 1 2

D2-15BGCLN 52 27 35 1 1 1 1 5

D2-PR6913-V 25 1 1 1 1 1 1 2

D2-PR1940-NV 21 1 1 1 1 23 22 4

GMT-D2 11 9 2 1 3 2 1 3

D4-H241G1 36 189 32 1 31 1 1 9

D4-10BG3L1 35 44 1 1 70 60 1 9

D4-10BG3L2 77 151 23 1 84 27 1 18

GMT-D4 46 108 9 1 57 12 1 12

ZIKV MR766 1 1 1 1 1 1 1 1

ZIKV PRVABC59 1 1 1 1 1 1 1 1

YF 17D 1 1 1 1 1 1 1 1

IMOJEV 1 1 1 1 1 1 1 1

GMT 1 1 1 1 1 1 1 1

GMT-flavi:NAOther

Flaviviruses

GMT-D2 strains:3

(<20-184)

GMT-D4

strains:12

(<20-275)

Virus

Heterologous

serotype

GMT-heterologous

serotype= 5.5 (<20-

184)

Geometric Mean Titre

Study No./ MPI

Homologous

serotype


serotype=172 (33-320+)

GMT-D1 strains :

5 (<20-320)

<1 year 1-2 year

GMT

Note:

Value 1=<20

Value 320=>320

194

Note:

Value 1=<20

Value 320=>320

Table 5. 10:-Anti-DENV-3 neutralising antibody titre at 75% reduction




FLV035

/ 8

FLV029

/12

FLV030 /

12

FLV033

/14

FLV037

/14

FLV038

/14

FLV038

/23

D3-H87G5 320 110 85 1 306 42 43 55

D3-15BG1 320 320 320 68 320 320 58 201

D3-UNC3002-V 320 72 22 23 187 39 30 59

D3-UNC3008-NV 320 70 212 40 320 61 63 113

GMT-D3 320 115 106 16 277 75 46 93

D1-HW2001G4 1 1 1 1 1 1 1 1

D1-10BG1L14 60 1 1 1 1 1 1 2

D1-15LG1 1 1 1 1 1 1 1 1

D1-10BG2L4 1 65 1 1 1 1 1 2

D1-10INDG3L4 1 25 1 1 1 1 1 2

D1-14PHLG4 20 40 1 1 1 1 1 3

GMT-D1 3 6 1 1 1 1 1 2

D2-NGCGAII 1 1 1 1 1 1 1 1

D2-10LGA1L9 46 39 1 1 23 1 1 5

D2-11BGCL4 1 1 1 1 1 1 1 1

D2-13MGCLN 1 1 1 1 1 1 1 1

D2-15BGCL4 1 1 1 1 1 1 1 1

D2-15BGCLN 1 1 1 1 1 1 1 1

D2-PR6913-V 1 1 1 1 1 1 1 1

D2-PR1940-NV 1 1 1 1 1 1 1 1

GMT-D2 2 2 1 1 1 1 1 1

D4-H241G1 1 69 1 1 1 1 1 2

D4-10BG3L1 1 1 1 1 1 1 1 1

D4-10BG3L2 32 62 1 1 50 1 1 5

GMT-D4 3 16 1 1 4 1 1 2

ZIKV MR766 1 1 1 1 1 1 1 1

ZIKV PRVABC59 1 1 1 1 1 1 1 1

YF 17D 1 1 1 1 1 1 1 1

IMOJEV 1 1 1 1 1 1 1 1

GMT 1 1 1 1 1 1 1 1

GMT-flavi:<20

Virus

Homologous

serotype

Heterologous

serotype

Other

Flaviviruses

<1 year

Study No./ MPI



serotype=93 (<20-320+)

GMT-D1

strains : 2

(<20-65)

GMT-heterologous

serotype= 1(<20-69)GMT-D2

strains:1

(<20-46)

GMT-D4

strains:2

(<20-69)

1-2 year

GMT

195

Note:

Value 1=<20

Value 320=>320





FLV035 /

8

FLV029

/12

FLV030 /

12

FLV033

/14

FLV037

/14

FLV038

/14

FLV038/

23

D3-H87G5 181 46 37 1 148 1 18 19

D3-15BG1 320 320 85 32 320 96 33 116

D3-UNC3002-V 170 40 1 14 96 25 21 24

D3-UNC3008-NV 320 37 93 13 320 34 38 67

GMT-D3 237 68 23 9 195 17 26 43

D1-HW2001G4 1 1 1 1 1 1 1 1

D1-10BG1L14 27 1 1 1 1 1 1 2

D1-15LG1 1 1 1 1 1 1 1 1

D1-10BG2L4 1 1 1 1 1 1 1 1

D1-10INDG3L4 1 1 1 1 1 1 1 1

D1-14PHLG4 1 23 1 1 1 1 1 2

GMT-D1 2 2 1 1 1 1 1 1

D2-NGCGAII 1 1 1 1 1 1 1 1

D2-10LGA1L9 1 23 1 1 1 1 1 2

D2-11BGCL4 1 1 1 1 1 1 1 1

D2-13MGCLN 1 1 1 1 1 1 1 1

D2-15BGCL4 1 1 1 1 1 1 1 1

D2-15BGCLN 1 1 1 1 1 1 1 1

D2-PR6913-V 34 1 1 1 1 1 1 2

D2-PR1940-NV 1 1 1 1 1 1 1 1

GMT-D2 2 1 1 1 1 1 1 1

D4-H241G1 1 30 1 1 1 1 1 2

D4-10BG3L1 1 1 1 1 1 1 1 1

D4-10BG3L2 1 1 1 1 1 1 1 1

GMT-D4 1 3 1 1 1 1 1 1

ZIKV MR766 1 1 1 1 1 1 1 1

ZIKV PRVABC59 1 1 1 1 1 1 1 1

YF 17D 1 1 1 1 1 1 1 1

IMOJEV 1 1 1 1 1 1 1 1

GMT 1 1 1 1 1 1 1 1

Other

Flaviviruses

Virus Geometric Mean Titre


serotype=43(<20-320+)

GMT-D1

strains : 1

(<20-23)

GMT-heterologous

serotype= 1 (<20-34)GMT-D2

strains:1

(<20-34)

GMT-D4

strains:1

(<20-30)

GMT-flavi: (<20)

<1 year

Homologous

serotype

Heterologous

serotype

1-2 year

Study No./ MPI

GMT

196




p-value:- Significance of effect of neutralisation compared with reference strains in each

serotype


Variable

Effect

(difference in

log(FRNT50))

Std. Err. tobit p-value (95%

Conf.interval)

Change per month -0.192 0.047 -4.07 0.010 -0.313 -0.071

Homologous serotype overall 3.723 1.554 2.4 0.062 -0.271 7.716

Homologous

serotype

D3-H87G5 0.000

D3-15BG1 2.687 0.866 3.1 0.027 0.462 4.912

D3-UNC3002-

V -0.781 0.420 -1.86 0.122 -1.861 0.298

D3-UNC3008-

NV 1.108 0.814 1.36 0.232 -0.984 3.199

Heterologous

serotype

D1-

HW2001G4 0.000

D1-10BG1L14 2.804 1.520 1.85 0.124 -1.103 6.711

D1-15LG1 1.005 0.587 1.71 0.148 -0.505 2.514

D1-10BG2L4 1.311 0.880 1.49 0.196 -0.951 3.573

D1-

10INDG3L4 0.867 0.505 1.71 0.147 -0.433 2.166

D1-14PHLG4 0.996 0.769 1.29 0.252 -0.982 2.973

D2-NGCGAII 0.000

D2-

10LGA1L9 1.520 0.902 1.69 0.153 -0.798 3.839

D2-11BGCL4 -0.388 0.435 -0.89 0.414 -1.506 0.730

D2-

13MGCLN 1.162 0.692 1.68 0.154 -0.617 2.942

D2-15BGCL4 -0.107 0.120 -0.89 0.414 -0.414 0.201

D2-15BGCLN 0.665 0.653 1.02 0.355 -1.013 2.343

D2-PR6913-V -0.167 0.524 -0.32 0.763 -1.515 1.181

D2-PR1940-

NV -0.022 0.572 -0.04 0.971 -1.493 1.449

D4-H241G1 0.000

D4-10BG3L1 0.003 0.867 0 0.997 -2.225 2.232

D4-10BG3L2 0.912 0.797 1.14 0.304 -1.137 2.961


197

i. Neutralisation among homologous DENV-3

Anti-DENV-3 antisera neutralised all homologous DENV-3, with varying magnitude.

Neutralization of four virus strains of DENV-3 serotype was assessed: WHO reference

DENV-3 strain (D3-H87G5); virulent and nonvirulent epidemic strains collected in Sri

Lanka in 1983 and 1989, respectively (UNC3002 and UNC3008); and Genotype 1

patient field isolate from 2015. A single patient isolate was tested because all DENV-3

imported by travellers to 2016 belong to Genotype 1.

Differential, strain-specific neutralisation within DENV-3 was observed (Figure 5.17).

Neutralisation of the field isolate D3-15BG1 was of significantly greater magnitude

than neutralization of the reference strain and epidemic virulent and non-virulent

strains. Comparison of confidence intervals for regression coefficients showed non-

virulent strain UNC3008 neutralisation magnitude was greater than virulent strain

UNC3002 (Table 5.9).

198

Study

no. MPI Color

code FLV 035 8 FLV029 12 FLV030 12 FLV033 14 FLV037 14 FLV038 14/23

Figure 5.17:- Homologous strain neutralisation by anti-DENV-3 antisera.

DENV-3 antisera differentially neutralized homologous DENV-3. Black lines are GMT at 95%CI. GMT is

greatest for field isolate D3-15BG1. The epidemic virulent reference strain D3-UNC3002-V was neutralized at

lowest magnitude. Among the study population, one individual (FLV033) at 14MPI has NT below GMT

against all the homologous strains. FLV038 at had NT at threshold (NT>3020) against field isolate D3-15BG1

at 14 MPI while NT dropped below GMT at 23 MPI.

199

ii. DENV-3 neutralisation over time

NT against homologous DENV-3 declined over time in the small number of antisera

tested. Patient FLV035 sampled 8 months post infection had NT for >320, two other

antisera samples at 12 months also showed NT of 320. Patient FLV038 was sampled 14

and 23 months after infection; in the second sample NT had declined from 320 (the

endpoint of the assay) to 113 (Table 5.9, Figure 5.18). This patient was infected with

D3-15BG1, the strain imported by all DENV-3 travellers and representing the dominant

strain circulating in the region at that time. Two patients sampled at 14 months

differentially neutralised DENV-3: one patient FLV033 showed low NT of 48 (against

epidemic virulent strain D3-UNC3002-V) while NT against the dominant virus

circulating in the region D3-15BG1 was maintained at the highest dilution (320); the

other patient FLV037 showed high magnitude NT at 320 against all DENV-3. As for

DENV-1 and DENV-2, DENV-3 homologous responses are patient- and virus-specific.

Figure 17 shows neutralisation of homologous strains over time. Overall comparison of

NT over time of DENV-3 antisera against DENV-3 is shown in heat map (Figure 5.19).

200

Figure 5. 18:- Strain specificity over time by DENV-3 sera panel, patient FLV-038.

Decline in neutralisation against all DENV-3, including autologous strain, is

apparent. Greatest decline was against epidemic virulent reference strain UNC-

3002-V.

201

Figure 5.19:- Change in homologous neutralisation over time, anti-DENV-3 antibody

Decrease in NT over time in DENV-3 sera panel across homologous DENV. Overall decrease

in NAb titre was seen from 8 to 23 months PI however responses were patient- and virus-

specific.

Stu

dy

no

(M

PI)

202

iii. DENV-3 cross-neutralisation against heterologous serotypes

FLV035 antiserum collected 8 months after infection neutralised 13 of 17 heterologous

DENV. There was an overall decrease in NT (<100x) against heterologous serotypes at

12 months post infection however in 1 of 3 patients (FLV037) cross-neutralisation was

maintained at 12 months against 13 of 17 viruses. Neutralisation against DENV-4 was

2-fold higher than against DENV-1 and 4-fold higher than against DENV-2. Strain-

specific variation in cross-NT was apparent (Figure 5.20). Among strains of DENV-1,

patient isolate D1-10BG1LG1 (Genotype 1 lineage 14, originating in Bali 2010) has

high (~3-fold) neutralisation (GMT) than D1-15LG1LH (Genotype 1, lineage H,

originating Bali 2015), however reference strain D1HW2001 (Genotype 4) was not

neutralised by any anti-DENV-3 antisera. Similarly, among DENV-2 strains

neutralisation against D2-10LGA1L9 (Asian I Genotype originating in Laos 2010) and

D2-13MGCLN (Cosmpolitan Genotype originating in Malaysia 2013) was ~2-fold

higher than other strains. Three of 6 anti-DENV-3 antisera had detectable neutralisation

(NAb>20) against the DENV-2 non-virulent epidemic reference strain PR1940 while

only 1 of 6 neutralised the DENV-2 epidemic virulent reference strain PR6913. Within

DENV-2 Cosmopolitan Genotype strains there was variation in NT: D2-11BGCL4

(originating Bali 2011) was <20, ranging to 83 (D2-13MGCLN, originating Malaysia

2013). Differential neutralisation was also observed against DENV-4, with high NT

against D4-10BG3L2 (originating Bali 2010) compared to D4-10BG3L1 (originating

Bali 2010), viruses which belong to different lineages of the same genotype.

As for DENV-1 and DENV-2, cross-neutralisation of heterologous DENV was patient-

and virus-specific. Variation in DENV-3 heterologous cross-neutralisation over time is

shown in Table 5.7 and in the heat map (Figure 5.21).

203

Study

no. MPI Color

code FLV 035 8 FLV029 12 FLV030 12 FLV033 14 FLV037 14 FLV038 14/23

Figure 5. 20:- Heterologous cross-neutralisation by DENV-3 antisera

DENV-3 antisera against heterologous strains showed GMT for each group was <100 while NT for certain

strains were >320. Differential NT was also patient-specific and >100 for some individuals. DENV-3

showed less cross-NT than DENV-1 and DENV-2.

204

Figure 5. 21:- Heterologous neutralisation over time by DENV-3 antisera.

Differential DENV-3 sera panel cross-neutralisation against heterologous strains over

time. The degree of cross-neutralisation maintained after 12 months was patient- and

virus-dependent. By 23 months post infection no cross-neutralisation was observed,

except for the non-virulent epidemic reference virus D2-PR1940-NV, by antisera from

the same patient.

Stu

dy

no

(M

PI)

205

iii. Neutralisation against autologous serotype

Decrease in neutralisation against autologous and homologous strains was seen 23

months post infection in one patient.

DENV was isolated from two patients, FLV037 and FLV038, who had travelled to Bali

Indonesia in 2015. Both isolates belong to Genotype 1 (D3-15BG1). Blood samples

were obtained 14 months after infection (FLV037) and 14 and 23 months after infection

(FLV038). Autologous and homologous viruses were equally neutralised by FLV037

antisera while FLV038 showed a decline in NT at 23 months, from >320 at 14

months to 113 at 23 months.(Figure 5.22).

iv. Neutralisation against related serogroups

Cross-reactivity against ZIKV, YF17D or IMOJEV was not detected for any DENV-3

antisera.

206

Figure 5.22:- Autologous strain neutralisation by DENV-3 antisera

Autologous virus was available for 2 patients from whom antisera were obtained 14

months; and 14 and 23 months, respectively. Responses in the two patients at 14 months

were distinct: FLV017 NT were maintained at high magnitude whereas FLV038 were of

lower magnitude and declined further at 23 months. NT against heterologous DENV were

always just above or below limits of detection.

207

d. Specificity of neutralising antibody response post DENV-4 infection

DENV-4 antisera neutralised homologous viruses at high magnitude (GMT 168)

compared to heterologous DENV (GMT 2). DENV-4 sera panel showed less cross-

neutralisation than DENV-1, DENV-2, and DENV-3. No cross reactivity against any

flavivirus serogroup was observed.

Six individuals were included (Table 4.4 also shown below). Longitudinal samples

were obtained from two individuals (FLV049 and FLV002) at different times post

infection, ranging from 8 to 72 months (six years). NT at three cut-offs (50%; 75%;

90%) are listed in Table 5. 12- Table 5. 14. At 75% and 90 % reduction NT against all

heterologous strains is <30.

208

Sample

ID

Gender Origin of infection Flavi

Vaccination


illnes Location Year of

visit

Duration of Stay

(Days)

Flavi

HI

NS1

result

Dengue

IgM

DENV

PCR^

FLV-002 M Bali 2010 8 No 20 Equivocal + + 24 60 72

FLV-049 F Amed; Ubud; Legian/

Bali

2015/2016 10 No <10 + + + 8 14

FLV-068 M Tuban/ Bali 2016 5 No <10 + + + 6

FLV-080 M Pak Thong Chai/

Thailand

2015 13 No <10 + - + 16

FLV-086 F Sri Lanka; Philippines 2015 10 No <10 + - + 21

FLV-099 M Kuta; Seminyak;

Ahmed

2016 14 No <10 + + + 9

Table 4. 4:- Demographic and laboratory findings in individuals with DENV -4 infection

“+”: - Positive




209

Figure 5.23:- Homologous and heterologous DENV neutralisation by anti-DENV-4

antibody, Patient FLV 049

Antisera from this patient showed high NT against homologous DENV within the first year of

infection; one heterologous virus was neutralized. Fourteen months after infection (6 months

after the first sample) no serotype cross-neutralisation was observed.

210




Note:

Value 1=<20

Value 320=>320

Table 5. 12:- Anti-DENV-4 neutralising antibody titre, 50% reduction

FLV068

/6

FLV049

/ 8

FLV099

/9

FLV049

/14

FLV080

/ 16

FLV086

/21

FLV002

/24

FLV002

/60

FLV002

/72

D4-H241G1 320 320 320 320 43 320 320 277 68 212

D4-10BG3L1 320 320 192 129 26 164 320 94 94 146

D4-10BG3L2 320 318 143 134 25 203 320 137 108 154

GMT-D4 320 319 206 177 30 220 320 153 89 168

D1-HW2001G4 1 1 1 1 1 1 1 1 1 1

D1-10BG1L14 1 1 1 1 1 1 1 1 1 1

D1-15LG1 1 1 21 1 1 1 1 1 1 1

D1-10BG2L4 1 1 1 1 1 1 21 22 24 3

D1-10INDG3L4 1 1 1 1 1 1 1 1 1 1

D1-14PHLG4 1 1 1 1 1 1 1 1 1 1

GMT-D1 1 1 2 1 1 1 2 2 2 1

D2-NGCGAII 36 1 1 1 1 46 1 1 1 2

D2-10LGA1L9 1 22 1 1 1 36 1 1 1 2

D2-11BGCL4 1 21 1 1 1 1 1 1 1 1

D2-13MGCLN 1 128 1 1 1 26 1 1 1 2

D2-15BGCL4 1 32 1 1 1 25 43 1 1 3

D2-15BGCLN 1 20 23 1 1 43 25 1 1 4

D2-PR6913-V 1 1 1 1 1 1 33 1 1 1

D2-PR1940-NV 1 22 1 1 1 1 29 1 1 2

GMT-D2 2 13 1 1 1 9 6 1 1 2

D3-H87G5 1 1 1 1 1 21 1 1 1 1

D3-15BG1 21 1 1 1 1 38 1 1 1 2

D3-UNC3002-V 1 1 1 1 1 31 1 1 1 1

D3-UNC3008-NV 23 30 35 1 1 21 46 23 1 9

GMT-D3 5 2 2 1 1 27 3 2 1 3

ZIKV MR766 1 1 1 1 1 1 1 1 1 1

ZIKV PRVABC59 1 1 1 1 1 1 1 1 1 1

YF 17D 1 1 1 1 1 1 1 1 1 1

IMOJEV 1 1 1 1 1 1 1 1 1 1

GMT 1 1 1 1 1 1 1 1 1 1

Study No./ MPI

Heterologous

serotype

GMT-heterologous

serotype= 2 (<20-128)

Homologous

serotypeGMT-homologous serotype=168 (25-320+)

GMT-D1 Strains:1

(<20-24)

GMT-D2 Strains:

2(<20-128)

GMT-D3 Strains:2

(<20-46)

Virus5-6 years1-2 year

GMT

< 1 year

Other

Flaviviruses


GMT-flavi:<20

211




Note:

Value 1=<20

Value 320=>320


FLV068

/36

FLV049

/ 8

FLV 099/

9

FLV049/

14

FLV080/

16

FLV086/

21

FLV002

/24

FLV002

/60

FLV002

/72

D4-H241G1 231 191 320 237 28 320 320 135 37 154

D4-10BG3L1 320 247 71 38 1 82 181 43 43 57

D4-10BG3L2 190 157 108 44 1 94 273 56 49 60

GMT-D4 241 195 135 73 3 135 251 69 43 80

D1-HW2001G4 1 1 1 1 1 1 1 1 1 1

D1-10BG1L14 1 1 1 1 1 1 1 1 1 1

D1-15LG1 1 1 1 1 1 1 1 1 1 1

D1-10BG2L4 1 1 1 1 1 1 1 1 1 1

D1-10INDG3L4 1 1 1 1 1 1 1 1 1 1

D1-14PHLG4 1 1 1 1 1 1 1 1 1 1

GMT-D1 1 1 1 1 1 1 1 1 1 1

D2-NGCGAII 1 1 1 1 1 1 1 1 1 1

D2-10LGA1L9 29 1 1 1 1 1 1 1 1 1

D2-11BGCL4 1 1 1 1 1 1 1 1 1 1

D2-13MGCLN 1 1 1 1 1 1 1 1 1 1

D2-15BGCL4 1 1 1 1 1 1 1 1 1 1

D2-15BGCLN 1 1 1 1 1 1 1 1 1 1

D2-PR6913-V 1 1 1 1 1 1 1 1 1 1

D2-PR1940-NV 1 1 1 1 1 1 1 1 1 1

GMT-D2 2 1 1 1 1 1 1 1 1 1

D3-H87G5 1 1 1 1 1 1 1 1 1 1

D3-15BG1 1 1 1 1 1 1 1 1 1 1

D3-UNC3002-V 1 1 1 1 1 1 1 1 1 1

D3-UNC3008-NV 1 1 1 1 1 1 1 1 1 1

GMT-D3 1 1 1 1 1 1 1 1 1 1

ZIKV MR766 1 1 1 1 1 1 1 1 1 1

ZIKV PRVABC59 1 1 1 1 1 1 1 1 1 1

YF 17D 1 1 1 1 1 1 1 1 1 1

IMOJEV 1 1 1 1 1 1 1 1 1 1

GMT 1 1 1 1 1 1 1 1 1 1


(GMT)

