Development of live bacterial delivery systems for presentation of dengue EDIII to the mucosal immune system 2

DEVELOPMENT OF LIVE BACTERIAL SYSTEMS
FOR PRESENTATION OF DENGUE EDIII TO THE
MUCOSAL IMMUNE SYSTEM






LAM JIAN HANG













NATIONAL UNIVERSITY OF SINGAPORE

2014
DEVELOPMENT OF LIVE BACTERIAL SYSTEMS
FOR PRESENTATION OF DENGUE EDIII TO THE
MUCOSAL IMMUNE SYSTEM

LAM JIAN HANG
B.SC (HONS), NUS





A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF
PHILOSOPHY

DEPARTMENT OF MICROBIOLOGY

NATIONAL UNIVERSITY OF SINGAPORE

2014
DECLARATION




I hereby declare that this thesis is my original work and it has been written by
me in its entirety. I have duly acknowledged all the sources of information
which have been used in the thesis.

This thesis has also not been submitted for any degree in any university
previously.




_______________________
Lam Jian Hang
20 August 2014





i

PUBLICATIONS

Hoo, R., Lam, J.H., Huot, L., Pant, A., Li, R., Hot, D., Alonso, S. (2014)
Evidence for a role of the polysaccharide capsule transport proteins in
pertussis pathogenesis. PLoS One. 9(12): e115243.

Ng, J.K., Zhang, S.L., Tan, H.C., Yan, B., Maria Martinez Gomez, J., Tan,
W.Y., Lam, J.H., Tan, G.K., Ooi, E.E., Alonso, S. (2014) First experimental
in vivo model of enhanced dengue disease severity through maternally
acquired heterotypic dengue antibodies. PLoS Pathog. 10(4): e1004031.



PRESENTATION AT INTERNATIONAL CONFERENCES

Lam, J.H., Alonso, S. (2012) Expression and delivery of dengue EDIII by
Bordetella pertussis BPZE1 via the BrkA autotransporter. Oral presentation
prize. In: 4th Australasian Vaccines and Immunotherapeutics Development
Meeting, Brisbane, Australia.















ii

ACKNOWLEDGEMENTS

I would like express to my heartfelt thanks to my supervisor Assoc. Prof.
Sylvie Alonso for first giving me the chance to join her lab in 2008, and
subsequently offering invaluable advice, guidance and opportunities to
develop my mind and skills as a researcher. Over the years, I have known her
to be the most patient and encouraging supervisor I could ask for and, once
again, I offer my sincere appreciation for all she has taught me.

To my lab mates, past and present, I thank you all for making the working
environment lively, entertaining, exciting and, frequently, edible. I really
treasure the friendships that we’ve built and the time we spent as a lab. A big
thank you to all the names listed in Table A below :)

Past
Present
Jowin Ng Kai Wei
Lin Wenwei
Regina Hoo May Ling
Julia Maria Martinez Gomez
Annabelle Lim Rui Fen
Michelle Ang
Xu Wei Zhen
Vanessa Koh
Grace Tan Kai Xin
Emily Ang
Khong Wei Xin
Ong Li Ching
Zarina
Chionh Yok Hian
Li Rui
Ng Sze Wai
Aakanksha
Issac Too

Anna Ker

Yeo Huimin

Eshele

Table A: List of awesome SA lab mates encountered over the course of
my PhD candidature.

I would also like to express my appreciation to my TAC Dr. Ooi Eng Eong
and Dr. Ratha Mahendran for offering useful comments and advice during our
meeting.

Lastly, I would like to express my deepest gratitude to my parents and my
sister who have been hugely supportive and understanding when I had to shift
my residential address to CeLS during my most intensive months. None of this
work would have been possible without them.
iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii
TABLE OF CONTENTS iii
SUMMARY xii
LIST OF TABLES xiv
LIST OF FIGURES xv
LIST OF ABBREVIATIONS xviii

CHAPTER 1: INTRODUCTION 1
1.1. DENGUE: VIROLOGY, DISEASE
AND EPIDEMIOLOGY 1
1.1.1. Virion structure and assembly 1
1.1.2. Disease and epidemiology 5