Study No./ MPI

Homologous

serotype

Heterologous

serotype

Other

Flaviviruses

Virus< 1 year 1-2 year 5-6 years

GMT-flavi:NA

GMT-homologous

serotype=80(<20-320+)

GMT-D1

Strains:<20

GMT-

heterologous

serotype= 1

(<20-29)

GMT-D2

Strains: 1

(<20-29)

GMT-D3

Strains:

<20

GMT

212

Note:

Value 1=<20

Value 320=>320





FLV068

/6

FLV049 /

8

FLV 099/

9

FLV049

/14

FLV080

/ 16

FLV08

6/21

FLV002

/24

FLV002

/60

FLV002

/72

D4-H241G1 107 81 215 111 22 179 238 61 25 88

D4-10BG3L1 192 113 42 25 1 46 71 29 29 34

D4-10BG3L2 72 77 57 27 1 51 118 28 30 33

GMT-D4 114 89 80 42 3 75 126 37 28 46

D1-HW2001G4 1 1 1 1 1 1 1 1 1 1

D1-10BG1L14 1 1 1 1 1 1 1 1 1 1

D1-15LG1 1 1 1 1 1 1 1 1 1 1

D1-10BG2L4 1 1 1 1 1 1 1 1 1 1

D1-10INDG3L4 1 1 1 1 1 1 1 1 1 1

D1-14PHLG4 1 1 1 1 1 1 1 1 1 1

GMT-D1 1 1 1 1 1 1 1 1 1 1

D2-NGCGAII 1 1 1 1 1 1 1 1 1 1

D2-10LGA1L9 1 1 1 1 1 1 1 1 1 1

D2-11BGCL4 1 1 1 1 1 26 1 1 1 1

D2-13MGCLN 1 1 1 1 1 25 1 1 1 1

D2-15BGCL4 1 1 1 1 1 1 1 1 1 1

D2-15BGCLN 1 1 1 1 1 1 1 1 1 1

D2-PR6913-V 1 1 1 1 1 1 1 1 1 1

D2-PR1940-NV 1 1 1 1 1 1 1 1 1 1

GMT-D2 1 1 1 1 1 2 1 1 1 1

D3-H87G5 1 1 1 1 1 1 1 1 1 1

D3-15BG1 1 1 1 1 1 1 1 1 1 1

D3-UNC3002-V 1 1 1 1 1 1 1 1 1 1

D3-UNC3008-NV 1 1 1 1 1 1 1 1 1 1

GMT-D3 1 1 1 1 1 1 1 1 1 1

ZIKV MR766 1 1 1 1 1 1 1 1 1 1

ZIKV PRVABC59 1 1 1 1 1 1 1 1 1 1

YF 17D 1 1 1 1 1 1 1 1 1 1

IMOJEV 1 1 1 1 1 1 1 1 1 1

GMT 1 1 1 1 1 1 1 1 1 1

GMT

Other

Flaviviruses

Study No./ MPI


GMT-homologous serotype=46

(<20-238+)

GMT-D1 Strains:

<20

GMT-

heterologous

serotype= 1

(<20-26)

GMT-D2 Strains:

1(<20-26)

GMT-D3

Strains:<20

GMT-flavi: (<20)

1-2 year 5-6 yearsVirus

Homologous

serotype

Heterologous

serotype

< 1 year

213

Table 5. 16:-DENV-4-Sera panel statistical verification

Variable

Effect

(difference in

log(FRNT50))

Std. Err. tobit p-value (95% Conf.interval)

Change per month -0.012 0.004 -3.27 0.022 -0.021 -0.003


Homologous

serotype

D4-H241G1 0.000

D4-10BG3L1 -2.209 0.858 -2.57 0.050 -4.415 -0.002

D4-10BG3L2 -2.214 0.972 -2.28 0.072 -4.712 0.283

Heterologous

serotype

D1-HW2001G4 0.000

D1-10BG1L14 0.000 0.000 0 1.000 0.000 0.000

D1-15LG1 0.057 0.066 0.87 0.426 -0.112 0.227

D1-10BG2L4 0.303 0.260 1.17 0.296 -0.364 0.970

D1-10INDG3L4 0.000 0.000 0 1.000 0.000 0.000

D1-14PHLG4 0.000 0.000 0 1.000 0.000 0.000

D2-NGCGAII 0.000

D2-10LGA1L9 -0.229 0.359 -0.64 0.551 -1.152 0.693

D2-11BGCL4 -0.576 0.506 -1.14 0.307 -1.878 0.725

D2-13MGCLN 0.096 0.703 0.14 0.896 -1.712 1.905

D2-15BGCL4 0.127 0.578 0.22 0.834 -1.358 1.612

D2-15BGCLN -0.010 0.402 -0.02 0.982 -1.042 1.022

D2-PR6913-V -0.351 0.631 -0.56 0.602 -1.974 1.271

D2-PR1940-NV -0.295 0.616 -0.48 0.652 -1.877 1.288

D3-H87G5 0.000

D3-15BG1 0.285 0.277 1.03 0.349 -0.426 0.997

D3-UNC3002-V 0.183 0.211 0.87 0.426 -0.360 0.725

D3-UNC3008-

NV 1.081 0.287 3.77 0.013 0.343 1.819



Effect: - Difference in log FRNT50 with censoring cut-offs set to 2.99-5.77

p-value: - Significance of effect of neutralization compared with reference strains in each serotype

95%CI: - robust variance estimation of CI for regression coefficients.

214

i. Longitudinal DENV-4 antisera against homologous virus

DENV-4 sera panel showed significantly greater homologous serotype neutralisation

compared with heterologous DENV. One reference strain (D4-H241G1) and two field

isolates D4-10BG3L1 and D4-10BG3L2, both originating in Bali Indonesia in 2010 and

representing lineage 1 and lineage 2 of Genotype 3 were tested. NT varied across the 3

viruses and were highest for the reference strain, which belonged to Genotype 1 (Figure

5.24); p-value for difference in log FRNT-50 was significantly lower (Table 5.16).

NTs against homologous DENV were maintained at high magnitude for antisera from 4

of 5 patients, up to 2 years after infection (Table 5.12). Antisera from the 5th

patient

(FLV080) sampled 16 months after infection neutralised all four DENV-4 at low

magnitude. This patient was infected in the Thailand in 2015, E gene sequence could

not be obtained.

Increase in specificity with increased time post infection was seen in the 2 patients for

whom consecutive samples were available and neutralisation potency per month among

the individual showed significant variation (p<0.022). Homologous neutralisation

declined overall, in a patient- and virus-specific manner (Figure 5.25andFigure 5.26)

215

Study no. MPI Color

code FLV 068 6

FLV049 8/14 FLV099 9 FLV086 21 FLV080 16 FLV002 24/60/72

Figure 5. 24:- Homologous virus neutralisation by DENV-4 antisera

DENV-4 sera panel against homologous strains shows differential neutralisation of reference vs

field strains. NT against reference virus D4-H241G1, which belongs to a different genotype

than the traveller field isolates, is of greater magnitude. Scatter plot is from 9 plasma sample

represented by dots from 6 individuals. Each dot is color coded as represented in legend. NAb

against DENV-4 reference strain was mostly >320 compare to patient isolates. Individual at 16

MPI (FLV080) showed constant low neutralisation against all homologous strains of DENV-4.

216

Figure 5. 25:- Homologous strain neutralisation by DENV-4 (FLV049)

Differential neutralisation of DENV-4 reference and field viruses by

FLV049 anti-DENV-4 antisera 8 and 14 months post infection.

8 months post infection.

14 months post infection.

217

Figure 5. 26:- Variation in homologous strain neutralisation by DENV-4 antisera over time.

DENV-4 sera panel against homologous strains at different times post infection. NT are of high

magnitude within the first 2 years, with the exception of patient FLV080 at 16 months. FLV002

antisera at 21, 60 and 72 months show an overall decline in NT. Thus, NT responses are patient-

and virus-specific.

Stu

dy

no

(M

PI)

218

ii. Heterologous cross-neutralisation by anti-DENV-4 antisera

DENV-4 antisera sampled within the first year of infection (FLV068, 6; FLV049, 8; and

FLV099, 9 months) cross-neutralised a number of heterologous DENV in a patient-

dependent manner. FLV068 and FLV 099 differentially neutralized DENV-1, DENV-2

and DENV-3 at low magnitude (NT 21 – 36), only 1 of 1 of 5 viruses was neutralized

by all three patient antisera (Table 5.13). Patient FLV049 antisera neutralised 7 of 18

heterologous viruses; NT against DENV-2 strains were of greatest magnitude (GMT 13,

range 1-128). Between 1-2 years after infection most heterologous cross-neutralisation

had declined to undetectable levels, with the exception of FLV086 which neutralised 5

of 8 DENV-2 and 4 of 4 DENV-3. Five to 6 years after infection responses had declined

further – 3 antisera sampled from the same patient (FLV002) 24, 60, and 72 months

after infection neutralised 6, 2, and 1 virus(es) respectively, mostly against DENV-2 (4

viruses) at the earliest timepoint.

Thus, as for DENV-1, DENV-2 and DENV-3, anti-DENV-4 antisera neutralise

heterologous DENV in a patient- and virus-specific manner.

iii. Neutralisation against autologous serotype

No autologous virus was isolated from this serogroup.

iv. Neutralisation against related serogroups

The related flaviviruses ZIKV, YF17D and IMOJEV were not neutralised by any anti-

DENV-4 antisera; all NT were <20.

219

Study no. MPI Color

code FLV 068 6 FLV049 8/14 FLV099 9 FLV086 21 FLV080 16 FLV002 24/60/72

Figure 5. 27: -Heterologous strains neutralisation by anti-DENV-4 antisera

Mean neutralisation titre across DENV-4 sera panel against heterologous strains was <30 against all

strains. Interestingly, individual (FLV080) with low NT across homologous strains had higher GMT

against one of the heterologous strain of DENV-2. This individual was infected in Thailand and has

highest GMT against heterologous strain from Laos.

220

Figure 5. 28: - Heterologous neutralisation over time by anti-DENV-4 antisera.

NAb titre was <30 for all time point. No cross reactivity was observed even at 6 months post

infection. However, one of the patients at 16 MPI had NAb >100 against DENV-2 Laos

strain

Stu

dy

no

(M

PI)

221

5.4.2. Magnitude of neutralising antibody over time

Analysis of neutralising antibody responses of DENV-specific antisera against

homologous and heterologous DENV showed increasing specificity against homologous

serotype viruses over time. This pattern was seen in all the patients irrespective of

infecting serotype.

Comparison of neutralisation potency of antisera from individuals infected with any

DENV serotype was visualised using principle coordinate analysis (PCoA). The

antigenic distance between antisera is derived from differential neutralisation capacity

against all DENV strains. Each PCoA plot consists of distribution of sera over span of

six years post infection. Numbers in plot represent patient study code (eg: 11 is FLV

011). Serotype infecting individuals is represented as different coloured circle around

the patient number.

Analysis of individuals’ immune responses within a year of infection showed variation

among serotypes (Figure 5.29). DENV-1 and DENV-2 antisera clustered together;

individuals 11 & 12 were sampled within 1-week post onset of acute illness (OAI) and

showed the greatest degree of heterologous cross-neutralization. DENV-3 and DENV-4

anti-sera, collected 6 months - 1-year post-OAI, clustered within the homologous

serotype and were distant from heterologous DENV.

Twelve months after infection increased clustering of DENV-specific neutralising

responses was apparent (Figure 5.30). DENV-2 antisera consistently neutralized

homologous and heterologous DENV at high magnitude; whereas DENV-1 antisera

differentially neutralized homologous and heterologous DENV. DENV-3 antisera

neutralize homologous and heterologous DENV at a similar magnitude as DENV-4

antisera.

222

Three to 6 years after infection DENV antisera clustered by type (Figure 5.31). Antisera

from 2 individuals 5 and 6 years post-infection with DENV-1 or DENV-2 showed

similar positioning in the plot, showing that neutralization capacity remained constant.

223

Figure 5.31:- Magnitude of anti-DENV 3-6 years post

infection

PCoA post three years after infection shows DENV

antisera clustered by infecting serotype type. Stability of

neutralization after 5-6 years was seen in two

individuals (1 and 2).

Figure 5. 30:- Magnitude anti-DENV 1-2 years post

infection

PCoA at 1-2 years post infection shows increased

clustering of neutralising antibody responses.

Figure 5. 29:- Magnitude of anti-DENV within 1

year of infection

PCoA of individuals less than 1 year post infection

shows anti-DENV response is cross-reactive.

Analysis was derived based on responses against

different viruses. DENV-3 and 4 show clustering of

homologous sera panel while DENV-1 and DENV-2

cross-reactivity was apparent.

224

5.4.3. Summary of neutralisation in DENV monotypic infection

Figure 5.32:- Summary of homologous and heterologous NAb responses

225

5.4.4. Total anti-DENV antibody:

Total anti-DENV antibody (TAb) responses in monotypic DENV-1-DENV-4 infection

were assessed to determine specificity and to serve as reference for parallel analysis of

neutralising antibody. Antibodies induced by DENV infection that are not neutralising

may bind to homologous and heterologous DENV.

Cross-recognition among DENV serotypes was assessed by haemagglutination

inhibition (HI) test. This test is based on the principle that sialic acid molecules on the

surface of red blood cells (RBC) bind to E glycoprotein on the DENV virion surface;

the agglutinated complex will form a lattice that appears as a smooth mat in a microtitre

plate well. Anti-DENV antibody that binds to the virion surface will inhibit

agglutination and RBC will drop to the bottom of the well, forming a red button. To

assess anti-DENV antibody titres, serum is serially diluted, and each dilution is added to

a constant amount of virus and RBC. The HI titre is the highest dilution which causes

total inhibition of agglutination.

Antisera from 34 individuals with monotypic DENV infection were tested against 25

strains of DENV. Not all the sample points of antisera tested in neutralisation were

tested for total antibody response. The closely related flavivirus Kunjin virus (KUNV)

was used as reference antigen to assess specificity.

226

a. Total antibody responses, DENV-1 infection

Twelve sera from 9 DENV-1-infected individuals sampled 9 days – 72 months post-

infection are included in the DENV-1 sera panel. Sera were tested against 6 homologous

DENV-1 strains and 18 heterologous DENV strains; data are expressed as GMT shown

in Table 5.17. Among homologous DENV-1, strain D1-HW2001G4 was recognised at

high magnitude compared to other DENV-1 strains isolated from travellers (Figure

5.33). Heat map analysis of HI titres showed that antisera from 2 individuals did not

recognise strains D1-14PHLG4 (belonging to the same genotype as D1-HW2001G4) or

D1-15LG1 (belonging to a different genotype, Genotype 1). An order of magnitude in

total antibody responses against homologous DENV-1 was apparent: D1-HW2001G4

>D1-10BG1L14>D1-10INDG3L4>D1-15LG1>D1-10BG2L4>D1-14PHLG4 that was

patient/antisera-specific.

Anti-DENV-1 antisera differentially recognised the majority of heterologous DENV

(Figure 5.34). Antisera from 4 individuals had <10 HI titre against heterologous strains.

Highest magnitude responses were directed against the non-virulent DENV-3 epidemic

reference virus D3-UNC3008NV and lowest responses were directed against D4-

14BG3L2. Five and 6 years after infection, TAb against selected strains – D2-

10LGA1L9, and the dominant Cosmopolitan strains D2-15BGCLN and D2-11BGCL4 –

were maintained at high magnitude (GMT:170) whereas TAb against related lineages -

D2-11BGCL isolated from a different traveller – were of lower magnitude (GMT:71).

Responses against the array of DENV-2, DENV-3 and DENV-4 strains varied

dependent on the virus, and the antisera, with mean GMT of 109, 155, 31 respectively.

227

Table 5. 15:- Total antibody response in DENV-1 sera panel

*= additional sample in HI test

Colour coding represents same patient at different time points

Values: 1 is <10 and 10240 is >10240

Antisera at all time points were not tested for TAb

FLV011/

9 daysFLV018/ 2 FLV017/2

*FLV076/

6

FLV022/

14

FLV026/

14

*FLV042

/16

*FLV085/

18

FLV018 /

20

FLV017/

20

FLV011/

34

FLV001/

60

FLV001 /

72

Flavi ref *KUNV 1280 320 160 160 40 40 40 40 80 40 40 80 80 89

D1-HW2001G4 10240 640 640 640 640 320 80 320 640 320 320 640 640 545

D1-10BG1L14 2560 320 160 160 320 80 40 80 120 640 80 160 160 184

D1-15LG1 640 80 40 80 80 40 240 40 40 1 20 80 80 53

D1-10BG2L4 10240 160 80 160 80 20 1 20 40 40 10 80 160 60

D1-10INDG3L4 640 160 40 120 160 20 20 40 80 80 40 160 160 83

D1-14PHLG4 640 160 80 40 80 20 1 20 20 10 20 40 40 34

GMT-D1 2032 202 101 136 160 45 16 50 76 43 40 127 143 98

D2-NGCGAII 10240 480 480 160 160 40 1 40 80 240 20 80 120 110

D2-10LGA1L9 5120 320 320 160 160 80 40 80 160 80 160 160 320 188

D2-11BGCL4 2560 320 160 80 160 40 1 20 40 80 40 80 80 71

*D2-11BGCL4 2560 640 320 160 160 80 20 80 80 160 80 240 240 170

D2-13MGCLN 1280 320 160 80 80 40 1 80 40 80 40 80 80 71

D2-15BGCL4 2560 160 160 40 160 160 20 20 40 160 40 160 160 104

D2-15BGCLN 640 640 160 40 80 40 1 20 80 80 80 160 320 79

*D2-15TLS/IDNGCLG 2560 640 320 160 160 80 80 80 80 160 80 320 20 160

D2-PR6913-V 1280 160 80 20 40 20 20 40 80 80 40 80 160 68

D2-PR1940-NV 1280 320 320 80 320 80 320 20 80 160 40 160 160 152

GMT-D2 2229 357 220 80 130 57 10 40 70 118 53 135 132 109

D3-H87G5 10240 320 320 80 320 160 640 160 40 320 160 160 160 259

D3-15BG1 2560 320 160 80 160 40 20 40 160 80 40 80 160 110

*D3-15BG1 320 240 160 80 160 40 20 40 160 80 30 80 160 90

D3-UNC3002-V 2560 320 320 80 80 40 40 40 320 80 40 80 160 123

D3-UNC3008-NV 2560 640 640 160 80 80 640 40 640 640 80 320 320 288

GMT-D3 2229 347 279 92 139 61 92 53 184 160 57 121 184 155

D4-H241G1 1280 160 320 80 160 160 1 40 20 160 40 80 80 79

D4-10BG3L2 1280 40 40 20 1 80 20 20 20 1 20 40 40 24

*D4-14BG3L2 960 40 20 20 1 40 1 1 20 20 20 20 40 16

GMT-D4 1163 63 63 32 5 80 3 9 20 15 25 40 50 31

GMT

Homologous

serotype

Heterologous

serotype

Virus

Study No/MPI

<1 year 1- 2 year 3- 6 yearsGeometric Mean Titer (GMT)

GMT-D3 strains

acutes: 599

(160-10240)

GMT-D3 strains

convalescent:

104 (20-640)

GMT-D3 all

strains:155

(20-10240)

GMT-

heterologous

serotype= 80

(<10-10240)

GMT-D2 strains

acutes: 559

(80-10240)

GMT-D2 strains

convalescent: 67

(<10-320)

GMT-D4 strains

acutes: 167

(40-1280)

GMT-D4 strains

convalescent: 17

(<10-160)

GMT-D4 all

strains:31

(10-1280)

GMT-D1 strains

acutes: 346

(40-10240)

GMT-D1 strains

convalescent:67

(<10-640)

GMT-homologous serotype=

98(<10-10240)

GMT-D2 all

strains:

109 (<10-

10240)

228

Figure 5. 33:- Total antibody responses against homologous DENV-1

(A) GMT at 95% CI of TAb, anti-DENV-1 antisera against homologous DENV-1. Responses

against D1-HW2001G4 were of greatest magnitude; anti-D1-114PHLG4 responses were lowest.

(B) Heatmap shows TAb over time, from 2-72 months post infection. “1” boxes represent HI

values <10. Responses are virus- and patient-specific.

(A)

(B)

Stu

dy

no

(M

PI)


FLV018 2/20

FLV017 2/20

FLV076 6

FLV022 14

FLV026 14

FLV042 16

FLV085 18

FLV 011 34

FLV001 60/72

229

(B)

Figure 5. 34: - Total antibody responses, anti-DENV-1 antisera against heterologous DENV

(A) GMT at 95% CI of TAb, anti-DENV-1 antisera against heterologous DENV. Highest magnitude

responses were directed against the non-virulent DENV-3 epidemic reference virus D3-UNC3008NV

and lowest responses were directed against D4-14B. (B) Heatmap shows TAb over time, 2 – 72 months

post infection. Empty boxes represent values <10. Differential cross-recognition of heterologous DENV

is apparent 5 and 6 years after infection, with TAb titres ranging from 20 – 320. Responses are virus-

and patient-specific.

(A)

Mo

nths

post

infe

ctio

n

(A)


FLV018 2/20

FLV017 2/20

FLV076 6

FLV022 14

FLV026 14

FLV042 16

FLV085 18

FLV 011 34

FLV001 60/72

Stu

dy

no

(M

PI)

230

b. Total antibody responses, DENV-2 infection

Antisera from 10 individuals infected with DENV-2 sampled five days to 51 months

post infection were assessed for total anti-DENV against 10 homologous DENV-2 and

14 heterologous DENV-1, DENV-3 and DENV-4 strains . Differential responses against

homologous DENV-2 were measured (Figure 5.35). Highest magnitude responses were

directed against the epidemic non-virulent strain D2-PR1940NV, contrasting with low

magnitude TAb against the epidemic virulent strain D2-PR6913V. Responses against

traveller field isolates varied; most (5 of 6) belonged to the Cosmopolitan Genotype, of

which 2 lineages were represented, and interestingly differential TAb magnitude against

these viruses was not clearly associated with virus lineage but was strain-dependent:

D2-11BGCL4 induced high magnitude (80-640, 204) responses whereas D2-10LGAIL9

induced low magnitude (20-160, 142) across all antisera.

All homologous DENV-2 were recognised by all antisera, albeit at levels (20) close to

the assay cut-off for D2-15BGCLN 51 months after infection. Mean TAb titres against

all homologous DENV are shown in Figure 35 (A). A heatmap analysis showing TAb

titres over time is shown in Figure 35 (B). Differential responses, that are virus- and

patient- specific, occur over time, and at the latest timepoint (51 months) a clear

distinction among virus-specific responses is apparent with high (80-160) and low (20)

magnitude homologous TAb against 4 of 10 and 3 of 10 viruses, respectively. TAb

titres for DENV-2 sera panel are shown in Table 5.18.

Responses amongst 9 of 10 individuals (excluding individual at 5 days PI) against

heterologous DENV-1, DENV-3 and DENV-4 viruses were of low magnitude with

overall GMT-17; GMT against individual viruses ranged from <10-320. Heatmap

analysis shows that most antisera produced low or negative TAb titres against

231

heterologous DENV, particularly DENV-1 (Figure 36 (B). Responses were clearly

virus-and patient-specific, but nonetheless had declined to low magnitude TAb titres

(<10 for 12 of 30 viruses; GMT 12 and 5) at 51 months post infection.

232

Table 5. 18:- Total antibody response DENV-2 sera panel



Values: 1 is <10 and 10240 is >10240

Not all sample time points tested for total antibody

FLV012 /

5 days

FLV014 /

7

*FLV070/

7

*FLV074/

13

*FLV072/

15

*FLV073/

15

FLV004 /

16

FLV005 /

16

FLV009 /

17

FLV010 /

19

FLV012 /

33

FLV009

/36

FLV010

/36

FLV014

/36

FLV010

/48

FLV004

/51

FLV005

/51

Flavi ref *KUNV 1280 80 30 160 30 160 20 20 40 20 40 40 20 80 20 20 20 46

D2-NGCGAII 10240 160 320 40 80 40 160 160 160 80 320 160 80 80 80 80 40 136

D2-10LGA1L9 5120 320 160 80 160 160 80 80 160 80 160 80 80 160 80 80 80 142

D2-11BGCL4 2560 160 40 40 80 40 40 40 40 20 80 40 20 80 20 40 20 53

*D2-11BGCL4 2560 640 160 160 320 80 160 160 320 80 320 160 80 640 80 160 80 204

D2-13MGCLN 1280 640 80 80 80 40 160 80 160 80 320 80 40 640 80 160 40 130

D2-15BGCL4 1280 160 80 40 10 10 40 40 80 20 160 80 20 160 20 80 40 55

D2-15BGCLN 1280 160 40 20 40 40 40 40 40 20 80 40 20 80 20 40 20 47

*D2-15TLS/IDNGLG 2560 320 160 160 480 160 160 80 160 40 320 40 40 320 80 160 160 164

D2-PR6913-V 1280 80 20 80 20 20 20 40 40 20 40 40 20 40 20 40 20 38

D2-PR1940-NV 2560 320 320 80 320 320 80 160 160 80 80 160 80 320 160 160 80 181

GMT-D2 2389 243 98 65 89 57 75 75 106 43 149 75 40 171 49 86 46 98

D1-HW2001G4 10240 160 160 40 40 40 40 40 80 40 80 80 40 80 40 80 40 80

D1-10BG1L14 10240 80 80 30 80 1 20 20 40 20 40 20 1 40 1 20 40 26

D1-15LG1 320 40 1 1 1 1 1 1 40 1 20 20 1 20 1 1 1 4

D1-10BG2L4 2560 40 120 1 1 40 1 1 20 1 20 20 1 15 1 1 1 6

D1-10INDG3L4 1280 20 120 1 1 1 1 1 20 1 1 1 1 1 1 1 1 3

D1-14PHLG4 320 40 1 1 1 1 1 1 20 1 20 20 1 20 1 1 1 4

GMT-D1 1810 50 24 3 4 3 3 3 32 3 17 15 2 16 2 3 3 9

D3-H87G5 10240 160 80 320 80 320 80 80 160 40 160 160 20 40 40 80 1 93

D3-15BG1 2560 80 20 20 20 80 20 20 40 20 40 40 1 40 1 20 20 26

*D3-15BG1 320 60 20 20 20 80 20 20 40 20 40 40 1 40 1 20 20 23

D3-UNC3002-V 5120 320 20 40 40 20 20 20 80 20 40 20 20 40 1 40 20 36

D3-UNC3008-NV 10240 160 20 40 80 20 20 20 40 1 80 20 1 80 1 40 20 28

GMT-D3 3378 132 26 46 40 61 26 26 61 13 61 40 3 46 2 35 11 35

D4-H241G1 640 40 40 80 40 40 40 80 80 30 40 40 1 40 1 80 30 35

D4-10BG3L2 640 20 40 20 1 1 20 40 40 20 20 40 1 20 1 40 1 12

*D4-14BG3L2 320 20 20 1 1 1 1 20 1 1 20 1 1 1 1 1 1 3

GMT-D4 508 25 32 12 3 3 9 40 15 8 25 12 1 9 1 15 3 11

Heterologous

serotype

Homologous

serotype

Virus

Study No./ MPI

< 1 year 1-2 year 2-3 years 4-5 years

Geometric Mean Titer (GMT)GMT

GMT-D4

strains for

acute sera :

508 (320-

640)

GMT-D4 strains

for convalescent

sera : 8 (<10-80)

GMT-D4

strains

total: 11

(<10-640)

GMT- total sera

heterologous

serotype= 15 (<10-

10240)

GMT-D3

strains for

acute sera :

3378 (320-

10240)

GMT-D3 strains

for convalescent

sera :27 (<10-320)

GMT-D3

strains

total : 35

(<10-

10240)

GMT-D1

strains for

acute sera :

1810 (320-

10240)

GMT-D1 strains

for convalescent

sera : 6 (<10-160)

GMT-D1

strains

total : 9

(<10-

10240)

GMT-D2

strains for

acute sera:

2389 (1280-

10240)

GMT-D2

convalescent

sera: 88 (10-640)


serotype= 106 (10-10240)

233

Figure 5. 35:- Total antibody responses, anti-DENV-2 antisera against homologous DENV-2

(A) GMT at 95% CI of TAb against homologous DENV-2. TAb titres against 3 of 6 Cosmopolitan

genotype viruses were of greater magnitude compared to other closely-related viruses that belonged to the

same lineage. Highest magnitude responses were directed against the epidemic non-virulent reference

virus D2-PR1940NV, contrasting with low magnitude TAb against the epidemic virulent D2-PR6913V.

(B) Heatmap shows TAb from 7 – 51 months post infection. Differential responses, that are virus- and

patient- specific, occur over time, and at 51 months TAb titres against 6 0f 10 viruses have declined to

low levels.

(A)

(B)

Stu

dy

no

(M

PI)


FLV014 7/36

FLV070 7

FLV074 18

FLV072 15

FLV073 15

FLV004 16/51

FLV005 16/51

FLV009 17/36

FLV010 19/36/48

FLV 012 33

234

A)

Figure 5. 36:- Total antibody responses, anti-DENV-2 antisera against heterologous DENV.

(A) GMT at 95% CI of TAb against heterologous DENV-1, DENV-3 and DENV-4. Overall, TAb titres

against DENV-3 were greater than for DENV-1 and the small number of DENV-4 assessed here. (B)

Heatmap shows TAb titres over time, from 7 - 51 months post infection. Responses are clearly patient-

and virus-specific, and anti-DENV-2 cross-recognition of heterologous viruses is below limits of detection

of the HI assay in 30% of tests. Cross-recognition of the closely related flavivirus KUNV occurs for all

antisera in a patient-specific manner, with TAb titres ranging from 20 – 160.

B)

(B)

(A)

Stu

dy

no

(M

PI)


FLV014 7/36

FLV070 7

FLV074 18

FLV072 15

FLV073 15

FLV004 16/51

FLV005 16/51

FLV009 17/36

FLV010 19/36/48

FLV 012 33

235

c. Total antibody response post DENV-3 infection

Antisera from 10 individuals sampled 8 - 19 months post infection were tested against 5

homologous DENV-3 and 19 heterologous DENV-1, DENV-2 and DENV-4 viruses.

Homologous DENV-3 viruses included D3-10BG1 and D3-15BG1, which represented

the same lineage within Genotype 1, the virus imported by all DENV-3-infected

travellers during the study period 2010-2015. Two viruses, isolated from travellers

infected in 2015 and originating in Bali, Indonesia were selected. Anti-DENV-3 antisera

recognised these traveller strains with similar mean TAb titres (39, 40). TAb titres

against the epidemic virulent and non-virulent viruses D3-UNC3002V and D3-

UNC3008NV were similar. Highest magnitude responses were directed against the

prototype reference virus D3-H87G5 (Figure 5.37 (A)). Heatmap analysis of

longitudinal homologous responses showed patient-specific variation in recognition of

traveller viruses which all belonged to the same lineage (Figure 5.38 (B)). TAb titres are

shown in Table 5.19.

236



Values: 1 is <10 and 10240 is >10240


FLV035

/ 8

*FLV044/

8

*FLV045/

8

FLV029

/12

FLV030 /

12

*FLV047/

12

FLV033

/14

FLV037

/14

FLV038

/14

*FLV046/

18

Flavi ref *KUNV 40 160 40 40 10 60 10 20 20 40 32

D3-H87G5 640 320 640 640 160 80 40 640 160 640 279

D3-15BG1 40 160 40 160 40 40 20 40 10 20 40

*D3-15BG1 40 120 40 160 40 40 20 40 10 20 39

D3-UNC3002-V 160 160 80 160 20 160 20 80 40 160 80

D3-UNC3008-NV 160 160 40 160 20 160 20 80 40 160 75

GMT-D3 121 174 80 211 40 80 23 92 30 92 76

D1-HW2001G4 160 20 10 80 80 20 320 160 20 80 57

D1-10BG1L14 160 15 1 40 40 15 240 80 20 120 35

D1-15LG1 80 1 1 20 10 40 1 80 1 40 9

D1-10BG2L4 40 20 20 20 20 10 40 20 20 40 23

D1-10INDG3L4 40 10 1 20 40 10 40 20 1 20 12

D1-14PHLG4 10 1 1 20 20 40 80 40 1 40 11

GMT-D1 57 6 2 28 28 19 46 50 4 48 19

D2-NGCGAII 160 20 10 40 40 10 320 40 20 40 37

D2-10LGA1L9 160 40 20 160 160 40 160 80 160 160 92

D2-11BGCL4 160 20 20 80 40 10 320 40 20 40 43

*D2-11BGCL4 160 20 20 80 80 20 320 40 40 80 57

D2-13MGCLN 80 10 1 40 40 20 80 20 20 40 22

D2-15BGCL4 160 20 20 40 40 20 160 40 80 160 53

D2-15BGCLN 80 20 40 20 20 1 80 20 20 40 22

*D2-15TLS/IDNGCLG 320 40 40 160 160 80 160 80 80 160 106

D2-PR6913-V 80 40 20 20 40 10 80 20 20 20 28

D2-PR1940-NV 160 40 40 80 80 80 80 160 40 40 70

GMT-D2 139 25 17 57 57 17 149 43 37 61 46

D4-H241G1 160 40 1 1 160 40 80 80 80 80 33

D4-10BG3L2 20 20 20 1 1 1 20 1 1 80 5

*D4-14BG3L2 20 20 1 1 1 1 1 20 1 20 3

GMT-D4 40 25 3 1 5 3 12 12 4 50 8

GMT Geometric Mean Titre

GMT-D1 strains

:19 (<10-320)

GMT-D2 strains:

46 (<10-320)

GMT-D4 strains: 8

(<10-160)


serotype=76 (10-640)

GMT-heterologous

serotype= 19

Heterologous

serotype

VirusStudy No./ MPI

<1 year 1-2 year

Homologous

serotype

Table 5. 19:- Total antibody response of DENV-3 sera panel

237

(A)

(B)

Figure 5. 37: - Total DENV-3 antibody response against homologous DENV

(A) GMT at 95% CI of TAb anti-DENV-3 antisera against homologous virus. TAb against

traveller viruses, which both belong to Genotype 1 and to the same lineage, were of

equivalent magnitude and were lower than against the reference virus D3-H87, which

belongs to Genotype 5. TAb against the epidemic virulent and non-virulent viruses D3-

UNC3002V and D3-UNC3008NV, were similar and were of higher magnitude than against the

traveller viruses. (B) Heatmap shows TAb over time, 8 - 91 months post infection. Responses

were virus- and patient-specific, and at 19 months post infection ranged from 20 – 640.

Stu

dy

no

(M

PI)

Study no. MPIColor

code

FLV 035 8

FLV044 8

FLV045 8

FLV029 12

FLV030 12

FLV047 12

FLV033 14

FLV037 14

FLV038 14

FLV046 18

238

(B)

(A)

Figure 5. 38:- Total antibody responses, anti-DENV-3 antisera against heterologous DENV

(A) GMT at 95% CI of TAb against heterologous DENV. Responses were virus- and patient-specific,

even among closely-related viruses. DENV-1 viruses belonged to 4 different genotypes; GMT-TAb

against the two Genotype 1 viruses were distinct (35 and 9) as were GMT-TAb against the 2 Genotype 4

viruses (57 and 11). Similarly, among DENV-2, 7 of 8 belonged to Cosmopolitan genotype yet TAb

responses varied widely. (B) Heatmap analysis shows change in TAb over time, 8 - 18 months post

infection. Anti-DENV-3 antisera recognised all heterologous DENV at 18 months post infection, with

TAb ranging from 20 – 160.

Study no. MPIColor

code

FLV 035 8

FLV044 8

FLV045 8

FLV029 12

FLV030 12

FLV047 12

FLV033 14

FLV037 14

FLV038 14

FLV046 18

Stu

dy

no

(M

PI)

239

d. Total antibody responses post DENV-4 infection

Anti-DENV-4 antisera from 6 individuals sampled 6 – 72 months post infection were

tested against 3 homologous DENV-4 and 21 heterologous DENV-1, DENV-2, and

DENV-3. TAb titres against all DENV are listed in Table 5.20.

Two traveller DENV-4 viruses which belong to Genotype 3, sampled in 2010 and 2014

and originating in Bali, Indonesia, and the prototype reference virus DENV-4 H241

were assessed. In the period of study DENV-4 was rarely identified in travellers

returning form the Asia Pacific region and thus is not as well represented in this cohort

as the other 3 serotypes. TAb against traveller viruses were of lower magnitude than

TAb against the reference virus, which belonged to Genotype 1. Mean TAb is shown in

Figure 5.39 (A). Heatmap analysis shows TAb over time, 6 -60 months post infection.

Responses were patient- and virus-specific: FLV-002 TAb titres at 60 months were of

greater magnitude than antisera from the other 5 individuals, sampled within the first 2

years of infection (Figure 5.39 (B) )

Cross-recognition of heterologous DENV was of low magnitude (Figure 5.40) and was

virus-and patient-specific. Responses were of greater magnitude, overall, against

DENV-2 (5) and had declined to undetectable levels in most viruses 60 months after

infection although 4 of 21 viruses, representing 3 heterologous serotypes, were still

recognised (TAb range <10-160).

240

FLV068

/6

FLV049 /

8

FLV080/

16

FLV086/

21

FLV002

/60

FLV002

/72

Flavi ref *KUNV 20 10 60 30 20 40 26

D4-H241G1 80 80 40 80 320 320 113

D4-10BG3L2 40 40 10 20 160 160 45

*D4-14BG3L2 40 40 10 40 160 160 50

GMT-D4 50 50 16 40 202 202 63

D1-HW2001G4 40 40 40 40 20 80 40

D1-10BG1L14 20 20 20 40 10 40 22

D1-15LG1 1 1 20 10 1 20 4

D1-10BG2L4 10 10 20 10 1 40 10

D1-10INDG3L4 1 1 10 10 1 40 4

D1-14PHLG4 1 1 20 20 1 20 4

GMT-D1 4 4 20 18 2 36 9

D2-NGCGAII 40 20 40 40 80 40 40

D2-10LGA1L9 40 40 80 160 1 20 27

D2-11BGCL4 10 10 40 40 20 30 21

*D2-11BGCL4 20 20 80 80 20 40 36

D2-13MGCLN 10 10 20 20 1 40 11

D2-15BGCL4 80 80 80 80 10 40 50

D2-15BGCLN 10 10 40 40 1 30 13

*D2-15TLS/IDN/C/G 20 160 80 80 20 40 50

D2-PR6913-V 1 1 80 80 1 30 8

D2-PR1940-NV 10 1 40 20 1 40 8

GMT-D2 15 14 53 53 5 34 21

D3-H87G5 80 80 80 320 60 80 96

D3-15BG1 20 1 40 20 1 40 9

*D3-15BG1 20 1 40 20 1 40 9

D3-UNC3002-V 20 40 40 80 20 40 36

D3-UNC3008-NV 160 160 40 40 1 40 34

GMT-D3 40 14 46 53 4 46 25

GMT

GMT-

heterologous

serotype= 17(<10-

320)

GMT-D1 Strains:9

(<10-80)

GMT-D2 Strains:

21(<10-160)

GMT-D3

Strains:25 (<10-

320)

GMT-homologous serotype=63 (10-

320)


Heterologous

serotype

Homologous

serotype

Virus

Study No./ MPI


Table 5. 20:- Total antibody response in DENV-4 sera panel



Values: 1 is <10 and 10240 is >10240


241

(A)

(B)

Figure 5.39: - Total DENV-4 antibody against homologous DENV

(A) GMT at 95% CI of TAb against homologous DENV-4. TAb against Genotype 3

traveller viruses were of lower magnitude than TAb against the Genotype 1 reference virus.

(B) Heatmap shows change in TAb titres over time, 6 – 60 months post infection.

Responses were patient- and virus-specific; TAb decline to levels close to the limit of

detection within 2 years (FLV-xxx) or may be maintained at high titres 5 years after

infection (FLV-002).

Stu

dy

no

(M

PI)

Study no. MPIColor

code

FLV 068 6

FLV049 8FLV080 16FLV086 21FLV002 60/72

242

Figure 5.40: - Total antibody responses, anti-DENV-4 antisera against heterologous DENV

(A) GMT at 95% CI of TAb against heterologous DENV-1, DENV-2, and DENV-3. Anti-DENV-4

antisera recognized heterologous DENV at low magnitude. (B) Heatmap analysis shows TAb over

time, 6 -60 months post infection. Responses were patient- and virus-specific: FLV-002 TAb titres at

60 months were of greater magnitude than antisera from the other 5 individuals, sampled within

the first 2 years of infection. TAb declined to undetectable levels in most viruses 60 months after

infection.

(B)

(A)

Stu

dy

no

(M

PI)

Study no. MPIColor

code

FLV 068 6

FLV049 8FLV080 16FLV086 21FLV002 60/72

243

5.4.5. Summary of total antibody in DENV monotypic infection

Figure 5. 41: - Summary of homologous and heterologous anti-DENV TAb

244

5.4.6. Longitudinal variation of neutralising and recognising anti-DENV antibody

Specificity and cross-reactivity of neutralising and total anti-DENV-1-4 antisera varied

over time. Acute phase (<2 months post infection) sera were highly cross-reactive

irrespective of infecting serotype. Increase in neutralisation specificity towards infecting

serotype was seen for antisera collected 1-2 years after infection although cross-

reactivity against heterologous DENV was still prevalent. By 2 years post infection

neutralisation was highly specific towards infecting serotype. Broad cross-recognition

of all four serotypes is still apparent after 2 years and after 4 years responses were

largely but not exclusively restricted to homologous serotype.

These longitudinal data was used to map antigen and antibody specificity in an

antigenic map as previously described for other viruses (323). DENV sera panels were

differentiated into five groups based on the time post infection. These maps represent

corresponding positioning of virus and antisera based on neutralisation and cross

recognition. Briefly, a target distance from a serum to each virus is derived by

calculating the difference between the logarithm (log2) reciprocal antibody titre for that

particular virus and log2 reciprocal maximum titre achieved by that serum (against any

virus). A 2-fold change in titre will equate to a single grid in a graph which is denoted

as Antigenic Unit (AU); antigens and the antisera that bind or neutralise them cluster

together, at a distance that is proportional to the magnitude of the response.

Neutralising and total antibody titre changed for each isolate at different time points of

infection. These viruses were derived from the same batch of titrated stock and used

throughout. Thus, variation in antibody responses can be attributed to differential

epitope recognition. Antigenic maps below illustrate change in specificity of

neutralising and cross-recognising antibody responses with the progression of time post

infection. These maps also show the variation of antigenicity among the DENV isolates.

245

These data show that homologous and heterologous isolates are not equally recognised

or neutralised by anti-DENV antisera.

a. Cross-reactive anti-DENV responses within 2 months post infection

Antibody repertoire activated against DENV within two months of infection does not

recognise antigenic diversity among DENV isolates. Response were highly cross-

reactive irrespective of infecting serotype. Both neutralising and total antibody showed

high antibody titres against all tested isolates.

Four plasma samples collected within 2 months of infection were assessed. The

antigenic map shows a high degree of cross-reactivity (Figure 5.42). Neutralisation

against all DENV strains was at the maximum detection limit of the FRNT (>320) and

antigenic distance could not be differentiated; this is represented as a straight line in the

map. A wider range of titres was seen for HI tests, ranging from 320-10240, and

antigenic distances were more dispersed. However, in both cases antigenic diversity

among homologous and heterologous strains could not be identified; no clustering was

observed.