1.2. THE ADAPATIVE IMMUNE RESPONSE
FOLLOWING A DENV INFECTION 7
1.2.1. The anti-DENV immune response in
protection and disease enhancement 7
1.2.1.1. Antibodies 8
1.2.1.1.1. Antibody-dependent enhancement
(ADE) of infection 8
1.2.1.2. T cells 9
1.3. PROSPECTS FOR A DENGUE VACCINE 10
1.3.1. Challenges of vaccine development 10
1.3.1.1. Unbalanced immune responses 11
1.3.1.2. Immune correlates of protection 11
1.3.1.3. Animal model 12
1.3.2. Vaccine candidates in development 15
1.3.2.1. LATVs 15
1.3.2.2. Subunit vaccines as an alternative 15
1.4. DENV ENVELOPE GLYCOPROTEIN 19
1.4.1. Structural and serological characteristics 19
1.4.2. EDIII as a vaccine candidate: some considerations 20
1.4.2.1. Production and protective efficacy 20
iv

1.4.2.2. Relevance to the human disease 20
1.4.2.3. Quality of the anti-EDIII antibody response 21
1.5. LIVE BACTERIAL VECTORS FOR
ANTIGEN DELIVERY 22
1.5.1. Rationale for using a live bacterial vector 22
1.5.2. Attenuated pathogens 24
1.5.2.1. B. pertussis as a live vector for
nasal delivery of heterologous antigens 25

1.5.2.1.1. Resurgence of pertussis despite vaccination 25
1.5.2.1.2. Developing a live attenuated B. pertussis vaccine 26
1.5.2.1.3. Virulence factors 26
1.5.2.1.3.1. FHA 27
1.5.2.1.3.2. PTX 28
1.5.2.1.3.3. BrkA 30
1.5.2.1.4. B. pertussis as a live delivery system 31
1.5.2.1.4.1. Integration of foreign genes into B. pertussis genome 31
1.5.2.1.4.2. Virulence factors as carriers for antigen presentation 33
1.5.3. Lactic acid bacteria 36
1.5.3.1. L. lactis as a live vector for delivery of
heterologous antigens 36
1.5.3.1.1. Characteristics of L. lactis 36
1.5.3.1.2. The L. lactis molecular toolbox 38
1.5.3.1.2.1. Promoters 38
1.5.3.1.2.1.1. Constitutive promoters 38
1.5.3.1.2.1.2. Inducible promoters 39
1.5.3.1.2.2. Protein targeting 41
1.5.3.1.2.2.1. Cytoplasmic expression 41
1.5.3.1.2.2.2. Secretion 41
1.5.3.1.2.2.3. Cell wall associated 42
1.5.3.1.3. L. lactis as live mucosal vaccine 46
1.6. HETEROLOGOUS PRODUCTION OF EDIII
IN B. PERTUSSIS AND L. LACTIS – SOME
CONSIDERATIONS 48
1.6.1. B. pertussis – a gram negative organism 48
v

1.6.2. L. lactis – a gram positive organism 49
1.7. OBJECTIVES OF THIS PROJECT 51

CHAPTER 2: MATERIALS AND METHODS 52
2.1 ASSESSING THE SUITABILITY OF EDIII
AS A SUBUNIT VACCINE CANDIDATE 53
2.1.1. Escherichia coli work 53
2.1.1.1. E. coli strains, plasmids and culture conditions 53
2.1.1.1.1. Bacterial strains and plasmids 53
2.1.1.1.2. Culture conditions 54
2.1.1.2. Molecular biology 54
2.1.1.2.1. List of primers 54
2.1.1.2.2. DNA amplification 54
2.1.1.2.3. Restriction enzyme digest, agarose gel
electrophoresis, gel extraction and generation
of recombinant DNA 55
2.1.1.2.4. E. coli transformation, plasmid extraction and
DNA sequencing 55
2.1.1.3. Production and purification of recombinant
EDIII (rEDIII) 55
2.1.1.3.1. Expression of ediii by IPTG induction 55
2.1.1.3.2. Inclusion body isolation 55
2.1.1.3.3. Solubilisation of inclusion body and refolding of rEDIII 56
2.1.1.3.4. Purification of rEDIII using Ni-NTA chromatography 56
2.1.2. Animal work 57
2.1.2.1. Mouse strains 57
2.1.2.2. Subcutaneous injection 57
2.1.2.3. Assessment of serum antibody responses 57
2.1.2.3.1. Enzyme-linked immunosorbent assay (ELISA) 57
2.1.2.3.1.1. Preparation of coating antigens 57
2.1.2.3.1.2. Indirect ELISA 58
2.1.3. Virus work 58