246

A

Figure 5. 42:- Anti-DENV response and antigenic diversity at less than two months post infection

Maps were constructed based on the neutralising and total antibody titre of 4 travellers with in week to 2 months post infection. All the filled shapes represent virus and

clear square boxes are the plasma sample

Figure A) represents neutralisation and figure B) represents cross recognition. Both antibody responses are highly cross-reactive at this time point as shown by close

positioning of the serum in map and no clear distinction among serotypes was observed.

Total antibody

a

Neutralisation B

A

247

b. Anti-DENV antibody responses 2-12 months post infection

Antisera collected between 2 and 12 months after infection displayed greater specific

neutralisation for homologous serotype, compared to earlier acute phase antisera.

However, cross-neutralisation of heterologous serotypes still occurred.

Four plasma samples collected between 2 and 12 months after infection were tested for

NAb and 12 samples for total antibody. In the antigenic map based on the NAb titre

(Figure 43(A)) increased homologous virus neutralisation compared to acute phase

responses was seen and clusters could be distinguished. However, a number of DENV-

1, DENV-2, and DENV-3 isolates and antisera continued to cluster with heterologous

DENV. DENV-4 isolates form a distinct cluster, unlike the other 3 serotypes.

Total anti-DENV antibody responses continued to be highly cross-reactive across all

four DENV serotypes. No antigenic clustering was apparent.

248

A

A

B

A

Figure 5. 43:- Anti-DENV antibody response with a year of infection

Serotype specific cross-neutralisation increased with separation of four different serotypes as shown in neutralisation map. However, DENV-1 and DENV-2

isolates and antisera still showed close proximity to heterologous DENV. Total antibody cross-recognition was still apparent, and no clustering was apparent.

Neutralisation Total antibody

a

249

c. Anti-DENV antibody responses 1-2 years post infection

Neutralising antibody responses 1 year after infection were highly specific towards

infecting serotype. Neutralisation mapping showed clustering of homologous antisera

with infecting serotype indicating the antibody repertoire has increased specificity

compared to earlier time points. Total antibody responses also increased in specificity.

Antigenic maps were derived from 18 antisera. Four distinct clusters were observed,

however there were outliers for each serotype. D1-10BG2L14 was antigenically closer

to D2-10LGA1L9 and distant to the homologous cluster. Furthermore, DENV-2

Cosmopolitan genotype isolates do not form a distinct cluster but instead form 2 groups,

some of which are closer to DENV-1 than to the homologous DENV-2. In addition, 2

isolates of DENV-1 which both belong to Genotype 1 and isolated in different years

(2010 and 2015) and distant from each other; the 2015 virus is antigenically closer to

Genotype 4. In summary, although neutralising antibody responses become more

specific 1-2 years after infection, heterologous cross-neutralisation still occurs.

Total antibody titres derived from 24 antisera were used to construct an antigenic map

(Figure 44(B). Less antigenic overlapping among heterologous isolates was observed

compared to previous time point but there was no clustering was observed in

neutralisation maps. Total antibody mapping showed that DENV-1 and DENV-2

formed clusters with their respective antisera, unlike DENV-3 and DENV-4.

250

Figure 5. 44:- Specificity of anti-DENV antibody 1-2-year post infection

Anti-sera collected 1 year after infection showed increased neutralisation specificity towards the infecting serotype. Four distinct clusters were observed,

however there were outliers for each serotype. Total antibody was highly cross-reactive.

A

A

B

A


a

251

d. Anti-DENV antibody responses 2-4 years post infection

Neutralising and total antibody responses two year after infection were highly specific

towards infecting serotype. All antisera clustered with homologous isolates.

Five antisera (4 DENV-2 and 1 DENV-1) were available for testing at this time point.

Distinct clustering was observed for both neutralisation and total antibody maps. The

random positioning of DENV-3 and DENV-4 is due to lack of anti-DENV-3 antisera.

(Figure 5. 45 A and B).

e. Anti-DENV antibody responses 4 years after infection

Six antisera (2 each of DENV-1, DENV-2 and DENV-4) were available (Figure 5.46).

DENV-1, DENV-2 and DENV-4 formed distinct clusters in both maps. In both maps,

DENV-1 and DENV-2 clusters are closer to each other than they are to DENV-4, which

is the most distant. The random positioning of DENV-3 is due to lack of respective

antisera.

A

A

252

Total antibody B

A

Figure 5. 45:- Anti-DENV-4 antibody at 2-4 years post infection

After 2 years anti-sera are specific to infecting serotype hence in antigenic map clustered towards infecting serotypes shown by both cross-

neutralisation and cross-recognition antibody tests. At this time point, there was no DENV-3 anti-sera available. All the positioning of DENV-3

strains are based on antibody titre derived from DENV-1/2/4 antisera thus DENV-3 strains are at random positioning along with DENV-1 and 2.

A

A

Total antibody

A

A

Neutralisation

253

B

A

Neutralisation

Total antibody

Figure 5. 46:- Anti-DENV-4 antibody 4 years post infection.

Clustering of anti-sera toward homologous strains in DENV-2 was seen. Due to lack of enough number of data sets (anti-sera) for all the

serotypes antigenic distance among strains could not be derived.

A

A

B

A

A

A


254

5.5. Summary discussion

5.5.1. Neutralisation endpoint stringency and increased specificity

In this traveller cohort with well-defined monotypic DENV infection, neutralisation

titres obtained at 50% reduction of infection do not exclude heterologous serotype

cross-neutralisation. Increasing stringency to 75% and 90% reduction increased

specificity to homologous virus however there was a corresponding decrease in

sensitivity and false-negative tests were noted. Heterologous cross-reactivity decreased

by 85% for anti-DENV-1 antisera and by 70% for anti-DENV-2 antisera at 75%

reduction. Applying 90% reduction decreased cross-neutralisation across DENV-1 and

DENV-2 by 92% and 80% respectively (Table 5.1-5.6). DENV-3 and DENV-4

heterologous cross-reactivity was of low magnitude compared to DENV-1 and DENV-

2, even when 50% reduction was applied. For these reasons the 50% reduction endpoint

was applied in analysis of anti-DENV responses.

Increasing neutralisation stringency to 90% reduction differentiates the DENV and

ZIKV sero complexes in monotypic DENV infection. However, this distinction is only

relevant to ZIKV MR766; no traveller antisera neutralised ZIKV PRVABC59. Cross-

neutralisation of ZIKVMR766 at 50% reduction was serotype-specific: the majority of

anti-DENV-1 and anti-DENV-2 antisera, from acute phase up to 72 months neutralized

this ZIKV strain; no anti-DENV-3 or anti-DENV-4 antisera (up to 72 months)

neutralised this virus. The reasons for the high magnitude neutralising antibody

responses directed against the archival ZIKV MR766, but not the contemporaneous

epidemic virus ZIKV PRVABC59, are unclear but may be associated with evolution of

a distinct viral genotype and phenotype with multiple passage through a range of

animals and cell lines.

255

5.5.2. Anti-DENV antibody responses are virus- and time-dependent

Anti-DENV neutralising and total antibody responses in 35 travellers with monotypic

DENV infection varied among individuals infected with same serotype of DENV.

Antisera from each individual, at similar times post infection, differentially neutralised

homologous and heterologous viruses (Table 5.1-5.9).

Homologous anti-DENV1 antisera from 5 of 6 individuals neutralised D1-15LG1

(originating in Laos in 2015) at high magnitude while acute and convalescent antisera

from one individual consistently neutralised this virus at low magnitude (table 5.1). This

last individual was infected in Bali, Indonesia as were the other 5 people. This pattern of

differential neutralisation was also apparent among heterologous viruses. Similarly, 4

DENV-2 (Cosmopolitan genotype, lineage 4) viruses were differentially neutralised by

homologous and heterologous antisera. These data strongly suggest that the DENV-

specific antibody repertoire varies significantly between individuals infected with

highly similar strains. In addition, the non-virulent epidemic DENV-2 and DENV-3

reference strains were neutralised at greater magnitude by homologous antisera

compared to their corresponding virulent epidemic viruses, which differ by 2 and 3

amino acids, respectively, in the E proteins.

Anti-DENV-1 and anti-DENV-2 antisera cross-neutralise heterologous DENV at high

magnitude whereas anti-DENV-3 and anti-DENV-4 antisera did not. These responses

were highly strain-specific, and this study was limited by the smaller number of DENV-

3 and DENV-4 and antisera available. DENV infections imported into WA by infected

travellers are dependent on endemic and epidemic DENV activity. In recent years the

predominant viruses circulating in neighbouring Indonesia, where most travellers were

infected, were DENV-1 and DENV-2. DENV-3 and DENV-4 circulate and are

imported less frequently. Unique confirmation of DENV-4 has been reported (344) and

256

variation in virus envelope neutralising domain may contribute to the DENV-3 and

DENV-4-specific immune repertoire. However, this pattern was not observed for total

antibody responses derived by hemagglutination inhibition test, which were highly

cross-reactive across all DENV.

Acute phase monotypic anti-DENV antisera are highly cross-reactive and neutralise all

homologous and heterologous DENV. Responses restrict with time and cross-

neutralisation has largely declined by one year. However, heterologous cross-

neutralisation was maintained up to 6 years post infection; antisera differentially

neutralised and recognised distinct virus strains.

Limitation of this study is the small sample size and the convenience sampling of

antisera dictated by this traveller cohort. Enrolment into the study is ongoing and more

recently travellers have returned with more diverse DENV infections, including DENV-

3 and DENV-4 which have not circulated as widely in the past. Future research

directions will focus on characterisation of the anti-DENV B cell repertoire; data from

this present study will form the basis of this work.

5.6. Research outcomes

Anti-DENV antisera differentially neutralise homologous virus strains, including

strains that belong to the same lineage within a genotype.

Anti-DENV antisera differentially neutralise heterologous viruses including

isolates that belong to the same serotype.

Anti-DENV antisera neutralise autologous virus at greater magnitude than

homologous serotype; homologous and heterologous neutralisation declines with

time whereas autologous neutralisation is maintained in most.

Acute phase monotypic anti-DENV antisera are highly cross-reactive and

neutralise all homologous and heterologous DENV.

257

Anti-DENV responses restrict with time and cross-neutralisation largely decline

by one year. However, heterologous cross-neutralisation may be maintained up

to 6 years post infection; antisera differentially neutralise and recognise distinct

virus strains.

258

Chapter 6: Antibody immune response post ZIKV infection

6.1 Introduction

ZIKV belongs to Spondweni serogroup of genus Flavivirus. This serogroup includes

ZIKV and Spondweni virus (SPONV), both virus show high serological cross-

reactivity and similar clinical presentation (125). Discovery of ZIKV dates back 70

years, when it was first identified in the Ziika forest better known as “Zika forest” in

Uganda. First isolation was in 1947 from a sentinel rhesus monkey, followed by

isolation from Aedes africanus in 1948 from the same jungle. SPONV was first

isolated in Nigeria in 1952; first isolated strain was Chuku strain (125). This strain

was originally misidentified as ZIKV. Both viruses are transmitted by mosquito of

Aedes genus, while SPONV has been isolated from multiple other mosquito genera.

ZIKV has a wide geographic distribution; SPONV has been reported from sub-

Saharan Africa. The open reading frame (ORF) of the SPONV Chuku strain

exhibited a 68.3% -69.0% and 74.6% -75.0% nucleotide and amino acid identity

respectively, when compared to geographically and genetically distinct strains of

ZIKV (345). Genetic, geographic and vector variation between SPONV and ZIKV

support that these viruses are separate species although they exhibit misleading

serological and clinical similarities.

ZIKV is reported to exist as single serotype that has evolved into 2 major lineages

differentiated based on its geographic origin: African (MR766 prototype cluster), and

Asian genotype (140, 346). Phylogenetic study of ZIKV was done after 2007 Yap

outbreak by Lanciotti et al. African lineage diverged to East African (MR766

prototype cluster) and West African (Nigerian cluster). Asian lineage diverges to

Southeast Asia and the Pacific subclades (126, 131). Phylogenetic study based on E-

and NS5 gene sequence of 43 ZIKV strains isolated from 1947 to 2007 in Africa,

Asia and the Pacific supports the divergence of African ancestor strains to two

259

clusters, suggesting ZIKV emerged from East Africa (Uganda) and moved to West

Africa (126, 347). The same study shows the ZIKV 2007 Micronesian and Malaysian

strains belong to Asian clade. This finding was also confirmed by E-and NS3

sequence analysis. ZIKV strains within the same lineage are less divergent whereas

there is greater divergence between the two main lineages. There is less than 12%

divergence at the nucleotide level among all ZIKV strains (348). ZIKV strains

reported in the French Polynesia (FP) and Brazil epidemics were closely related and

belonged to the Asian lineage. ZIKV isolates from FP, Brazil, Columbia, and Puerto

Rico showed 99% nucleotide identity within the Asian lineage and ~89% identity to

East and West African lineages (13).

Zika was a silent infection for years, with only a small number of human cases

reported (349, 350). It has been postulated that the virus originated in East Africa and

then spread into both West Africa and Asia approximately 50–100 years ago (351).

Since the outbreak in Yap 2007, a series of outbreaks in different years have been

reported (349, 350). WHO reports till 18 Aug 2016, 70 countries and territories have

evidence of ZIKV transmission since 2007 and 53 countries reported outbreaks from

2015 onwards. In February 2016, 11 countries have reported person-to-person

transmission, 4 with possible endemic transmission or evidence of local infection

(352). Clinical symptoms of Zika include mild fever, arthralgia and conjunctivitis,

with recent reports of association with Guillain Barr Syndrome and Microcephaly

(353).

Epidemiological data suggests the epidemic Asian strain that circulated in FP has

greater virulence than the original Yap strain that emerged in 2007. This FP ZIKV

was associated with neurological complications and was also reported in epidemics

in Brazil followed to other American countries, where it is also associated with

neurological complications. Adaptive genetic changes including glycosylation

260

patterns has associated with evolution of virulent ZIKV strains (140, 351). Lack of

whole genome sequence data limits analysis of virus evolution and divergence

(348).

The spread of ZIKV to dengue-endemic areas has the potential for increased impact

in human health, owing to the high degree of homology between these two

flaviviruses and the potential for immune enhancement. ZIKV is closely related to

DENV with approximately 43% amino acid divergence across the viral polyprotein

as well as the ectodomain of the envelope (E) protein (354). ZIKV E-structure has

been shown to resemble other flaviviruses but contains a unique positive charge

adjacent to the fusion loop believed to influence host attachment (355). The

flavivirus E glycoprotein is the principal antigen in induction of neutralising

antibodies (NAb) that may contribute to protection and which are a marker for

vaccine-induced immunity. The cross-reactive but not necessarily the cross-

protective nature of E-protein-initiated antibody-mediated immune responses has

complicated assessment of ZIKV-induced immunopathology, especially in

populations where DENV is endemic.

Epitopes that elicit potent cross-neutralising antibodies against both ZIKV and

DENV have been described (356). The functional significance of such cross-reactive

antibodies has been investigated and a role for cross-neutralisation as well as

enhancement has been proposed (258, 357).

This study aimed aim to assess the capacity of anti-ZIKV antisera to cross-neutralise

and/or cross-recognise ZIKV, DENV, YFV and JEV. Antisera were obtained from

individuals with monotypic ZIKV infection; recent ZIKV infection in a background

of previous DENV infection; and ZIKV infection in individuals who received YFV

vaccine, from soon after presentation with febrile illness, to 605 days post

261

presentation. Samples were obtained from West Australian travellers who visited

countries with known ZIKV transmission or with a history of ZIKV transmission in

the past. Zika and dengue are not endemic in Western Australia. Samples were tested

against sentinel ZIKV MR766, human Puerto Rico 2015 isolate ZIKV PRVABC59);

DENV (all four serotypes:- DENV-1-HW2001, DENV-2-NGCAII, DENV-H87GV

and DENV-4-H241GI); YF 17D and IMOJEV.

6.2. Aims of study

To assess specificity of anti-ZIKV neutralising antibody responses in

monotypic ZIKV infection and in ZIKV-infected individuals with previous

DENV infection or flavivirus vaccination

To assess duration of ZIKV-specific antibody responses

To identify an anti-ZIKV protein response that may differentiate ZIKV and

DENV infection

262

6.3. Results

6.3.1. Neutralising antibody response

Antisera from 16 individuals sampled within the first month of infection and up to

605 days after presentation to their health care professional were assessed. Eight

individuals were enrolled into the WA Traveller Study (samples were coded “FLV-

[study number]”) and were available for recall and recollection, and 8 presented for

diagnosis of febrile illness (samples coded as “ZIKV-[study-no])” (Table 4.5 also

shown below).

ZIKV infection was classified as ‘confirmed’ or ‘probable’ based on the WHO case

(352). Confirmed cases were those in which ZIKV RNA was detected by PCR (in

urine); probable cases were those in which anti-ZIKV IgM and/or increased HI titres

(more than 4-fold) were detected. All individuals had a history of recent travel to

countries where ZIKV was known to be circulating (South Pacific nations;

Americas) or where sporadic Zika cases been reported (Indonesia; Philippines, India,

Thailand) and presented with clinical symptoms similar to Zika and not explained by

another etiology. ZIKV is not endemic in Western Australia.

Thirty-four serum or plasma samples from 16 individuals were tested. Nine

individuals were sampled on 2 or more occasions from acute phase (< 30 days) to

605 days post presentation (range 2-6 samples). Four individuals received yellow

fever vaccination. Serum or plasma assessed for anti-ZIKV NAb was diluted starting

at 1:10 or 1:40 depending on available sample volume and the maximum dilution

was 1:640. Unless sample volume was limited, all sera were tested against 2 strains

of ZIKV (MR766 and PRVABC59); DENV (all four serotypes when sample volume

permitted; or DENV-4 only when sample volume was restricted); YF17D; and

263

IMOJEV. Neutralising antibody titres are listed in table 6.1. Neutralisation curves for

each individual are shown in Appendix 6.1 and an example is shown in Figure 6.1.

Comparison of 50%, 75% and 90% reduction in interpretation of neutralisation titre

showed that applying 90% reduction produced false negative results in individuals

with confirmed ZIKV infection and thus FRNT-50 was routinely applied here.

264

ZIKV case

classification

Sample

ID Gender

Origin of infection

Flavivirus

Vaccination

Diagnostic tests Samples available at

Location Year of

visit

Duration

of Stay

(Days)

Flavi HI ZIKV

IgM

ZIKV

PCR Presentation

Days pos t

onset of illness

Confirmed

FLV-064 M Indonesia 2014 10 No <10 + + no 605

FLV-087 F El Salvador 2015 105 No 640 -/weakly

+ + yes 12/39/425

FLV-093 M Mexico 2016 60 YF-2015 N/A N/A + no 330

ZIKV 01 M South Pacific 2015 N/A Yes (date N/A) 20-640 + + yes 210

ZIKV 02 F Fiji 2016 N/A N/A 20 + + yes 11

Probable

FLV-032 F Colombia 2015 N/A YF-2015 160 + N/A yes 7/37/90/240/390

FLV-088 M El Salvador 2015 60 Not sure >640-320 - N/A yes 23/425

FLV-089 F El Salvador 2015 60 Not sure 320 equivocal ND yes 23/425

FLV-090 F Tonga End

2015 14 No

<10

+ ND

yes 425

FLV-091 M Mexico 2016 30 YF-2015 160 + ND yes 3/240

ZIKV 03 F Colombia 2016 N/A N/A 1280 - N/A yes 38

ZIKV 04 M Philippines 2016 N/A N/A <10 + N/A yes N/A

ZIKV 06 F Tonga End

2015 N/A N/A

<10

+ N/A

yes N/A

ZIKV 07 F Tonga End

2015 N/A N/A

<10

+ N/A

yes N/A

ZIKV 09 F India 2016 N/A N/A 10 equivocal N/A yes N/A

ZIKV011 M Thailand 2017 N/A N/A <10 + N/A yes N/A

Table 4. 5: - Demographic and laboratory findings of individuals with ZIKV infection



ND: Not done

Flavi HI : MVE 151 antigen

ZIKV PCR: Urine

“+”: - Positive


265

*:- Confirmed ZIKV cases

NA: “Not available” due to lack of sample these samples could not be tested against ZIKV PRVABC59

Detectable neutralising antibody response

None of anti-ZIKV antisera showed cross reactivity against IMOJEV

Table 6. 1:- Neutralising antibody response in ZIKV infection

FRNT50 FRNT90 FRNT50 FRNT90 FRNT50 FRNT90 FRNT50 FRNT 90 FRNT50 FRNT90 FRNT50 FRNT90 FRNT50 FRNT90

FLV-064* Indonesia 2014 No 605 65 22 23 11 <10 <10 <10 <10 <10 <10 <10 <10 <40 <40

<30 ≥640 89 NA NA NA NA NA NA NA NA 76 <40 <40 <40

12 ≥640 ≥640 572 <40 NA NA NA NA NA NA ≥640 ≥640 <40 <40

39 ≥640 185 224 <40 170 46 ≥640 160 ≥640 134 ≥640 160 <40 <40

425 457 109 115 33 46 11 639 153 252 46 279 49 <40 <40

FLV-093* Mexico 2016 Yes (2014) 330 322 92 48 19 <10 <10 <10 <10 <10 <10 111 <10 236 16

<30 ≥640 324 NA NA <40 <40 <40 <40 <40 <40 95 <40 ≥640 73

210 ≥640 ≥640 223 62 <40 <40 <40 <40 <40 <40 <40 <40 322 43

<30 ≥640 125 ≥640 148 NA NA NA NA NA NA 94 <40 <40 <40

11 ≥640 216 ≥640 235 NA NA NA NA NA NA 198 <40 <40 <40

<30 176 48 NA NA NA NA NA NA NA NA 55 <30 195 <40

7 619 30 NA NA NA NA NA NA NA NA <40 <40 <40 <40

37 ≥640 78 85 <40 <40 <40 <40 <40 <40 <40 <40 <40 <40 <40

90 523 116 274 46 <40 <40 <40 <40 <40 <40 <40 <40 <40 <40

240 379 94 116 37 <40 <40 <40 <40 <40 <40 <40 <40 <40 <40

390 381 92 129 38 <40 <40 <40 <40 <40 <40 <40 <40 <40 <40

<30 ≥640 ≥640 43 <40 ≥640 ≥640 ≥640 ≥640 ≥640 <40 ≥640 ≥640 <40 <40

23 ≥640 ≥640 106 <40 ≥640 ≥640 ≥640 ≥640 ≥640 98 ≥640 ≥640 <40 <40

425 431 48 57 17 ≥640 108 ≥640 123 208 18 ≥640 116 <40 <40

<30 ≥640 ≥640 194 20 ≥640 159 ≥640 271 331 58 ≥640 ≥640 <40 <40

23 ≥640 ≥640 48 <40 605 124 ≥640 102 519 40 ≥640 ≥640 <40 <40

425 ≥640 110 47 11 292 58 563 59 152 15 ≥640 198 <40 <40

<30 ≥640 ≥640 148 <40 NA NA NA NA NA NA <40 <40 <40 <40

425 82 29 40 15 <10 <10 <10 <10 <10 <10 <10 <10 <40 <40

<30 ≥640 ≥640 349 38 <40 <40 ≥640 133 63 <40 306 60 ≥640 109

3 ≥640 215 275 <40 <40 <40 348 82 40 <40 ≥640 107 618 92

240 235 85 105 30 <20 <20 <20 <20 <20 <20 193 14 ≥320 30

<30 ≥640 ≥640 252 48 ≥640 518 ≥640 626 ≥640 323 ≥640 ≥640 353 <40

38 ≥640 ≥640 107 42 ≥640 182 ≥640 470 ≥640 55 ≥640 363 <40 <40

ZIKV 04 Philippines 2016 N/A <30 ≥640 222 ≥640 <40 NA NA NA NA NA NA <40 <40 <40 <40

ZIKV 06 Tonga End 2015 N/A <30 136 36 NA NA NA NA NA NA NA NA <40 <40 <40 <40

ZIKV 07 Tonga End 2015 N/A <30 ≥640 11 NA NA NA NA NA NA NA NA <40 <40 <40 <40

ZIKV 09 India 2016 N/A <30 573 57 NA NA NA NA NA NA NA NA <40 <40 <40 <40

ZIKV011 Thailand 2017 N/A <30 ≥320 157 ≥160 23 <40 <40 <40 <40 <40 <40 <40 <40 <40 <40

ZIKV 03 Colombia 2016 N/A

FLV-090 Tonga 2015 No

FLV-091 Mexico 2016 Yes(2015)

FLV-088El

Salvador2015 No

FLV-089El

Salvador2015 No

ZIKV 02* Fiji 2016 N/A

FLV-032 Colombia 2015 Yes-2015

FLV-087*El

Salvador2015 No

ZIKV 01*South

Pacific2015

Yes(date

N/A)

Anti DENV FRNTAnti YF-17D

ZIKV MR766 ZIKV PRVABC59 Anti DENV-1-FRNT Anti DENV-2-FRNT Anti DENV-3-FRNT Anti DENV-4-FRNTSample ID Location

Date of

travel

Vaccinatio

n History

Time post

presentati

on of

illness

(days)

Anti-ZIKV FRNT

266

Figure 6. 1:-Neutralising antibody response post Zika

Non-linear regression curve showing neutralisation of flavivirus infection by anti-ZIKV antibody. (A) FLV090 with monotypic ZIKV

infection; (B and C) FLV087 and FLV089 with DENV cross-neutralisation; both individuals had lived in a dengue-endemic country (El

Salvador) for most of their lives before emigrating to Western Australia. They presented with Zika-like illness on their return from visiting

El Salvador in 2015.