2.1.3.1. Virus strain, cell lines, growth conditions and
virus quantitation 58
vi

2.1.3.1.1. Virus strain and cell lines 58
2.1.3.1.2. Growth conditions 59
2.1.3.1.3. Virus quantitation 59
2.1.3.1.4. Plaque reduction neutralisation test (PRNT) 60
2.1.4. Statistical analysis 60
2.2. BORDETELLA PERTUSSIS AS A LIVE VECTOR
FOR THE PRODUCTION AND MUCOSAL
DELIVERY OF DENV2 EDIII 61
2.2.1. Escherichia coli work 61
2.2.1.1. E. coli strains, plasmids and culture conditions 61
2.2.1.1.1. Bacterial strains and plasmids 61
2.2.1.1.2. Culture conditions 63
2.2.1.2. Molecular biology 63
2.2.1.2.1. List of primers 63
2.2.1.2.2. Polymerase chain reaction (PCR) 64
2.2.1.2.2.1. DNA amplification for cloning work 64
2.2.1.2.2.2. Colony PCR screening 65
2.2.1.2.3. Restriction enzyme digest 65
2.2.1.2.4. Agarose gel electrophoresis 65
2.2.1.2.5. Gel extraction 66
2.2.1.2.6. Generation of recombinant DNA 66
2.2.1.2.7. E. coli heat shock transformation 66
2.2.1.2.8. Plasmid extraction 67
2.2.1.2.9. DNA Sequencing 67
2.2.2. Bordetella pertussis work 67
2.2.2.1. B. pertussis strains and culture conditions 67

2.2.2.1.1. Bacterial strains 67
2.2.2.1.2. Culture conditions 68
2.2.2.2. Bacterial transformation 69
2.2.2.2.1. Preparation of electrocompetent cells 69
2.2.2.2.2. Electroporation 69
2.2.2.2.3. Double homologous recombination 69
2.2.2.3. Analysis of protein production 70
2.2.2.3.1. Western blot 70
vii

2.2.2.3.1.1. Preparation of bacterial lysate and
culture supernatant 70
2.2.2.3.1.2. Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) 70
2.2.2.3.1.3. Coomassie Blue staining 71
2.2.2.3.1.4. Electroblotting 71
2.2.2.3.1.5. Immunoblotting 71
2.2.2.3.2. Flow cytometry 72
2.2.2.4. Animal work 73
2.2.2.4.1. Mouse strains 73
2.2.2.4.2. Intranasal inoculation 73
2.2.2.4.3. Lung colonisation study 73
2.2.2.4.4. Immunisation study 74
2.2.2.4.4.1. Immunisation and blood collection schedule 74
2.2.2.4.4.2. Assessment of serum IgG response by
enzyme-linked immunosorbent assay (ELISA) 74
2.2.2.4.4.2.1. Preparation of coating antigens 74
2.2.2.4.4.2.2. Indirect ELISA 74
2.2.3. Statistical analysis 75
2.2.4. Virus work 75

2.2.4.1. Virus strain, cell lines, growth conditions,
virus quantitation and PRNT 75
2.3. LACTOCOCCUS LACTIS AS A LIVE VECTOR
FOR THE PRODUCTION AND MUCOSAL
DELIVERY OF DENV2 EDIII 76
2.3.1. Escherichia coli work 76
2.3.1.1. E. coli strains, plasmids and culture conditions 76
2.3.1.1.1. Bacterial strains and plasmids 76
2.3.1.1.2. Culture conditions 77
2.3.1.2. Molecular biology 78
2.3.1.2.1. List of primers 78
2.3.1.2.2. DNA amplification and colony PCR screening 79
2.3.1.2.3. Restriction enzyme digest, agarose gel
electrophoresis, gel extraction and generation
viii

of recombinant DNA 79
2.3.1.2.4. E. coli heat shock transformation, plasmid extraction
and DNA sequencing 79
2.3.2. Lactococcus lactis work 79
2.3.2.1. L. lactis strains and culture conditions 79
2.3.2.1.1. Bacterial strains 79
2.3.2.1.2. Culture conditions 80
2.3.2.2. Bacterial transformation 80
2.3.2.2.1. Preparation of electrocompetent cells 80
2.3.2.2.2. Electroporation 81
2.3.2.2.3. Selection of antibiotic resistant transformants 81
2.3.2.3. Analysis of protein production 82
2.3.2.3.1. Western blot 82
2.3.2.3.1.1. Nisin induction 82

2.3.2.3.1.2. Preparation of bacterial lysate and culture supernatant 82
2.3.2.3.1.3. SDS-PAGE, Coomassie Blue staining,
electroblotting and immunoblotting 82
2.3.2.3.2. ELISA 83
2.3.2.3.2.1. Preparation of recombinant L. lactis culture supernatant 83
2.3.2.3.2.2. Indirect ELISA 83
2.3.2.3.3. Flow cytometry 84
2.3.2.3.3.1. Preparation of gfp-expressing L. lactis strains for analysis 84
2.3.2.3.3.2. Preparation of LL-EDIII(CWA) strain for analysis 84
2.3.2.4. Animal work 85
2.3.2.4.1. Mouse strain 85
2.3.2.4.2. Immunisation study 85
2.3.2.4.2.1. Preparation of L. lactis inoculum 85
2.3.2.4.2.2. Immunisation and blood collection schedule 85
2.3.2.4.2.3. Assessment of serum IgG response by ELISA 86
2.3.2.4.2.3.1. Preparation of coating antigens 86
2.3.2.4.2.3.2. Indirect ELISA 86
2.3.2.4.2.4. Splenocyte restimulation assay 87
2.3.2.4.2.4.1. Preparation of single cell suspension 87
2.3.2.4.2.4.2. Measurement of cytokines by ELISA 87
ix

2.3.3. Statistical analysis 87

CHAPTER 3: RESULTS 88
3.1. ASSESSING THE SUITABILITY OF EDIII
AS A SUBUNIT VACCINE CANDIDATE 88
3.1.1. Production of rEDIII and immunisation of BALB/c mice 88
3.1.2. Assessment of serum antibody responses towards
EDIII and DENV2 88

3.2. BORDETELLA PERTUSSIS AS A LIVE VECTOR
FOR THE PRODUCTION AND MUCOSAL
DELIVERY OF DENV2 EDIII 91
3.2.1. Recombinant BPZE1 producing FHA-EDIII
chimeric protein 91
3.2.1.1. Construction of BP-FHA-EDIII, BP-FHA-EDIII(1Cys)
and BP-FHA-EDIII(0Cys) strains 91
3.2.1.2. Production and secretion of the FHA-EDIII chimera 92
3.2.2. Recombinant BPZE1 producing BrkA-EDIII chimeric protein 94
3.2.2.1. Construction of BP-BrkA-EDIII strain 94
3.2.2.2. Production of the BrkA-EDIII chimera 94
3.2.2.3. Surface exposure of EDIII was confirmed by
flow cytometry 96
3.2.2.4. The in vitro and in vivo fitness of BP-BrkA-EDIII
was not impaired 98
3.2.2.4.1. In vitro growth kinetics 98
3.2.2.4.2. Lung colonisation 99
3.2.2.5. Serum IgG response against DENV2 was not detected
after nasal immunisation with BP-BrkA-EDIII 99
3.2.3. Recombinant BPZE1 producing BrkA-EDIII-S1 chimeric protein 102
3.2.3.1. Construction of BP-BrkA-EDIII-S1 strain 102
3.2.3.2. Fusing EDIII and S1 to BrkA resulted in severe
degradation of the chimeric protein and the secretion
of a proteolytic fragment 103
3.2.3.3. SecP33 appeared to compete with native S1 for
the same secretory pathway 105
x

3.2.3.4. Secretion of SecP33 might involve the Ptl-machinery 107
3.2.3.5. Fitness of BP-BrkA-EDIII-S1 strain was significantly

impaired 109
3.2.3.6. Serum IgG response against DENV2 was not detected
after nasal immunisation with BP-BrkA-EDIII-S1 110
3.2.4. Recombinant BPZE1 producing S1-EDIII chimeric protein 112
3.2.4.1. Construction of BPSE3 strain 112
3.2.4.2. Production of S1-EDIII chimeric protein by BPSE3 strain
was confirmed by western blot 113
3.2.4.3. Growth impairment was not observed in BPSE3 115
3.2.4.4. Serum IgG response against DENV2 was not detected
after nasal immunisation with BPSE3 116
3.3. LACTOCOCCUS LACTIS AS A LIVE VECTOR
FOR THE PRODUCTION AND MUCOSAL
DELIVERY OF DENV2 EDIII 120
3.3.1. Recombinant L. lactis producing cytoplasmic EDIII 120
3.3.1.1. Construction of gfp-expressing L. lactis strains 120
3.3.1.2. Effect of nisin concentration on the yield of EDIII-GFP 122
3.3.1.3. Comparing the effects of semi-aerobic and aerobic culture
conditions on the yield and folding of cytoplasmic EDIII 123
3.3.1.3.1. Yields of cytoplasmic EDIII were comparable after
semi-aerobic or aerobic induction 123
3.3.1.3.2 Folding of EDIII seemed to be improved after
aerobic induction 124
3.3.2. Recombinant L. lactis producing secreted or cell wall
associated-EDIII 128
3.3.2.1. Construction of LL-EDIII(Sec) and LL-EDIII(CWA)
strains 128
3.3.2.2. Production of EDIII(Sec) and EDIII(CWA) proteins
were confirmed by western blot 130
3.3.2.3. Presence of EDIII in culture supernatant of LL-EDIII(Sec)
was further confirmed with ELISA 132