267

Anti-ZIKV, anti-DENV, anti-YFV and anti-JEV antibody responses were assessed

(a) within 1 month; (b) 1-12 months; and (c) more than 12 months following

presentation with febrile illness.

a. Within 1 month

Samples were available from 11 of 16 individuals within 1 month of presentation.

High magnitude (>640) anti-ZIKV-MR766 FRNT-50 NAb titres >640 were detected

for 10 of 11 individuals. Samples from 7 individuals were available in sufficient

volume to allow assessment of anti-ZIKV-PRABC59; a minority (2 of 7) neutralised

this strain at high magnitude. Anti-ZIKV-MR766 GMT was greater than anti-ZIKV-

PRABC59 GMT (Figure 6.2). Two antisera produced low anti-ZIKV-MR766 NAb

titres; both were probable (IgM positive) cases. One of these cases submitted a

second sample 7 days later at which time NAb titre increased to 619.

ZIKV antisera from 9 of 11 individuals cross-neutralised DENV. When serum

volume permitted, all four DENV serotypes were tested; DENV-4 alone was tested

when sample volume was limited. Sera were available from 3 ZIKV confirmed cases

(FLV087; ZIKV01; ZIKV02); all 3 antisera neutralised DENV (Table 6.1). FLV087

reported a previous confirmed DENV infection while traveling in El Salvador 5

years earlier and her anti-DENV NAb respones were maintained in samples collected

following her enrolment into the Traveller Study (Table 6.1). Anti-DENV NAb titres

for ZIKV01, with no history of previous DENV infection, declined when tested at

210 days while titres were maintained at day 11 for ZIKV02, the latest sample that

was available. For both ZIKV01 and ZIKV02, FRNT90 titres were <40 and thus,

below the limits of detection of this test.

268

Anti-ZIKV antisera from 5 of 16 individuals cross-neutralised YFV17D. Of the 5

individuals, 4 were known to have previously received yellow fever vaccination.

Follow-up samples were available for 4 individuals, 3 of whom were previously

vaccinated, for between 38 – 390 days. Anti-YFV FRNT50 titres were maintained

for 210 and 240 days for 2 vaccinated individuals and declined to undetectable levels

for the other 2, one of whom was vaccinated, 7 and 38 days after the sample

collected at presentation. In both cases FRNT90 at the first, positive, sample was <40

and thus below levels of detection. Comparative FRNT50 and FRNT90 for two

strains of ZIKV is shown in figure 6.2.

b. 1 – 12 months post presentation

Sera from 6 individuals collected 30 – 365 days after presentation were assessed.

Anti-ZIKV MR766 GMT declined from 532 to 473 (12% decline), and anti-ZIKV

PRVABC59 GMT also declined, from 242 to 128 (47% decline). (Figure 6.3).

Anti-ZIKV antisera cross-neutralisation of DENV was maintained in 5 of 7 antisera

from 6 individuals with known previous DENV infection or who had lived in

dengue-endemic countries into adulthood before migrating to Western Australia.

Then 2 individuals in whom anti-DENV titres were not maintained (FLV032,

ZIKV01) were travellers who returned to WA from countries where ZIKV was

known to be circulating and who presented soon after with febrile illness, ZIKV

RNA was detected in one of their acute phase samples, and anti-ZIKV IgM was

detected in the other. Neither had a history of previous DENV infection. Samples

from all individuals neutralised DENV in the first moth after presentation.

NAb responses against YFV17D were maintained in vaccine recipients, with the

exception of FLV-032 whose anti-YFV titres had declined to undetectable levels by

269

day 37. She received her YF vaccine in Australia in October 2015, soon before

traveling to Columbia.

c. More than 12 months post presentation

Sera from six individuals were available more than one year after presentation (390 –

365 days). Five of the 6 were sampled at earlier time points.

Anti-ZIKV MR 766 FRNT50 GMT decreased from 473 (1-12 months post

presentation) to 248 and anti -ZIKV PRVABC59 GMT declined from 128 to 58

(Figure 6.4).

Serum from 3 of the 6 individuals (FLV087; FLV088; FLV089) cross-neutralised

DENV-4 with FRNT50 ranging from maximum >640 to lowest279; similarly,

FRNT90 ranged from >640-49. FLV087 was previously infected with DENV,

FLV088 and FLV089 had both lived in El Salvador for most of their lives before

migrating to WA as adults and although they do not recall being diagnosed with

dengue while living there, previous DENV infection in the endemic country is not

unlikely.

Antisera from the remaining 3 individuals (FLV064; FLV032; FLV090) did not

cross-neutralise DENV, and all 3 were never previously diagnosed with dengue and

had lived in WA all their lives. FLV064 was only sampled 605 days after presenting

with a febrile illness after returning from Indonesia, and ZIKV RNA was detected in

his urine.

Persistence of anti-ZIKV neutralising antibody in history of flavivirus exposure is

illustrated by heat map Figure 6.5 (A, B, and C).

270

Study ID

Color code

FLV-087

ZIKV 01

ZIKV 02

FLV-032

FLV-088

FLV-089

FLV-090

FLV-091

ZIKV 03

ZIKV 04

ZIKV 06

ZIKV 07

ZIKV 09

ZIKV011

Figure 6. 2:-ZIKV neutralisation within 30 days post presentation of illness.

271

Study ID Color code

FLV-087

FLV-093

ZIKV 01

FLV-032

FLV-091

ZIKV 03

Figure 6. 3:- ZIKV neutralisation 1-12 months post presentation of illness.

272

Study ID Color code

FLV-064

FLV-087

FLV-032

FLV-088

FLV-089

FLV-090

Figure 6. 4: ZIKV neutralisation more than 12 months post presentation of illness.

273

Figure 6. 5:-Variation in ZIKV neutralising antibody concentration over time based on the flavivirus background

Values on heat map represent antibody titre at respective days post infection. After one year post infection neutralisation of ZIKV in the

absence of previous flavivirus background was significantly lower than in the presence of anti-flavivirus background.

(A) Anti-ZIKV NAb from patients with ZIKV only infection 4 patients at different time points were tested (B) Anti-ZIKV NAb from

patients with DENV cross reactivity, one of the patient has confirmed DENV previous infection, while rest has DENV NAb when tested

at the given time point but no record of past DENV infection (C) NAb from patients with known history of YF 17D vaccination. NAb titre

was maintained high in individual with flavivirus exposure in compare to flavivirus naïve population.

(B) (C) ZIKV with DENV ZIKV with YF vaccination

(A) ZIKV only

Stu

dy n

o. (D

ays

post

in

fect

ion

)

(A) ZIKV only

6 4 0

6 4 0

6 4 0

6 4 0

1 7 6

6 1 9

6 4 0

6 4 0

2 3 5

3 7 9

3 2 2

3 8 1

6 4 0

2 5 2

3 4 9

2 7 5

3 8

3 0

8 5

2 2 3

1 0 5

1 1 6

4 8

1 2 9

ZIK

V M

R766

ZIK

V P

RV

AB

C59

ZIK V 0 1 (< 3 0 )

ZIK V 0 3 (< 3 0 )

F L V 0 9 1 (< 3 0 )

F L V 0 9 1 (3 )

F L V 0 3 2 (< 3 0 )

F L V 0 3 2 (7 )

F L V 0 3 2 (3 7 )

ZIK V 0 1 (2 1 0 )

F L V 0 9 1 (2 4 0 )

F L V 0 3 2 (2 4 0 )

F L V 0 9 3 (3 3 0 )

F L V 0 3 2 (3 9 0 )

2 0 0

4 0 0

6 0 0

274

6.3.2. Total anti-ZIKV antibody

Total anti-ZIKV antibody (TAb) was assessed by Hemagglutination inhibition (HI) test

to assess magnitude of responses against ZIKV MR 766 and ZIKV PRVABC59.

Sera from 14 of 16 individuals were assessed at least once post presentation. Among 4

Zika confirmed cases, 2 demonstrated a >4-fold difference in HI titres between the

strains. In Zika probable cases, 3 of 9 demonstrated a differential HI pattern. All four

showed HI titre against MR766 >4-fold greater compared to PRVABC59. Decline in HI

titres against both viruses with progression of time post infection was observed in

patients in whom consecutive samples were available.

Furthermore, high HI titre (>100) against both ZIKV MR 766 and ZIKV PRVABC59

was observed in individuals with previous DENV infection whereas HI titre was <100,

against ZIKV PRVABC59, in individuals with no history of previous DENV infection,

at the same timepoint after presentation (Table 6.2). ZIKV PRVABC59 is therefore a

more specific target for anti-ZIKV antisera induced by contemporaneous ZIKV

infection.

275

Case definition Patient ID Number of visits Days post diagnosis HI titre

MR766 PRVABC59

Confirmed FLV-064 1 605 80 80

FLV-087 4 0 NA NA

12 40960 10240

39 2560 2560

425 1280 1280

ZIKV 01 2 0 <10 <10

210 640 640

ZIKV 02 2 0 80 320

11 320 2560

Probable FLV-032 6 0 NA NA

7 NA NA

37 320 320

90 320 320

240 160 160

390 160 160

FLV-088 3 0 NA NA

23 10240 1280

425 1280 1280

FLV-089 3 0 20480 640

23 10240 640

425 1280 1280

FLV-090 2 0 320 80

425 1280 1280

FLV-091 3 0 NA NA

3 NA NA

240 640 1280

FLV-092 1 330 640 640

ZIKV 03 2 0 10240 2560

38 2560 640

ZIKV 04 1 0 320 320

ZIKV 05 1 0 >40960 20480

ZIKV 06 1 0 NA NA

ZIKV 07 1 0 NA NA

ZIKV 08 1 0 NA NA

ZIKV 09 1 0 80 80

ZIKV 010 1 0 NA NA

ZIKV 011 1 0 NA NA

NA: - “Not Available” due to insufficient sample

Table 6. 2: - Total antibody titre against ZIKV

276

6.3.3. Immunoblot analysis, ZIKV and DENV

The high degree of amino acid similarity among flaviviruses, including is preliminary

study to possible differential serological diagnosis post DENV and ZIKV infection. In

this study antisera from WA travellers with monotypic DENV or ZIKV infection was

applied as probe to identify viral protein targets in Western immunoblot analysis and to

assess whether a specific marker for ZIKV infection could be identified. Two ZIKV,

ZIKV MR766 and ZIKV PRVABC59; four DENV serotype reference viruses (DENV-1

HW2001G4, DENV-2-NGCAII, DENV-3-H87G5 and DENV-4-H241GI) and YF17D

were used to identify possible virus-specific binding sites.

Viral protein preparation and determination of binding sites is described in detail in

Chapter 3. Briefly, virus whole cell lysates were prepared from infected Vero cells.

Infected monolayers were scraped with cell scrappers and fixed with 0.025%

glutaraldehyde. Fixed infected cells were sonicated to prepare whole virus lysate. Cell

lysate for mock infections was prepared in a similar way as virus-infected cells. Lysates

were quantified for protein content using bicinchoninic acid assay (BCA assay). Equal

quantities (20ug/ul) of crude protein cell lysate were denatured with loading buffer (5X

laemmili sample buffer) at 950C and then subjected to 12% SDS-PAGE, followed by

transfer to nitrocellulose membrane. After transfer membrane was blocked with 5%

non-fat milk in PBST (PBS with 0.05% Tween 20, pH 7.4). Membranes were then

probed overnight with antisera as primary antibody, diluted in blocking buffer. Titration

of the correct serum dilution was determined by dot blot assay. Anti-human antibody

labelled with horseradish peroxidase was used as secondary antibody, incubated with

ECL substrate and visualised using FujiFilm Imager 3000.

277

Protein band position was visualised with Coomassie stained gels. Band size was

determined with reference to 15-250 protein ladder. Based on published data (358-361)

a band between 80-46KDa was identified as E-protein fraction and between 22-11KDa

as capsid/prM protein. No purification of protein lysate or primary antibody was done

and therefore background was high.

Two antisera from individuals (FLV064; FLV090) with confirmed ZIKV infection and

with no other detectable anti-flavivirus antibody (no vaccination and no

DENV/YF17D/IMOJEV neutralisation); one sample from an individual with previous

ZIKV and DENV infection (FLV087); one sample from an individual with ZIKV

infection, YF17D vaccination, no DENV cross-neutralisation (FLV032) and 2 samples

with monotypic DENV infection and no ZIKV cross-neutralisation (FLV030 and

FLV002) were assessed for their capacity to bind to flavivirus proteins. The flavivirus-

specific monoclonal antibody 4G2 was included as Positive control that recognises E

protein; pooled flavivirus seronegative sera served as Negative control. Presence of

protein bands was confirmed by Coomassie stain (Figure 6.6).

6.3.3.1. Positive and negative control

Purified 4G2 bound to ZIKV and DENV; a clear band at 80-56KDa was seen and there

was no binding with mock lysate (Figure 6.7 A and B).

Negative Control produced faint bands at 80KDa and numerous faint bands were seen

with the mock lysate sample.

6.3.3.2. Anti-ZIKV antisera

A band at capsid/prM protein at 22-11KDa was specific to ZIKV only and was present

for both ZIKV MR766 and ZIKV PRVABC59. In addition to this band the 80-56KDa

E-protein was observed for all the flavivirus lysates (Figure 6.8 A and B). E-protein

278

binding was seen in the mock lysate and this might be due to addition of FBS during

lysate preparation (this has been widely reported in Immunoblotting discussion sites and

in Immunoblot kit manufacturer FAQ sites); no binding to mock lysate was seen in dot

blots.

6.3.3.3. Anti-ZIKV/DENV antisera

The 22-11 KDa band specific to ZIKV capsid/prM protein was present in ZIKV lysates

alone, along with high intensity 80-56KDa E protein for all viruses (Figure 6.9).

6.3.3.4. Anti- ZIKV/YF17D

No WB bands for any viral proteins could be identified in multiple trials. The same

batch and amount of protein was used, as for other assays, but no specific bands were

seen. Binding did occur in dot blot analysis where spots were obtained at 1:50 plasma

dilution however when the same dilution was used to develop the post-transfer

membrane no bands appeared (Figure 6.10). This might be due to lack of primary

antibody binding to separated protein fragments.

The individual from whom this antiserum was obtained (FLV032) clearly developed

anti-ZIKV neutralising antibody on her return from travel to Columbia and was

vaccinated against yellow fever before she left Australia. Serum collected at her first

visit neutralised YF17D, but subsequent samples collected 7, 37, 90, 240, and 390 days

after her first visit did not. It is possible that WB is not sufficiently sensitive in this

individual’s case.

6.3.3.5. Anti-DENV

Anti-DENV-3 and DENV-4 antisera from 2 travellers with monotypic DENV-3 or

DENV-4, respectively, did not cross-neutralise ZIKV MR766 or ZIKV PRVABC59, or

heterologous DENV.

279

Anti-DENV-4 specifically bound to DENV-4 specific capsid/prM only. Faint bands

were seen across E-protein. Anti-DENV-3 sample showed a faint band at 22-11KDa

specific to DENV-3 despite the high degree of background non-specific binding in this

immunoblot (Figure 6.11).

280

Figure 6. 6:-Coomassie stain.

Coomassie stain (CS) SDS-PAGE shows lysate position after

electrophoresis

281

Figure 6. 7:-Positive and Negative control

(A) Immunoblot of purified 4G2 monoclonal antibody against DENV/ZIKV and mock lysate.

Bands produced after Coomassie stain are shown. A specific E protein band at 80-56KDa was

seen, with no non-specific binding to mock vero cell lysate. (B) Immunoblot of Negative Control

(pool of flavivirus-seronegative sera). A band at 80-46KDa was visible for mock lysate and a few

other faint bands are present.

B

A

282

Figure 6. 8:-Monotypic anti-ZIKV antisera (FLV-064/FLV090)

ZIKV-specific prM/Capsid protein band at 22-11KDa in monotypic anti-ZIKV antisera. E

protein band at 80-46KDa seen across all virus lysates. Variation in band intensity is apparent.

Dot blot at varying plasma dilution (1:50/1:100/1:200) against protein lysate ZKM (ZIKV

MR766), ZKP (ZIKV PRVABC59), DENV 1-4 (D1-D4), YF17D(YF) and Vero mock lysate

(V).

Dot blot for Plasma

titration

A

283

Figure 6. 9:- Anti-ZIKV/DENV antisera (FLV-087)

ZIKV-specific prM/Capsid protein band was visible. E protein band was

more intense than for anti-ZIKV or anti-DENV alone. Dot blot at varying

plasma dilution (1:50/1:100/1:200) against protein lysate ZKM (ZIKV

MR766), ZKP (ZIKV PRVABC59), DENV 1-4 (D1-D4), YF17D(YF) and

Vero mock lysate (V).

Dot blot plasma titration

284

Dot blot for 1o Ab titration

Figure 6.10:-Anti-ZIKV/YFV antisera (FLV-032)

A) No bands were visible in immunoblot of FLV0321 antisera, although binding was seen in dot

blot analysis using the same lysate.

B) (B) Dot blot at varying plasma dilution (1:50/1:100/1:200) against protein lysate ZKM (ZIKV

MR766), ZKP (ZIKV PRVABC59), DENV 1-4 (D1-D4), YF17D(YF) and Vero mock lysate

(V). Distinct binding at 1:50 dilution for all lysates, except mock, was obtained. It is possible

that WB is not sufficiently sensitive in this individual’s case.


285

Figure 6. 11:- Anti-DENV antisera(FLV002/FLV030)

a)Anti-DENV-4, dot blot and immunoblot. Distinct band at 22-11KDa corresponding to

prM/Capsid protein specific to DENV-4 is seen as is faint E protein band across DENV 1-4.

b) Very faint band at DENV-3 prM/Capsid protein (visible only with high contrast). Dot blot at

varying plasma dilution (1:50/1:100/1:200) against protein lysate ZKM (ZIKV MR766), ZKP

(ZIKV PRVABC59), DENV 1-4 (D1-D4), YF17D(YF) and Vero mock lysate (V).


286


In this Chapter, acute and convalescent anti-ZIKV total and neutralising antibody

responses following presentation for Zika-like illness were assessed. Cross-

neutralisation was serogroup-specific. In the absence of previous DENV infection

convalescent anti-ZIKV antisera do not necessarily cross-neutralize or cross-recognize

DENV, and DENV was not cross-neutralised by anti-ZIKV antisera from individuals

who had received YFV vaccination. The magnitude of anti-ZIKV NAb responses

differentially declined with time: responses in individual with previous DENV

infection declined slowly while responses in individuals with no previous infection

declined to very low magnitude after 1 year, to titres considered negative according to

WHO criteria for ZIKV laboratory diagnosis. ZIKV MR766 and ZIKVPRVABC59

were differentially neutralised and this finding highlighted the importance of strain

selection in ZIKV diagnostic and research approaches.

6.4.1. Anti-ZIKV specificity and cross-reactivity

Sero diagnosis of ZIKV infection in dengue endemic areas is confounded by cross-

recognition of DENV antigens by anti-ZIKV antibodies. ZIKV-infected travellers who

are not from dengue-endemic areas allow investigation of sensitivity and specificity of

humoral immune responses in ZIKV infection. Cases of travel associated ZIKV

infection has been studied before. The first Zika case in Australia was reported was in a

previously healthy 52-year old Australian woman who became ill after returning from

Jarkarta (322). She was provisionally diagnosed with dengue but following a negative

dengue-specific RT-PCR test, sequencing of flavivirus-specific PCR product identified

the infecting virus as ZIKV. Prior to this, in 2008 ZIKV was first reported in USA in a

scientist returning from Senegal (361). This was also the first report of possible sexual

transmission of ZIKV. Leung et al proposed that an animal bite was a plausible route of

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transmission in the case of Zika in a traveller returning to Australia from Indonesia,

where he was bitten by a monkey (362). However, the authors did not consider the

possibility that ZIKV was transmitted to this individual via an infected mosquito and

that ZIKV was present in Indonesia but not yet identified in humans. Other cases of

ZIKV imported to countries without known ZIKV circulation as listed in WHO reports

include Germany (2013, 2014), Canada (2014), Japan (2014), and Finland (2015) (363).

Although many traveller-associated cases have been reported there are no analyses

available from these presumably monotypic ZIKV infections regarding serum anti-

ZIKV antibody responses against DENV in vitro. Analysis of human anti-ZIKV

monoclonal antibodies (mAbs) has been reported; Wang et al (2017) described 460

mAbs directed to E-protein and compared neutralisation mechanisms and potential viral

epitopes to obtain insights for possible therapeutic approaches(364). Steller et al

isolated a panel of 119 mAbs from epidemic plasma samples, assessed their specificity

for ZIKV, and showed that the most potent neutralising antibodies were targeted against

ZIKV EDIII and NS1 protein. ZIKV EDIII monoclonal specificity has also been

reported in other studies (365).

In this study, serum samples collected from travellers with monotypic ZIKV infection

or ZIKV infection with known or likely previous DENV infection and/or YFV

vaccination were assessed for cross-neutralization and cross-recognition of DENV,

YFV and JEV, and ZIKV strain-specific responses were also examined. Using

polyclonal human sera allowed analysis of the natural history of ZIKV-specific humoral

immune responses. A major observation was that specificity of ZIKV-specific

neutralisation and binding is time- and flavivirus background-dependent. Anti-ZIKV

cross-neutralised DENV, YF17D, and JEV in early acute phase. Responses restricted

with time to become more ZIKV-specific, in the absence previous flavivirus exposure.

This pattern was patient-specific; certain individuals who had been immunised against

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YFV maintained ZIKV-specific NAb even as their YFV-specific responses declined. A

second major observation was the dramatic decline in anti-ZIKV NAb, to titres

considered negative for ZIKV infection, more than one year after monotypic ZIKV

infection.

6.4.2. Anti-ZIKV antisera cross-neutralisation of DENV is time- and flavivirus

background-dependent

The capacity of anti-DENV antisera to cross-neutralise and cross-recognise ZIKV has

been assessed, but the capacity of anti-ZIKV antisera to cross-neutralise and cross-

recognise DENV and other flaviviruses, particularly the vaccine viruses, is not

understood. A major finding in the present study is that anti-ZIKV antisera

neutralisation and recognition of DENV is highly patient- and time-specific. Among

confirmed Zika cases cross-neutralisation against DENV was seen only in individuals

with known or likely DENV infection and similarly, cross-reactivity against YF17D

was seen only in the context of known YF vaccination history. Epitope-specific

neutralisation potency of anti-ZIKV antibody is a possible explanation for the varying

neutralisation pattern observed among our cohort. Anti-ZIKV antibody directed towards

DENV E-protein DI/DII was shown to be poorly neutralising while antibody towards

EDIII and NS1 was highly specific (365). Among 400 donors from dengue epidemic

areas of Mexico and Brazil, ZIKV and DENV neutralisation potencies varied over 2

logs. B cell clones expressed E-protein specific to ZIKV and DENV-1 neutralising

antibody in 4 out of 6 individuals (258). Analysis of the ZIKV-specific B cell repertoire

is the target of research in the WA Traveller cohort and will be based on data generated

in the current study.

In summary the present study in WA Travellers showed that convalescent anti-ZIKV

antibody response after monotypic ZIKV infection is specific to ZIKV. DENV cross-

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neutralisation occurred in the context of previous DENV infection and was limited to

the DENV serocomplex; no cross reactivity against YFV or JEV was measured.

6.4.3. Persistence of anti-ZIKV NAb in individuals with previous flavivirus infection

High magnitude (FRNT50 >640) anti-ZIKV NAb responses were present in all

individuals in acute infection. In convalescence, ~13-14 months post presentation, high

magnitude responses against ZIKV MR766 and ZIKV PRVABC59 were measured for

antisera from individuals with YFV vaccination or previous/likely DENV infection

whereas in monotypic ZIKV infection FRNT50 titres were low (82 and ~40 respectively

against ZIKV MR766 and ZIKV PRVABC59) at 425 days for patient FLV090 and

similarly low titres for patient FLV064 (65 and 23 against ZIKV MR766 and ZIKV

PRVABC59, respectively) at 605 days. Anti-DENV-1 antibody has been proposed to

prime subsequent ZIKV infection resulting higher neutralisation, compared to a naïve

ZIKV population (258) in a mouse model.

In summary this study demonstrates that previous flavivirus exposure prolongs the anti-

ZIKV antibody response, whereas in monotypic ZIKV infection anti-ZIKV NAb titres

decline to levels considered to be negative.

6.4.4. Differential ZIKV MR766 and anti-ZIKV PRVABC59 neutralisation

Differential neutralisation against ZIKV MR766 and anti-ZIKV PRVABC59 by anti-

ZIKV antisera was observed. The majority of antisera (89%) neutralised ZIKV MR766

at high magnitude (FRNT50>640) while a minority (< 20%) showed FRNT50>640

against ZIKV PRVABC59. No individuals had undetectable neutralisation against

ZIKV MR766 (by the stringent WHO criteria for ZIKV serodiagnosis FRNT90<40)

while most did not neutralise ZIKV PRVABC59 (FRNT90<40) even during the acute

phase. To assess potential assay bias traditional PRNT was also performed for these

samples and confirmed the pattern of strain-specific neutralisation.

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Total anti-ZIKV antibody assessed by HI test showed that a subset of antisera

differentially recognised the 2 ZIKV strains, at least 4-fold, indicating the presence of

cross-recognising but not necessarily cross-neutralising antibody elicited after ZIKV

infection. A study comparing four strains of ZIKV reported ~6-fold higher

neutralisation against ZIKV MR766 compared to ZIKV PRVABC59(366), similar to

the results of the present study. Differential neutralisation by DENV immune sera

against ZIKV HD78788 and PF13 has been reported (260). It has also been shown that

not all mAbs equally neutralise all ZIKV strains. On evaluation of 6 mAbs, 4 out of 6

showed higher FRNT 50 for African strain compared to Asian strain; 3of 6 had more

than 4-fold difference in FRNT titre (367). Phenotypic difference between Asian (CPC-

0740, SV0127-14) and African (ArD 41525) lineages of ZIKV were compared for vivo

and vitro phenotypic difference and it was reported that African isolates at low passage

had higher infection and dissemination rates compared with Asian isolates (368). In

contrast, a study on neutralisation of ZIKV MR766 and ZIKV (H/PF/2013) French

Polynesian strain by sera or plasma samples from eight ZIKV-infected individuals

showed that both ZIKV strains showed similar (< 3-fold) sensitivity to neutralisation

(369).

Identification of conserved amino acid residues that differentiate the Asian strain,

including American strain, from the ancient African strains has been reported (370).

Glycosylation in the envelope (E) protein has been suggested to contribute to

dominance of the Asian lineage in the recent Oceania epidemics (131). The recent Asian

epidemic virus contains an N-Linked glycosylation signal, whereas the majority of other

strains do not, suggesting a potential role for glycosylation in ZIKV disease severity

(371). Addition of 12 nucleotides in E, in the Asiatic lineage from the Yap ZIKV 2007

outbreak is also believed to be noteworthy as 4 E amino acids corresponding to an E

145 glycosylation motif described in flaviviruses has been associated with virulence

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(131). Another candidate for neutralisation resistance among flaviviruses is substitution

in E Ser279 (372).

Differential cross neutralisation among the two ZIKV strains MR766 and PRVABC59

measured in this study may be a consequence of genetic variation among ZIKV strains

or differing patterns of NAb recognition of ZIKV replicating in Vero cells; maturation

of virus may also affect neutralisation. The membrane protein (prM) content of virions

may contribute to cell-type-dependent patterns of antibody neutralization (369, 373-

375).

Further epitope analysis and antibody repertoire analysis, beyond the scope of the

present study, will be undertaken as a future research to investigate differential ZIKV-

strain-specific antibody responses.

6.4.5. Virus protein binding analysis by immunoblot

The most common flavivirus antigenic target, the E protein, has more than 50% amino

acid homology between DENV and ZIKV. The E protein plays a central role in virus

entry and is major target of neutralising antibodies and therefore of vaccine

development. Other potential differential binding sites include prM and NS1 protein

(376-378).

This study aimed to assess if it was possible to differentiate DENV ZIKV binding sites

recognised by anti-ZIKV and anti-DENV polyclonal antisera. Dot blot followed by

Western immunoblot was undertaken to identify antisera binding sites against flavivirus

lysates. The 2 methods did not always align - non-specific binding was not seen against

mock cell lysate for any of the test sample in dot blot analysis while bands were visible

in mock cell lysate when electrophoresed through SDS-PAGE and blotted. Furthermore,

two anti-DENV antisera did not show any binding to ZIKV protein lysate in dot blot but

bands were seen WB after SDS-PAGE.

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Antisera from two confirmed monotypic ZIKV infections (FLV-064/FLV090) bound to

ZIKV capsid/prM protein in both strains. E protein bands were seen for all flaviviruses.

Intensity of ZIKV-specific band at 22-11KDa varied for different antisera although the

amount of protein lysate was consistent throughout. Similarly, ZIKV capsid/prM protein

binding alone was seen in antisera from an individual with ZIKV infection and with

history of DENV infection (FLV-087); the E-protein band was of high intensity. This

individual’s DENV infection was thought to occur in 1990, 25 years before her ZIKV

infection. Anti-DENV NAb titres ranged from 46 – 640 across the four serotypes; lack

of anti-DENV antibody binding to DENV lysates may be related to assay sensitivity.

In summary this study demonstrated that antibody binding to ZIKV capsid/prM protein

can differentiate recent ZIKV infection, in individuals with previous DENV infection.

There are no reports of ZIKV capsid/prM protein-specific binding in assessment of Zika

serodiagnosis.

Differential binding to other proteins has been demonstrated. NS1 was shown to

distinguish between serogroups (DENV and WNV) (359); anti-prM antibody cross-

reacted among viruses of the same serogroup (WNV and SLEV). In the current

immunoblot system, NS1 was not visualised. Cardosa and others have shown that prM

recognition may serologically differentiate individuals infected with JEV and DENV

(376).

In the present study all antisera bound to all viral E proteins, with varying intensity.

Highly cross-reactive antibodies induced by flavivirus infection recognise heterologous

E proteins; cross-reactive E-protein was shown in the analysis of WNV, SLEV and

DENV (359) and thus recognition of E is not useful for differentiation of infecting

flavivirus.

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Validation of the preliminary finding that ZIKV capsid/prMprotein binding can

discriminate recent ZIKV infection in the setting of previous DENV infection is

ongoing. Technical enhancements include protein (virus lysate; primary antibody)

purification. This study provides a baseline from which to explore ZIKV and DENV

protein-specific serodiagnostic approaches.


Differential neutralisation between the prototype and recent strains of ZIKV was

seen.

Anti-ZIKV cross-reactivity against DENV was seen only in acute, past DENV

exposure and individuals with history of living or frequent travel to DENV

endemic areas.

Not all post ZIKV immune response is cross reactive to DENV or other

flavivirus vaccine strain.

Persistence anti- ZIKV NAb was observed to be associated with the history of

past heterologous flavivirus-specific immunity.

Possible capsid/prM protein based serological differentiation between DENV

and ZIKV.

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Chapter 7: - Enhancement of ZIKV infection by monotypic anti-DENV antibody

7.1. Preamble

This chapter assessed the capacity for anti-DENV antibodies to enhance ZIKV

infection. Anti-DENV antisera were obtained from WA travellers with well-defined

monotypic DENV infection with all 4 serotypes. Acute and convalescent antisera were

analysed. Pools of monotypic sera representing each DENV serotype were included to

emulate ZIKV infection in populations with hyperendemic DENV transmission. The

potential risk of developing severe dengue, for WA travellers with monotypic DENV

infection and who may be reinfected during subsequent travel is not understood.

Previous studies of ZIKV enhancement have been based in dengue endemic countries

with high seroprevalence and where the sequence of infections is not known, whereas

our study population allowed pre-existing DENV immunity to be defined.

7.2 Introduction

The humoral immune response to flavivirus infection is influenced by the presence of

pre-existing antibody to related flaviviruses. Suboptimal levels of neutralizing antibody

lead to poor viral clearance and these non-neutralizing antibodies can enhance the viral

replication thereby increasing disease severity (379). However, if the cross-neutralizing

antibodies are able to block infection this results in cross-protection. Thus, humoral

immune responses in consecutive flavivirus infection have two possible outcomes: -

protection or increased disease severity. The best studied mechanism is antibody

dependent enhancement (ADE).

ADE is an immunological complication reported in several viral infections including

DENV, HIV, Influenza A, Coxsackie virus B, RSV, Ebola and also in also in protozoan

parasites like Leshmania (380). ADE also has been shown to be associated with

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demographic and virulence factors such as age, sex, origin of population, past exposure

to infection and infecting strains (381).

7.2.1. Mechanism of ADE

ADE is based on the theory that the presence of non-neutralising antibody levels results

in opsonisation of immune complexes via Fc receptor (252). Ligation of monocyte or

macrophage with FcR receptor leads to biased immune responses enhancing

replication of infectious particles (247). Waning of primary anti-DENV Abs to the level

no longer sufficient to neutralise infection is thought to induce FcR-mediated

opsonisation. Molecular studies demonstrate FcR-mediated endocytosis triggers

cytoplasmic immuno-tyrosine activation motifs resulting in biased T-helper-1 (TH1)

response to TH-2 cells response resulting in increased infection. In THP-1 cells, after

ligation of FcRIa and FcRIIa, entry of infectious immune complexes activates

expression of the negative regulators dihydroxyaetone kinase and autophagy proteins

(cascade of down-regulatory pathways) that results in decreased production of type I

interferon-activated antiviral molecules supressing capacity of macrophage antiviral

mechanisms hence enhancing viral replication (247). Halstead et al have described two

principles of ADE: extrinsic ADE which results in FcR mediated entry of virus, and

intrinsic ADE that modifies innate and adaptive intracellular antiviral mechanisms and

enhances replication (247). Several studies have shown cross-linking of immune

complexes with FcR receptors increases cellular infectivity and results in disease

severity in dengue.

7.2.2. ADE in DENV and ZIKV infection

Anti-DENV antibodies induced in primary infection do not protect against secondary

infection with heterologous DENV (382, 383). Indeed, epidemiological observations

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indicate that secondary infection is associated with more severe disease. ADE has been

proposed as a mechanism for disease severity in secondary infection with heterologous

DENV serotypes (384, 385). ADE has been demonstrated in vitro in FcR-receptor

bearing cell lines including U937, K562 and THP-1 (253, 254) and in vivo in non-

human primates (386).

During a 1981 DENV-2 epidemic in Cuba with 300,000 cases, 10,000 severe cases and

98% of fatalities occurred in individuals with secondary infection (249, 387). DHF in

infants during the first year of life is also associated with ADE (388). A strong

correlation between passively transferred maternal neutralizing antibody and DHF in

infants has been reported (389). Maternal antibodies are protective during the first few

months, however waning antibody titres to non-neutralizing concentrations is proposed

to induce enhancement of DENV infection in the infant (389). In a study of 20 infants

infected with DENV-2 below age of 1, 8 showed DHF/DSS and the predominance

antibody found in mothers was against DENV-1 (390). Another study done in 168

children with acute dengue aged 18 months to 14 years reported that patients with

secondary antibody responses to multiple DENV serotypes were twice likely to have

severe dengue compared to those with single infection (391). A well-characterized

paediatric cohort (2-14 years) study in Nicaragua showed that risk of severe dengue

disease is within a narrow range of pre-existing DENV antibody titres. This study shows

the risk of severe dengue in DENV-naïve children and children with high anti-DENV

antibody titres (>1:1280) was similar, while presence of 1:21 to 1:80 pre-existing

DENV-Ab increased hazard risk by 7.64-fold. Furthermore, in this paediatric cohort the

DENV-Ab half-life was estimated as 4 years, and by 3 years the decrease in Ab titre to

levels that were suggested to have exacerbated disease among 22% of children (381).

Due to these immunological complications, production of a vaccine that provides

equivalent protective immunity against all four DENV serotypes and thus does not

297

increase the risk of severe disease is a major challenge. The first dengue licensed

vaccine, Dengvaxia® (CYD-TDV) developed by Sanofi Pasteur has been licensed for

use in individuals 9-45 years living in endemic areas. Phase-III trials across Asia and

Latin America demonstrated effective immune responses for the first 25 months post

dose I while long-term follow-up raised concerns about risk of hospitalization. Among

30,000 participants aged 2-16 years vaccine efficacy against primary series was 59.2%

with 79.1% protection against severe dengue. Efficacy was questioned after

hospitalization and severe dengue in vaccine recipients, with elevated risk of

hospitalized dengue in 2-5-year olds. Vaccine efficacy in DENV seropositive

individuals at baseline was 78.2% and in individuals seronegative at baseline was

38.1% (392). Concerns have been raised that individuals of any age with no prior

dengue exposure could be at risk of hospitalization when infected with dengue post

vaccine. Even though Dengvaxia has the potential to reduce the burden of disease in

endemic areas use of the vaccine should be limited to individuals who have at least one

dengue virus infection and a simple test such as DENV-IgG was suggested to identify

seropositive individuals to be vaccinated (393, 394).

Recent studies have shown a high degree of similarity in DENV and ZIKV E-protein

structure. Dai et al (2016) reported structure of ZIKV-E at 2.0 Å resembles all the

known flaviviruses E-structure and has a unique positively charged patch adjacent to the

fusion loop region (355). Similarly, another study at 3.8 Å resolutions supports the

similarity between ZIKV and other Flaviviruses E-protein and reports ~10 amino acid

difference in E-glycoprotein that forms the icosahedral shell (395).

ZIKV shares 55.6% amino acid sequence identity with DENV, 46% with yellow fever

virus, 56.1% with Japanese encephalitis virus and 57% with West Nile virus (139).

54% E-protein sequence identity has been reported between DENV-2 and ZIKV (357).

Amino acid identity in DI, DII and DIII of E-protein of 35%, 51% and 29% between

298

ZIKV and DENV has been shown (365). This similarity is sufficient to allow cross-

reactive immune responses between DENV and ZIKV. Anti-E antibodies in DENV are

neutralising antibodies which also proposed to enhance infection in heterotypic

secondary infection. Possible cross-reactivity between anti-DENV and ZIKV has posed

significant hurdles in understanding pathogenic outcomes in populations with DENV

immune backgrounds. With the several reports of possible ZIKV enhancement in the

presence of pre-existing anti-DENV antibody, this study assessed possible potential

complications for WA travellers with history of DENV infection who may be

subsequently infected with ZIKV.

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7.3. Results

U937 cell line (ATCC® CRL-1593.2™) was used as FcR receptor bearing cells to

detect enhancement of infection. This cell line is non-permissive to ZIKV. However, in

the presence anti-flavivirus antibody ZIKV has been reported to enter and replicate.

In this study, ZIKV PRVABC59 (Asian strain) enhancement by DENV antisera

obtained at different time points after presentation with febrile illness was tested.

Enhancement of ZIKV replication was determined by virus output detected by plaque

assay on Vero cell line. Virus production (as pfu/ml) for test antisera was compared to

virus production in the presence of the flavivirus-specific mAb 4G2 (Positive control) or

flavivirus-seronegative pooled sera (Negative control).

Mann-Whitney non-parametric t-test was used to define statistical significance of the

virus yield in terms of p-value. Difference in virus yield at different dilution for each

serum sample was compared with virus yield with positive and negative controls. The p-

values thus derived give the significance of difference (p<0.01) in enhancement. The

plasma sample with statistically significant difference from negative and insignificant

difference from positive (showing same enhancement as positive) is considered as

enhancing. Samples statistically closer to negative (insignificant p-value) are considered

non-enhancing. This statistical analysis was done to confirm enhancement as viral

output was not high compared with positive control (396-399).

Fit spline/LOWESS was used to generate curves to represent change in pfu/ml at

different plasma dilutions. Statistical analysis was done using GraphPad Prism 7.0.

Virus output from U937 cells after exposure of ZIKV in the presence of anti-DENV

antisera is listed in Table 7.2.

Six different combinations of monotypic anti-DENV antisera, and positive and negative

controls are listed in table 7.1.