3.3.2.4. Bicarbonate-buffered medium improved the yields of
EDIII protein 133
xi

3.3.2.5. EDIII was not detected on the surface of LL-EDIII(CWA) 136
3.3.2.6. Subcellular location determined stability of EDIII
during erythromycin and nisin deprivation 138
3.3.2.7. Immunogenicity study 140
3.3.2.7.1. Intratracheal immunisation of C57BL/6 with
LL-EDIII-GFP or LL-EDIII(CWA) but not
LL-EDIII(Sec) generated low-level IgG responses
against EDIII 140
3.3.2.7.2. Assessing the ability of ediii-expressing L. lactis to
prime the immune system to respond to DENV challenge
(preliminary work) 144

CHAPTER 4: DISCUSSION 146
4.1. ASSESSING THE SUITABILITY OF EDIII AS A
SUBUNIT VACCINE CANDIDATE 146
4.2. BORDETELLA PERTUSSIS AS A LIVE VECTOR
FOR THE PRODUCTION AND MUCOSAL
DELIVERY OF DENV2 EDIII 147
4.3. LACTOCOCCUS LACTIS AS A LIVE VECTOR
FOR THE PRODUCTION AND MUCOSAL
DELIVERY OF DENV2 EDIII 153

CHAPTER 5: GENERAL CONCLUSIONS 161

CHAPTER 6: FUTURE WORK 164

REFERENCES 166







xii

SUMMARY

As the target of strongly neutralising monoclonal antibodies, EDIII is a highly
popular subunit vaccine candidate. However, monomeric EDIII is poorly
immunogenic. A live bacterial vector that expresses and delivers EDIII to the
host immune system represents a potential means of augmenting the immune
response. BPZE1, the live attenuated vaccine strain of the respiratory pathogen
Bordetella pertussis, was used to deliver DENV2 EDIII to the mucosal
immune system of the respiratory tract. To facilitate interaction with B cells,
EDIII was produced as a fusion to the FHA (surface exposed and secreted),
BrkA (surface exposed) or PTX (secreted) virulence factor. The FHA-EDIII
chimera proved unsuitable as the disulphide bond within EDIII was
incompatible with FHA translocation across the outer membrane, causing a
severe reduction in yield of the chimeric protein. Abolishing the disulphide
bond via substitution of one or both Cys residues with Gly restored cellular
protein levels but not secretion, and also led to the loss of a major neutralising
epitope. On the other hand, fusing EDIII to either BrkA or PTX resulted in
successful surface exposure and secretion, respectively. Immunogenicities of
all ediii-expressing recombinant BPZE1 strains generated in this study were
assessed via intranasal immunisation of BALB/c mice. Strong serum antibody

responses against the bacterial vector were consistently detected by ELISA.
However, EDIII-specific responses were weak and no significant response was
detected against DENV2. Absence of viral-neutralising activity in the immune
sera was confirmed by the plaque reduction neutralisation test. These results
therefore indicated that BPZE1 was not ideal for the presenting EDIII to the
respiratory mucosal immune system. As an alternative approach, we chose the
lactic acid bacterium L. lactis for EDIII delivery. Three recombinant strains of
L. lactis were constructed, producing cytoplasmic, secreted or cell wall
associated EDIII. Gene expression was driven by the inducible nisA promoter
cloned into the replicative pMG36e plasmid. Production of EDIII in the
different cellular compartments was confirmed by western blot. While
increasing doses of nisin (5 to 20 ng/ml) did not lead to higher EDIII yields,
addition of bicarbonate buffer to control acidity and suppress the bacteria acid
tolerance response resulted in appreciable increases in yields of secreted and
xiii