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Table 7. 1:- Description of plasma samples used in ADE

Sample group Description Infecting serotype/

Study number (MPI)

Sample ID

used in ADE

Control 4G2 monoclonal antibody from

hybridoma cell line as positive

control

4G2

Pool of three flavivirus negative

plasma samples as negative control

Negative

DENV acute plasma i) pool of plasma samples less than

two months post infection

DENV-1:-FLV-011

0.3), FLV-017(2), FLV-

018(2)

DENV-2 -FLV-012(0.1)

DENV <2

MPI

ii) Plasma sample of one of the acute

patient

FLV-018 (2) DENV-1-018

DENV convalescent

plasma pool

pool of plasma sample at 1-6 years

post infection

DENV-1 FLV 018 (31)

/FLV 001 (60)

DENV-2- FLV 014

(36)/ FLV004 (51)

DENV-3-FLV 029 (12),

FLV 030 (12)

DENV-4 -FLV 049 (14)

FLV 002 (60)

DENV >12

MPI

DENV-1 plasma DENV-1 plasma post 12 months

post infection

FLV-022 (14)

FLV-026-(14)

DENV-1-022

DENV-1-026

DENV-1 plasma sample at 6 months

post infection

FLV-076(6)

FLV- 060(6)

DENV-1-076

DENV-1- 060

DENV-3 plasma DENV-3 plasma post 12 months

post infection

FLV-038(14)

FLV-037(14)

DENV-3-038

DENV-3-037

DENV-3 plasma at 6 months post

infection

FLV-050(6)

FLV-056(6)

DENV-3-050

DENV-3-056

Pooled sera from

DENV-1 and 3

patients

Pool of DENV-1 and DENV-3 six

months post infection

FLV-076/FLV- 060+

FLV-050, FLV-056

DENV 1&3 (6

MPI)

Pooled sera from

DENV-1 and 3

patients

Pool of DENV-1 and DENV-3 one-

year post infection

FLV-022/FLV-

026+FLV-038, FLV-

037

DENV 1&3

(12 MPI)

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Table 7. 2:- Virus output from U937 post exposure to respective anti-DENV - ZIKV immune complex

Sample group Sample ID* Virus yield at plasma dilution (pfu/ml) Average

pfu/ml

p-value

compared

with

positive

control

p-value

compared

with

negative

control 10^1 10^2 10^3 10^4 10^5 10^6

Controls 4G2 monoclonal (+ve) 7.50 x10^6 2.20 x10^6 9.00 x10^5 4.00 x10^5 1.00 x10^5 5.00 x10^3 1.85 x10^6

Negative 0 0 0 0 0 0 0

DENV acute

plasma

DENV <2 MPI 1.10 x10^4 3.30 x10^4 1.70 x10^4 1.40 x10^4 1.10 x10^4 1.30 x10^3 1.46 x10^4 0.036 0.0022

DENV-1-018 3.80 x10^5 5.10 x10^5 4.20 x10^5 4.90 x10^5 2.20 x10^5 2.00 x10^4 3.40 x10^5 0.2944 0.0022

DENV

convalescent

plasma pool

DENV >12 MPI 1.60 x10^4 1.50 x10^4 1.20 x10^4 5.00 x10^2 0 0 7.25 x10^3 0.0152 0.0152

DENV-1 plasma

DENV-1-022 3.90 x10^4 2.70 x10^4 2.80 x10^3 4.50 x10^3 0 0 1.22 x10^4 0.0087 0.0606

DENV-1-026 5.10 x10^3 2.70 x10^3 5.00 x10^2 0 0 0 1.38 x10^3 0.0043 0.1818

DENV-1-076 7.60 x10^2 0 0 0 5.20 x10^2 0 2.13 x10^2 0.0022 0.4545

DENV-1- 060 0 0 0 0 0 0 0

DENV-3 plasma

DENV-3-50 5.00 x10^2 6.00 x10^1 0 0 0 0 9.33 x10^1 0.0022 0.4545

DENV-3-056 2.00 x10^2 0 0 0 0 0 3.33 x10^1 0.0022 0.4545

DENV-3-038 1.50 x10^4 1.70 x10^4 3.70 x10^4 1.90 x10^3 5.00 x10^2 0 1.19 x10^4 0.0043 0.4545

DENV-3-037 4.40 x10^3 4.60 x10^2 0 0 0 0 8.10 x10^2 0.0152 0.0152

Pooled sera from

DENV-1 and 3

patients

DENV 1&3 (6 MPI) 1.30 x10^2 0 0 0 0 0 2.17 x10^1 0.0022 0.4545

DENV 1&3 (12 MPI) 3.10 x10^3 2.50 x10^4 0 0 0 0 4.68 x10^3 0.0043 0.4545

Note: - p<0.01 shows statistically significant distance compared with positive and negative control

Significant difference in virus output between positive control and sample: - no-enhancement

Significant difference in virus output between negative control and sample:- Enhancement

Antisera that significantly enhanced ZIKV infection are coloured in grey; non-conclusive results with significant difference from both positive and negative control are highlighted in light grey

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7.3.1. Acute phase plasma: DENV plasma sample within two months post infection

Acute phase anti-DENV antisera collected less than 2 months after infection enhanced

ZIKV infection. Differential enhancement was seen for individual monotypic antisera

and for the pooled antisera with variable virus yield, while enhancement by both

samples was statistically significant (p=0.2944 and p=0.036). Virus output was greater

than for negative control in all cases. Acute phase plasma from a patient with

neutralising antibody against ZIKV MR766 showed greater enhancement of viral

output.

This group of anti-DENV antisera includes a pool of four plasma samples obtained <2

months post infection: 3 DENV-1 (FLV-011, FLV-017, FLV-018) and 1 DENV-2

(FLV-012) antisera. None of the antisera neutralised ZIKV PRVABC59, however anti-

ZIKV MR766 FRNT50 was > 50. No patients were infected with ZIKV and there was

no ZIKV PRVABC59-specific neutralisation with antisera collected < 2 months or at

any subsequent time.

The 2 acute phase test samples enhanced ZIKV production while no virus was produced

with the negative control pooled serum. Virus production was enhanced at 1:100

dilution of the acute pooled antiserum and the individual antiserum (FLV018). Virus

production by FLV018 antiserum was 5-fold lower than for positive control while yield

with the pooled acute antisera was more than 100-fold less but was still statistically

significant. Similarly, virus output following exposure to FLV018 antiserum was 15-

fold higher than for pooled acute antisera.

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7.3.2. ZIKV enhancement by convalescent anti-DENV antisera collected 1-6 years post

infection

ZIKV production by pooled convalescent antisera was significantly lower than positive

control. This pool consisted of 8 anti-DENV antisera collected 1 – 6 years post

infection. Peak virus replication was obtained at the lowest antiserum dilution (1:10). At

higher dilutions of plasma (1x10^5 and 1x10^6) no viral replication was detected.

Average titre (pfu/ml) across dilutions with virus output was higher than for negative

control and was > 200-fold lower than for positive control, but the difference was not

statistically significant for either (p=0.0152).

Enhancement by pooled convalescent antisera is 2-fold lower than for acute phase

antisera. Viral yield at different plasma dilutions is illustrated in Figure 7. 1-A

Enhancement by pooled acute and pooled convalescent antisera, and positive and

negative controls, is shown in Figure 7. 1-B.

7. 3.3. DENV-1 plasma samples

No anti-DENV-1 antisera induced enhancement of replication of ZIKV PRVABC59.

Four DENV-1 plasma samples were tested representing six months and 12 months post

infection (MPI). Of two anti-DENV-1 antisera at 6 months, one induced virus yields at

10 and 10^5 dilution while no viral production was detected for the other antisera. Virus

output was obtained in both samples at 12MPI. Average pfu/ml for DENV-1-022

antisera was 9-fold higher than for anti-DENV-1-026 antisera.

Overall, anti-DENV-1 at 12 MPI had higher virus yield compared to anti-DENV at six

months post infection. Viral output in all cases was significantly lower (p=0.0022-

0.0087) than for 4G2 with more than 200-fold less virus production. Enhancement of

304

ZIKV infection was statistically comparable to negative control (p=0.0606-0.04545).

Viral output at different plasma dilution is shown as spline curve in Figure 7. 2(A) and

Enhancement by Anti-DENV-1 antisera, and controls, is shown in Figure 7. 2(B).

7.3.4. DENV-3 plasma samples

Similar to anti-DENV-1, no anti-DENV-3 antisera induced statistically significant

enhancement of ZIKV PRVABC59. This sample set consists of 4 anti-DENV-3 antisera

at six and 12 MPI. Compared to positive control, viral output is more than 200-fold less

for anti-DENV-3 antisera collected 12MPI, and more than 1000-fold less for anti-

DENV-3 antisera collected six MPI. Although there was virus output, it was not

statistically significant compared to negative control and thus, enhancement was not

induced. Viral output at different plasma dilution is shown as spline curve in Figure7.

3(A) and Figure7. 3 (B) Enhancement by Anti-DENV-3 antisera, and controls, is shown

in Figure 7. 3(B).

7.3.5. Pooled sera from DENV-1 and DENV-3 patients

a) At six months post infection

A pool of anti-DENV-1 and anti-DENV-3 antisera collected 6MPI did not induce

enhancement of ZIKV replication. Average pfu/ml was 4-fold lower compared to virus

output with individual antisera tested. Maximum yield with this pool was 130 pfu/ml

which was not statistically significant compared to 4G2.

b) At 12 months post infection

No statistically significant enhancement of infection by pooled DENV-1 and DENV-3

antisera obtained at 12 MPI. Maximum virus yield was obtained at 1:100 dilution. At

this dilution, viral output was higher than enhancement induced by 3 of 4 individual

305

antisera. Virus output with this pool was more than 400-fold less compared to 4G2.

Viral output was equivalent to negative control (p=0.4545).

Virus output at different dilution of this pool at 6 and 12 MPI is shown in Figure 7. 4

(A) and compared with positive and negative control in Figure 7. 4 (B).

306

Figure 7. 1:-ZIKV enhancement by monotypic anti-DENV pool at different stage post infection.

A) Spline curve of ZIKV output in the presence of acute phase anti-DENV antisera

(DENV<2MPI and DENV-1-018), convalescent phase anti-DENV antisera

(DENV>12MPI), 4G2 and negative pool. Highest viral output was induced by 4G2 and

lowest output was induced by convalescent antisera.

B) Viral output in the presence of anti-DENV antibody. Difference in average pfu/ml

between acute and convalescent anti-DENV antisera was not statistically significant by

Mann-Whitney non-parametric t-test. Virus production was significant for acute phase

antisera, and for convalescent antisera enhancement was inconclusive as all dilutions did not

induce virus replication.

A

B

307

A

B

Figure 7. 2:-ZIKV enhancement by anti-DENV-1 antisera

A) Spline curve of viral yield at different plasma dilutions of DENV-1 vs positive and

negative control. One of the DENV-1 at 6MPI antisera did not induce any infection

and a second anti-DENV-1 antisera showed higher virus replication at lower dilution.

Both DENV-1 at 12MPI showed yield of ZIKV.

B) No significant difference in enhancement by anti-DENV-1 antisera collected at 6

and 12 MPI. Viral output is higher than for negative control not statistically significant

as not all dilutions induced enhancement.

308

B

A

B

Figure7. 3:-ZIKV enhancement by anti-DENV-3 antisera

A) Spline curve of viral output at different plasma dilution of anti-DENV-3 antisera at 6

months and 12 months post infection. No antisera enhanced ZIKV infection.

B) No anti-DENV-3 antisera collected 6 and 12 MPI enhanced ZIKV infection.

309

B

Figure 7. 4 :-ZIKV enhancement based on French Polynesian co-circulation model.

A) Pooled anti-DENV-1 and anti-DENV-3 antisera collected 6 and 12 MPI did not enhance

ZIKV infection.

B) ZIKV production in the presence of pooled anti-DENV-1 and anti-DENV-3 antisera, 4G2

and negative control. Virus output is greater for 12 MPI antisera, compared to 6 MPI antisera,

but these values were not statistically significant compared to positive control.

A

310


Re-emergence of ZIKV and spread into DENV-endemic areas has raised concerns about

possible immunopathological outcomes due to pre-existing anti-DENV antibodies.

Severe dengue is postulated to be a consequence of anti-DENV antibody-mediated

immune enhancement. Enhancement of ZIKV infection in the setting of pre-existing

anti-DENV immunity is postulated as a mechanism for the severe Zika disease that

emerged in the recent epidemics beginning in 2007. This study assessed the capacity of

well-defined anti-DENV antisera to enhance ZIKV replication in vitro. In addition, the

sequence of ZIKV circulation in French Polynesia in 2013, in the background of prior

DENV-1 and DENV-3 epidemics, was modelled to determine if anti-DENV-1 and anti-

DENV-3 antisera enhanced ZIKV infection.

7.4.1. DENV and ZIKV in French Polynesia

Transmission of mosquito-borne viruses is well documented in the Pacific. A pattern

of single DENV serotype dominance has been reported in many South Pacific island

nations. The displacement of the circulating serotype by newly introduced DENV has

been regularly observed over time (118). Epidemiological studies report periodic cycles

of 12 years of DENV and 20 years of the other 3 serotypes (400). The Pacific had

predominant circulation of DENV-1 with co-circulation of DENV-4 in 2007 along with

DENV-2 sporadic outbreaks followed by DENV-3 in 2013 (327). DENV-1 was the

documented serotype in the first recorded dengue outbreak in French Polynesia (FP) in

the 1940s. After 20 years dengue reappeared with DENV-3 as dominant serotype which

was followed by the first cases of severe dengue in the 1970s. In 2009 after decades of

circulation of DENV-1, DENV-4 reappeared in FP after 20 years (401). In March 2013

after 18 years of absence, DENV-3 re-emergence was reported in FP (327). ZIKV was

introduced into FP while DENV-3 was circulating in 2013. Following re-emergence of

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ZIKV in Yap state in the Western Pacific in 2007 the largest ZIKV outbreak in the

Pacific occurred in FP (132). Later ZIKV infection was confirmed with sequencing

which showed ZIKV strain Cambodia as the closet strain. By 10 weeks of first case

reported 5,895 patients with suspected ZIKV infections were recorded and 294 patients

were ZIKV RT-PCR confirmed cases(132). At the same time the evidences of

concomitant circulation of ZIKV, DENV-1, and DENV-3 and increase in GBS also

raised question of impact of sequential infections and disease outcome (132).

7.4.2. ZIKV enhancement by monotypic anti-DENV from travellers

Given circulation of both ZIKV and DENV in Indonesia, presence of DENV immune

response background in WA travellers and high chances re-visit to endemic areas

influenced us to analyse possibility of DENV-1 and DENV-3 primed enhancement of

ZIKV. Furthermore, the fact that DENV-3 isolated in traveller’s population in our

cohort belongs to genotype-I encouraged us to start preliminary research on possibility

of ADE in case of non-endemic population with monotypic DENV infection based on

Pacific Island outbreak pattern. We based our study on this model to test whether either

of these serotype plasma samples from travellers may have potential to enhance ZIKV

replication in macrophages.

In this study, role of anti-DENV sera from monotypic infection to enhance ZIKV Asian

strain in vitro tested. Intrinsic ADE was tested to presume possibility of infection and

replication of virus in the presence of serum sample. Analysing total viral load output

from U937 FcR bearing cell line in the presence and absence of anti-DENV antisera

showed not all post DENV sera sample enhances viral replication. Effects of time post

infection were also studied. Viral output with acute samples pool (DENV1 and 2) was

higher than convalescent sample pool (1-6 years DENV1-4). Viral output was higher

compared to the negative tested in same experiment however was >100 fold less than

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positive control and this difference was statistically significant. Interestingly, DENV-1

individual (FLV018) who had cross reactivity with prototype ZIKV at acute and

convalescent phase showed significant enhancement of ZIKVPRVABV59. This

individual has no cross-reactivity against ZIKVPRVABC59 when tested with

neutralisation. In vitro study showed not all DENV 1 and 3 could enhance production of

viral load, in fact some of the sera did not support viral production post exposure. No

statistical significant difference (p>0.05) was obtained between individual samples and

pooled sera sample. Although there was yield of virus in the tested samples, but the

output was >1000 fold less than positive control hence statistically closer to negative

pool. Thus, based on this pilot study on viral output post exposure with DENV-1 and 3

does not support increase in viral load in the presence of anti-DENV antibody. While

there are also reports not all anti-DENV antibody supports the enhancement of ZIKV.

However, it cannot be denied that this study includes only limited size. Differential

pattern of ADE among the samples can be explained by variable epitope sensitivity of

anti-DENV in different individuals. Epitope sensitive binding has been previously

studied in mAbs derived studies. Monoclonal antibodies derived post DENV infection

shown to selectively bind to epitope footprint that spans certain E-protein dimers (402)

Dejnirattisai et al showed DENV derived mAbs that bind to linear epitopes were able to

promote ADE (260).

In our study, significant enhancement of ZIKV viral output from U937 cell line when

exposed to virus in the presence of 4G2 monoclonal was found. A related paper showed

similar enhancement of ZIKV prototype strain by 4G2 on THP-1 cell line (403). In

contrast to our finding in non-endemic population, enhanced infection of ZIKV

PRVABC59 in FcR receptor bearing cell was reported from cases in Thailand(357). In

the study form patients in Thailand both acute/convalescent sera and plasmablast-

313

derived mAbs were reported to enhance infection. Similarly, enhancement of ZIKV by

DENV derived mAbs and plasma samples have reported in several studies (155, 257).

This is the initial phase study to generate background for possible DENV-primed ZIKV

infection in travellers hence providing important information about possible

consequences in non-endemic population. However, this study needs more data to

predict the possible outcomes of secondary ZIKV in infected traveller population.

Further this report is based on the yield of infectious virus from Fc-R bearing cells in

the presence and absence of anti-DENV antibody, still the data about enhancement of

infection needs to be analysed with molecular PCR or flow-based techniques to predict

possible consequences of pre-DENV exposure in secondary ZIKV infection in

population from non-endemic regions.


Acute phase anti-DENV enhances ZIKV infection-monotypic anti-DENV-1 or

pooled acute DENV-1+DENV-2.

No convalescent (1-6 years) anti-DENV (all four) serotypes enhanced ZIKV

infection.

Anti-DENV-1 and 3 combinations did not prime ZIKV infection to U937 cells.

ADE was greatly dependent on time post infection and individual’s immune

response.

314

Chapter 8:- Summary and conclusion

Flavivirus-specific antibody responses were assessed in West Australian travellers

returning from dengue and Zika endemic or epidemic countries. The majority of study

volunteers had never lived in these countries and had well-defined monotypic infection

with DENV or ZIKV; some of them had received YFV vaccination. Dengue and Zika

do not occur in WA as the mosquito vectors are not present. Magnitude of neutralising

antibody, measured by focus reduction neutralisation test (FRNT); total antibody,

measured by haemagglutination inhibition (HI) test; as well as antibody mediated

enhancement (ADE) were assessed for antisera collected from study volunteers who

were sampled during acute phase (< 2months) up to 6 years after presentation for febrile

illness. The WA Traveller Study is not a true prospective cohort and includes

convenience samples obtained from volunteers at variable time intervals. Antisera were

tested against a collection of DENV isolates derived from travellers presenting with

febrile illness and who were diagnosed with DENV infection; the virus collection

includes representatives of all four serotypes collected between 2010 – 2015. This

collection is dependent on DENV circulating in the Asia Pacific region at the time and

thus is biased towards contemporaneous endemic or epidemic isolates, predominantly

DENV-1 and DENV-2. Phylogenetic analysis identified genotypes and lineages, and

representative isolates were used as targets. Other travellers returned from Zika

epidemic countries with confirmed or probable ZIKV infection; some had lived in

dengue endemic countries previously.

Anti-DENV responses were highly cross-reactive during acute phase and neutralized

heterologous DENV at high magnitude. Over time and up to six years after infection

responses became increasingly serotype-specific –this could be visualised on antigenic

maps - however heterologous cross-neutralisation was maintained in all individuals to

315

some degree, even at 6 years, and was virus- and patient-specific. Virus-specific

neutralisation was not genotype or lineage dependent; individual isolates were

differentially neutralised by antisera from individuals collected at the same time point

after infection. Antigenic mapping showed serotype clustering could be distinguished

after 1 year; however individual viruses were as close to heterologous serotype clusters

as they were to homologous clusters and this pattern persisted at 6 years. A second

major observation was that the magnitude of responses to homologous DENV in these

monotypic travellers also declined over time, including responses to autologous virus,

and this was true for all 4 serotypes.

The magnitude of ZIKV-specific neutralising antibody responses declined over time in

the small number of study volunteers with monotypic ZIKV infection. By 13 and 20

months after infection FRNT90 titres were below limits of detection (<40 as per WHO

criteria for sero diagnosis of ZIKV infection). However, ZIKV-infected individuals with

previous flavivirus infection showed prolonged anti-ZIKV antibody responses, up to 13

months after infection. This study demonstrated that antibody binding to ZIKV

capsid/prM protein can differentiate recent ZIKV infection, in individuals with previous

DENV infection.

Two ZIKV strains were assessed and showed a high degree of differential cross-

neutralisation, with high magnitude responses directed against the prototype ZIKV

MR766 isolate but not the contemporaneous ZIKV PRVABC59 in acute phase

monotypic DENV infection, highlighting the importance of strain selection in ZIKV

diagnostic and research approaches.

In the small number of antisera tested in the present study, ZIKV replication was

enhanced by acute phase (< 2months) antisera only. This finding contrasts with previous

reports of ZIKV enhancement by convalescent antisera, however those studies used

316

immune sera collected within 100 days of infection; convalescent antisera in the present

study were collected between 6 months and 6 years post presentation.

A major limitation of this study is the small sample size and the convenience sampling

of antisera dictated by this traveller cohort. Enrolment into the study is ongoing and

more recently travellers have returned with more diverse DENV infections, including

DENV-3 and DENV-4 which have not circulated as widely in the past. In addition,

ongoing molecular epidemiological analysis of traveller viruses has begun to delineate

genotypes and lineages and this information will be very informative for ongoing

phylogeographic antigenicity studies.

Future research directions will focus on characterisation of the anti-flavivirus B cell

repertoire; data from this present study will form the basis of this work.

317

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confer protection against antibody dependent enhancement in AG129 mice. PLOS Neglected Tropical Diseases. 2018;12(1):e0006191. 400. Aubry M, Teissier Y, Mapotoeke M, Teissier A, Giard M, Musso D, et al. High risk of dengue type 2 outbreak in French Polynesia, 2017. Eurosurveillance. 2017;22(14):30505. 401. Cao-Lormeau V-M, Roche C, Aubry M, Teissier A, Lastere S, Daudens E, et al. Recent Emergence of Dengue Virus Serotype 4 in French Polynesia Results from Multiple Introductions from Other South Pacific Islands. PLOS ONE. 2011;6(12):e29555. 402. de Alwis R, Smith SA, Olivarez NP, Messer WB, Huynh JP, Wahala WMPB, et al. Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(19):7439-44. 403. Charles AS, Christofferson RC. Utility of a Dengue-Derived Monoclonal Antibody to Enhance Zika Infection In Vitro. PLoS Currents. 2016;8:ecurrents.outbreaks.4ab8bc87c945eb41cd8a49e127082620.

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Appendixes

List of Appendixes

Appendix 3. 1:- Hemagglutination and NS1 antigen test

Appendix 3. 2:- TCID50 determination of virus

Appendix 3. 3:- Checker board assay for primary and secondary antibody

Appendix 3. 4:-Reagents for preparation of virus antigen

Appendix 4.1:- Information sheet

Appendix 4.2:- Travel and Medical History Questionnaire

Appendix 4.3:- Participant consent form

Appendix 5.1.:- Neutralising antibody response over time in individuals with DENV-1

infection


infection


infection


infection

Appendix 6.1.:- Neutralising antibody response over time in individuals with ZIKV

infection

339

Appendixes for Chapter 3

Appendix 3.1: Hemagglutination and NS1 antigen test

Protocol followed for HA and NS1 was established in PathWest reference laboratory.

a) Hemagglutination assay (HA)

The Haemagglutination assay is a pH dependent test for the detection of viral protein.

Dilution of virus supernatant was made up in BABS buffer made from 1: 5

concentration of bovine albumin and borate saline. For the dilution 1:1 serial dilution of

virus supernatant and BABS buffer was made in 96 well v-bottom plate. Each virus

sample was tested in pH buffer solution at pH’s 6.0, 6.2 and 6.4. Goose red blood cells

at the three pH’s was made and 50 µl goose cells was added to all wells. Plates were

gently tap to shake and sealed and read after an hour for presence or absence of cell

pellet. The presence of virus is indicated by the absence of a cell pellet and no virus by

the presence of a cell pellet at the bottom of the well.

b) NS1 antigen test

This test is a very sensitive and specific test for the detection of DENV only. This was

performed to confirm virus produced is dengue virus and detection of virus in HA

negative samples. NS1 antigen was carried out at PathWest using a commercial dengue

NS1 antigen-capture ELISA kit, PLATELIA DENGUE NS1 AG (Bio-Rad). This test is

based on a one-step sandwich microplate format. The test used murine monoclonal

antibody (MAb) for capture and revelation. If NS1 antigen was present in the sample an

immune complex MAb-NS1-MAb/peroxidase would be formed. The test was

performed and calculations were made according to manufacturer’s protocol.

340

Appendix 3.2 : Tissue Culture Infective Dose 50 Assay (TCID50)

TCID50 was done to quantity of virus that will produce a cytopathic effect (CPE) in

50% of the cultures inoculated to make sure virus cultured would give optimal CPE in

further tests. End-point determination was done by ELISA using 4G2 monoclonal

antibody and goat anti-mouse antibody labelled with horse radish peroxidase. Briefly,

serial dilution of virus supernatant was grown in 96 well round bottom plates. After 7

days cells were fixed with 20% PBS/Acetone for 90 minutes in 4oC. After fixing cells

were blocked with 2% casein solution for an hour at room temperature. Cells were then

labelled with primary antibody 4G2 monoclonal antibody followed by GAM labelled

secondary antibody. Plates were washed in between primary and secondary antibody for

4X with ELISA buffer. ABTS substrate with 30% H2O2 was used as substrate and

optical densities of plate were read. 4G2 and GAM concentration was used as

previously stated in lab.

Appendix 3.3: Checker board assay for determination of 4G2 and GAM concentration

for Focus assay.

Different dilution of GAM and 4G2 was titrated to get optimal concentration that would

give visible spots in focus assay. For assay, known concentration of virus (optimal

concentration as determined by TCID50 that will give 50% CPE) was grown on 24 well

plates. Each well was inoculated with same amount of virus and 2 wells were left as cell

control (no virus). After 5-7 days plates were fixed, blocked with blocking buffer and

stained with different concentration pair of primary and secondary antibody. TrueBlue

peroxidase was used as substrate. Optimal concentration of that gives clear distinct

spots was used in further assays.

341

Appendix 3.4: Reagents for preparation of virus antigen

All the reagents were prepared following standardised protocol from PathWest.

Preparation of 0.175N sodium hydroxide

0.70g NaOH was dissolved in 100ml ultrapure water. Solution was filter sterilised with

022µ filter and stored at room temperature for <= 3 months.

Preparation of 1M sodium thiosulphate

28.82gm of Na2O2S25.H20 was dissolved in 100ml ultrapure water and sterilised with

0.22µ filter. Solution was stored at 4oC for <3 months.

Preparation of 0.1M Binary Ethylenimine (BEI)

0.20489gm of BEI was dissolved in 10 ml 0.175N NaOH. Solution was incubated at

37oC water bath for 60 mins. BEI was stored in 4

oC and used within 24hrs.

342

Participant Information Sheet

CHIEF INVESTIGATOR

Dr Allison Imrie, University of Western Australia, School of Biomedical Sciences; Telephone: (618) 6457 1377; Fax: (618) 6457 2912; email: [email protected]

PROJECT TITLE

Immune Responses in Mosquito-Borne Virus Infection

PURPOSE

You are being asked to take part in this research study because you were previously diagnosed with a mosquito-borne viral infection. Around the time of your infection, your immune system responded by producing antibodies and forming memory T and B cells (white blood cells or lymphocytes) and these cells can be found in your blood for many years after your infection. The purpose of this study is to learn more about your immune responses to the mosquito-borne viruses, and how these responses may contribute to the diseases these viruses cause. We will test antibodies and lymphocytes taken from your blood to see how they recognize mosquito-borne viruses in vitro, that is, in the laboratory.

In order to decide whether or not you wish to be in this study, you will need to know about any good or bad things that could happen to you if you decide to join. This process is called ‘informed consent’. Once you understand the study, and if you agree to take part,

you will be asked to sign a consent form. You will be given a copy to keep. EXPECTED LENGTH OF STUDY

The estimated length of this study is 5 years, however you can stop participating at any time within this 5-year period. We may ask you to give blood each year so that we can look at how your immune responses change over time.

PROCEDURES

To enter into this study, you must be at least 18 years old and be willing to sign a consent form.

First Study Visit At this visit you will:

Talk about the study with the Investigator

Read and sign the consent form after all your questions are answered

Complete a brief questionnaire on your travel history Study staff will take 150 ml of blood (about 8 tablespoons; about 1/3 the volume of a standard blood bank donation), which will be used to look at how your immune cells


The Faculty of Health and Medical Sciences

The University of Western Australia

M502, 35 Stirling Highway

Crawley WA 6009 Australia

E: [email protected] CRICOS Provider Code: 00126G

Appendix 4.1:- Information sheet

Appendix

343

respond to mosquito-borne viruses, in the laboratory. Blood will be collected from your arm vein using the same method as for routine medical blood tests. This may be performed by an experienced blood collector or a trainee under the supervision of an experienced blood collector. If the person collecting your blood is a trainee, you will be informed and have the option of electing to have a more experienced person do that task. This visit will take an estimated 45 minutes. Follow-up Visits The blood sample you donate at your first visit will be tested in the laboratory, and depending on the results we may ask you to return for a follow-up visit at which:

We may ask you to donate a large (150 ml) or small (30 ml) volume of blood. The amount you donate will depend on the kind of immune response we have measured in your blood, which we need to analyze in greater detail.

You will complete a brief questionnaire on your travel history This visit will take an estimated 30 minutes.

Depending on the results of the laboratory tests, we may ask you to return each year, for up to 5 years.

Blood collected from you will only be used for the purposes described here. Analysis of your blood sample(s) for immune responses against mosquito-borne viruses can take several weeks to complete. The results from this study will be published in medical and scientific journals and presented at national and international scientific meetings. Study participants’ names are never used in any form of report or discussion associated with this project.

RISKS AND DISCOMFORTS

Taking blood may cause some soreness, bleeding and bruising, and (very rarely) infection where the needle enters the body. A person may (very rarely) feel faint or faint when blood is taken. Although 150 ml of blood may seem like quite a large amount of blood to give, this is about one-third of the amount collected from a blood donor at the Blood Bank.

Risk of infection is slight since only sterile one-time equipment is used.

BENEFITS

You may receive no benefit from this study. However, knowledge gained from this study may, in future, benefit others who are infected with mosquito-borne viruses, such as dengue, Zika, chikungunya, Ross River, and Murray Valley. It may also help in the development of a vaccine to prevent infection in future generations.

CONFIDENTIALITY

Your research records will be confidential to the extent permitted by law. You will not be personally identified in any publication about this study. A code, which will be known only to study personnel and you, will be used instead of your name on laboratory records in this study. The code will be stored in a safe place in the Chief Investigator’s office. Personal information about your test results will not be given to anyone without your written permission. Individual results may be provided upon request and where available, though any data generated from this study are for research purposes only and not for diagnostic purposes.

COMPENSATION FOR INJURY

344

Your participation in this study does not prejudice any right to compensation, which you may have under statute or common law.

VOLUNTARY CONSENT AND CERTIFICATION

You take part in this study of your own free will and you can stop at any time for any reason. Your consent does not take away any of your legal rights in case of negligence or carelessness on the part of anyone working on this project.

ETHICS

Approval to conduct this research has been provided by the University of Western Australia, in accordance with its ethics review and approval procedures. Any person considering participation in this research project, or agreeing to participate, may raise any questions or issues with the researchers at any time. In addition, any person not satisfied with the response of researchers may raise ethics issues or concerns, and may make any complaints about this research project by contacting the Human Ethics Office at the University of Western Australia on (08) 6488 3703 or by emailing to [email protected]. All research participants are entitled to retain a copy of any Participant Information Form and/or Participant Consent Form relating to this research project.

CONTACTS To contact the research team, please call the Mosquito-Borne Virus Study Phone: (08)

6457 1129.

mailto:[email protected]

345

Appendix 4.2:- Travel and Medical History Questionnaire

346

Participant Consent Form

Immune Responses in Mosquito-Borne Virus Infection

Chief Investigator: Associate Professor Allison Imrie

I (the participant) have read the information provided and any questions I have asked

have been answered to my satisfaction. I agree to participate in this activity, realizing

that I may withdraw at any time without reason and without prejudice.

I understand that all attributable information that I provide is treated as strictly

confidential and will not be released by the investigator in any form that may identify

me. The only exception to this principle of confidentiality is if documents are required

by law.

I have been advised as to what data is being collected, the purpose for collecting the

data, and what will be done with the data upon completion of the research.

I have been informed that individual results may be provided upon request and where

available, though any data generated from this study are for research purposes only and

not for diagnostic purposes.

I agree that research data gathered for the study may be published provided my name or

other identifying information is not used.

________________________________________________________________________

_

Participant Name Participant Signature Date

Approval to conduct this research has been provided by the University of Western

Australia, in accordance with its ethics review and approval procedures. Any person

considering participation in this research project, or agreeing to participate, may raise any

questions or issues with the researchers at any time.

In addition, any person not satisfied with the response of researchers may raise ethics

issues or concerns, and may make any complaints about this research project by

contacting the Human Ethics Office at the University of Western Australia on (08) 6488

3703 or by emailing to [email protected].

All research participants are entitled to retain a copy of any Participant Information Form

and/or Participant Consent Form relating to this research project


The Faculty of Health and Medical Sciences

The University of Western Australia

M502, 35 Stirling Highway

Crawley WA 6009 Australia

E: [email protected]

CRICOS Provider Code: 00126G

Appendix 4. 3.:- Participant consent

form

mailto:[email protected]

347

Appendix for Chapter 5 : Antibody mediated immune response in monotypic

dengue

This appendix consists of nonlinear regression curve and comparative mean reduction

analysis for each DENV individuals at different time post infection. Each graph title

represents the study number followed by their infecting serotype and time of sample

collection post infection. Each sera panel is separated by a page gap between the graphs.

Graphs are arranged according to their study number. Non-linear regression curve for

each sample is illustrated as its antibody response against each serotype of DENV. In

mean reduction curve x-axis represents strains of DENV tested and y-axis represents

mean reduction percentage for each strain of virus strains.

348

Appendix 5.1.:- Neutralising antibody response over time in individuals with

DENV-1 infection

DENV-1 SERA PANEL

Number of individuals: 6

Number of individuals with multiple

sampling points:-5

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HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

Au

tolo

go

us v

iru

s

0

1 0 0

1 5 0

F L V 0 2 2 V 2 (D 1 -2 y e a rs p o s t in fe c t io n )

Me

an

Re

du

cti

on

%

50

75

90

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

Au

tolo

go

us v

iru

s

0

1 0 0

1 5 0

F L V 0 2 2 V 1 (D 1 :1 y e a r 2 m o n th s )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

359

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 2 6 V 1 (D 1 :~ 1 y e a r -2 m o n th s )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

360


DENV-2 infection

DENV-2 SERA PANEL



sampling points:-6

362

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

Au

tolo

go

us v

iru

s

0

1 0 0

1 5 0

F L V 0 0 4 V 1 (D 2 : 1 y e a r -4 m o n th s )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

Au

tolo

go

us v

iru

s

0

1 0 0

1 5 0

F L V 0 0 4 V 2 (D 2 :4 y e a r s -3 m o n th s )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

364

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 0 5 V 1 (D 2 : 1 y e a r -4 m o n th s )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 0 5 V 2 ( D 2 :4 y e a rs -3 m o n th s )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

366

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0


V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 0 9 V 2 (D 2 :~ 3 y e a rs )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

368

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 1 0 V 1 (D 2 :1 y e a r -7 m o n th s )

V iru s ty p e

Me

an

Re

du

cti

on

%50

75

90

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 1 0 V 2 (D 2 :~ 3 y e a rs )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 1 0 V 3 (D 2 :~ 4 y e a rs )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

370

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 1 2 V 1 (D 2 :5 d a y s )

V iru s ty p e

Me

an

Re

du

ctio

n %

50

75

90

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 0 1 2 V 2 (D 2 :~ 2 y e a r s 9 m o n th s )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

372

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

Au

tolo

go

us v

iru

s

0

1 0 0

1 5 0

F L V 0 1 4 V 1 (D 2 :7 m o n th s )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

Au

tolo

go

us v

iru

s

0

1 0 0

1 5 0

F L V 0 1 4 V 2 (D 2 :~ 3 y e a rs )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

373


DENV-3 infection

DENV-3 SERA PANEL



sampling points:-1

374

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 2 9 V 1 (D 3 :1 y e a r )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

375

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 3 0 V 1 (D 3 :~ 1 y e a r )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

376

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0


V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

377

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 3 5 V 1 (D 3 :~ 8 m o n th s )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

378

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

Au

tolo

go

us v

iru

s

0

1 0 0

1 5 0


V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

380

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

Au

tolo

go

us v

iru

s

0

1 0 0

1 5 0


V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

Au

tolo

go

us v

iru

s (

15B

)

0

1 0 0

1 5 0

F L V 0 3 8 V 2 (D 3 :-2 y e a rs p o s t in fe c t io n )

Me

an

Re

du

cti

on

%

50

75

90

381


DENV-4 infection

DENV-4 SERA PANEL



sampling points:-2

383

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

1

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 0 2 V 1 (D 4 :2 y e a rs )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 0 2 V 2 (D 4 :~ 5 y e a rs )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 0 2 V 3 (D 4 :~ 6 y e a rs )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

385

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 4 9 V 1 (D 4 :~ 8 m o n th s p o s t in fe c t io n )M

ea

n R

ed

uc

tio

n %

50

75

90

386

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 6 8 V 1 (D 4 :~ 8 m o n th s )

V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

387

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0


V iru s ty p e

Me

an

Re

du

cti

on

%

50

75

90

388

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 8 6 V 1 (D 4 : 1 y e a r 6 m o n th s )

Me

an

Re

du

cti

on

%

50

75

90

389

D1-H

W2001G

4

D1-1

0B

G1L

14

D1-1

5L

G1

D1-1

0B

G2L

4

D1-1

0IN

DG

3L

4

D1-1

4P

HL

G4

D2-N

GC

GA

II

D2-1

0L

GA

1L

9

D2-1

1B

GC

L4

D2-1

3M

CL

N

D2-1

5B

GC

L4

D2-1

5B

CL

N

D2-

PR

6193V

D2 -

PR

1940N

V

D3-H

87G

5

D3-1

5B

G1

D3-

UN

C 3

002V

D3-

UN

C 3

008 N

V

D4-H

24G

1

D4-1

0B

G3L

1

D4-1

0B

G3L

2

ZIK

V M

R 7

66

ZIK

V P

RV

AB

C59

YF

17D

JE

IM

OJE

V

0

1 0 0

1 5 0

F L V 0 9 9 V 1 (D 4 -7 m o n th s p o s t in fe c tio n )

Me

an

Re

du

cti

on

%

50

75

90

390

Appendix 6.1.:- Neutralising antibody response over time in individuals with ZIKV

infection

Each graph is the analysis of non-linear regression of neutralisation at log 10 plasma

dilution. Number of virus tested and starting point of dilution varies according to

availability of sample. Each graph title represents patient code and time point of sample

collection. Graph title with no time point represents sample taken at point of diagnosis.

50,75,90% reduction is shown in each graph. Past flavivirus history of individual is

stated for each graph. Graphs are listed in no particular order

0 .5 1 .0 1 .5 2 .0 2 .5

0

1 0 0

1 5 0

F L V 0 6 4 (6 0 5 d a y s p o s t in fe c t io n )

P la s m a d ilu tio n (L o g 1 0 )

Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6

Z IK V P R V A B C 5 9

Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

1 .0 1 .5 2 .0 2 .5

0

1 0 0

1 5 0

F L V -0 9 3 - (3 3 0 d a y s p o s t in fe c t io n )


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

FLV-064:- Confirmed ZIKV, no previous flavivirus exposure

FLV-093: - Probable ZIKV, with known YF17D vaccination but no

known previous DENV exposure. Frequent traveller to endemic areas.

391

1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

F L V 0 8 7 (3 9 d a y s p o s t in fe c tio n )


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

F L V 0 8 7


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6

Y F 1 7 D

J E IM O J E V

D E N V -4 (H 2 4 1 )

1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0



Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -4 (H 2 4 1 )

0 .5 1 .0 1 .5 2 .0 2 .5

0

1 0 0

1 5 0

F L V 0 8 7 (4 2 5 d a y s p o s t in fe c tio n )

P la s m a d ilu tio n (L o g 1 0 )

Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

FLV-087: - Confirmed ZIKV, with known DENV exposure.

392

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

F L V 0 3 2 (7 d a y s p o s t in fe c t io n )


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6

Y F 1 7 D

D E N V -4 (H 2 4 1 )

J E IM O J E V

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

F L V 0 3 2


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6

Y F 1 7 D

D E N V -4 (H 2 4 1 )

J E IM O J E V

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0


P la s m a D ilu tio n

Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0



Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0


S e ru m D ilu t io n

Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 ) 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0



Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

FLV-032: - Probable ZIKV, with known YF17D vaccine but vaccine response was not detected later time points. FLV-032: - Probable ZIKV, with known YF17D vaccine but vaccine response was not detected later time points.

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0



Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

393

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0



Re

du

cti

on

%


D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

J E IM O J E V

Y F 1 7 D

Z IK V M R 7 6 6

50

75

90

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

F L V 0 8 8


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6

Z ik a P R V A B C 5 9

J E IM O J E V

Y F 1 7 D

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0

0

1 0 0

1 5 0

F L V 0 8 8 -(4 2 5 d a y s p o s t in fe c tio n )


Re

du

cti

on

%

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

50

75

90

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

F L V 0 8 9


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )0 .5 1 .0 1 .5 2 .0 2 .5

0

1 0 0

1 5 0

F L V 0 8 9 - (4 2 5 d a y s p o s t in fe c t io n )


Re

du

ctio

n %

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0



Re

du

cti

on

%

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )


50

75

90

Z IK V M R 7 6 6

Y F 1 7 D

J E IM O J E V

D E N V -4 (H 2 4 1 )

FLV-088: - Probable ZIKV, with unknown DENV frequent traveller to El Salvador.

FLV-089: - Probable ZIKV, with unknown DENV, frequent traveller to El Salvador.

394

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

F L V 0 9 1 (3 d a y s p o s t in fe c t io n )


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

1 .0 1 .5 2 .0 2 .5

0

1 0 0

1 5 0

F L V -0 9 1 (2 4 0 d a y s )


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

F L V 0 9 1


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

0 .5 1 .0 1 .5 2 .0 2 .5

0

1 0 0

1 5 0

F L V 0 9 0 - (4 2 5 d a y s p o s t in fe c t io n )


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

FLV-090: - Probable ZIKV only infection

FLV-091: - Probable ZIKV infection with known YF17D vaccination but no known DENV exposure. Frequent traveller to

endemic areas.

395

1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

Z IK V 0 3


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5 5 .0

0

1 0 0

1 5 0

Z IK V 0 1 (2 1 0 d a y s )


Re

du

cti

on

%


D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

J E IM O J E V

D E N V -4 (H 2 4 1 )

Y F 1 7 D

Z IK V M R 7 6 6

50

75

90

1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

Z IK V 0 1


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6

Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

Z IK V 0 2


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -4 (H 2 4 1 )

1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

Z IK V 0 2 (1 1 d a y s )


Re

du

ctio

n %

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -4 (H 2 4 1 )

ZIKV cases tested during diagnosis

396

1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

Z IK V 0 4


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

Z IK V 0 6


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6

Y F 1 7 D

J E IM O J E V

D E N V -4 (H 2 4 1 )

1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

Z IK V 0 7


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6

Y F 1 7 D

J E IM O J E V

D E N V -4 (H 2 4 1 )

397

0 .5 1 .0 1 .5 2 .0 2 .5

0

1 0 0

1 5 0

Z IK V 0 1 1


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6


Y F 1 7 D

J E IM O J E V

D E N V -1 (H W 2 0 0 1 )

D E N V -2 (N G C )

D E N V -3 (H 8 7 )

D E N V -4 (H 2 4 1 )

1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5

0

1 0 0

1 5 0

Z IK V 0 9


Re

du

cti

on

%

50

75

90

Z IK V M R 7 6 6

Y F 1 7 D

J E IM O J E V

D E N V -4 (H 2 4 1 )

Antibody mediated immune response against Flavivirus ...

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Transcript of Antibody mediated immune response against Flavivirus ...