cell wall anchored EDIII. Immunogenicity of each recombinant strain was
assessed by immunising C57BL/6 mice via the respiratory route. Strong serum
antibody responses against L. lactis were detected by ELISA. However,
EDIII-specific responses were weak and only a single mouse immunised with
the cytoplasmic EDIII strain exhibited significant activity against DENV2.
Despite the general absence of antibody responses against the virus,
recombinant L. lactis was able to efficiently prime the host immune system, as
evidenced by high levels of IFN-γ in the supernatants of rEDIII- or DENV2-
restimulated splenocytes isolated from mice immunised with the cytoplasmic
EDIII L. lactis strain. Altogether, our work indicates that B. pertussis and L.
lactis bacteria may not represent promising live vectors for the mucosal
delivery of EDIII. They may however allow efficient priming of the host
immune system, thereby providing some protection against a subsequent
DENV challenge.

xiv

LIST OF TABLES

Table 1.1: Dengue vaccines in development 17
Table 1.2: Immune response and protection studies
with recombinant B. pertussis 35
Table 1.3: Cytoplasmic production of foreign antigens by L. lactis 43
Table 1.4: Secretion of foreign proteins by L. lactis 44
Table 1.5: Surface display of foreign proteins by L. lactis 45

Table 1.6: Immune response and protection studies with
recombinant L. lactis 47
Table 2.1: E. coli strains and plasmids 53
Table 2.2: Primers used for E. coli work 54
Table 2.3: Virus strain and cell lines 59
Table 2.4: E. coli strains and plasmids 61
Table 2.5: Primers used for E. coli or B. pertussis work 64
Table 2.6: B. pertussis strains 67
Table 2.7: List of primary antibodies used in western blot 72
Table 2.8: E. coli strains and plasmids 76
Table 2.9: Primers used for E. coli or L. lactis work 78
Table 2.10: L. lactis strains 79
Table 2.11: List of primary antibodies used in western blot 83
Table 3.1: PRNT
50
titres of rEDIII-immunised mice 90
Table 5.1: Strengths and weaknesses of BPZE1 and L. lactis 162











xv

LIST OF FIGURES

Figure 1.1 (A): Schematic representation of the DENV genome 3
Figure 1.1 (B): Schematic representation of the mature DENV virion 3
Figure 1.1 (C): Organisation of the E glycoprotein on the surface of
the mature virion 3
Figure 1.1 (D): Schematic representation of the DENV E protein
primary structure 4
Figure 1.1 (E): The E homodimer 4
Figure 1.1 (F): Ribbon diagram of EDIII protein 4
Figure 1.2: Global distribution of DENV serotypes from 2000 to 2013 7
Figure 1.3: Model of FHA secretion 28
Figure 1.4: Schematic representation of PTX 30
Figure 1.5: Model BrkA translocation 31
Figure 1.6: Schematic representation of homologous
recombination 32
Figure 3.1: Induction of neutralising antibody responses with the
administration of rEDIII 89
Figure 3.2: Schematics of fhaB-ediii cloning 92
Figure 3.3: Fusing EDIII to FHA was detrimental to production
and secretion of the chimeric protein 93
Figure 3.4: Schematics of brkA-ediii cloning 94
Figure 3.5: Production of BrkA-EDIII chimeric protein by recombinant
BPZE1 was confirmed by western blot 96
Figure 3.6: Surface exposure of BrkA-EDIII protein was confirmed by
flow cytometry 97
Figure 3.7: In vitro growth of BP-BrkA-EDIII was not impaired 98
Figure 3.8: BP-BrkA-EDIII retained its lung colonisation ability 99
Figure 3.9: Serum IgG response against DENV2 was not detected
despite strong activity towards B. pertussis 100

Figure 3.10: Schematics of brkA-ediii-ptxS1 cloning 102
Figure 3.11: Fusing EDIII and S1 to BrkA resulted in severe
degradation and the secretion of a protein fragment 104
Figure 3.12: SecP33 appeared to compete with native S1 for the same
xvi

secretory pathway 106
Figure 3.13: Secretion of SecP33 appeared to be associated with the
secretion of B oligomer subunits 108
Figure 3.14: Fitness of BP-BrkA-EDIII-S1 was severely compromised 109
Figure 3.15: Serum IgG response against DENV2 was not detected in
mice immunised with BP-BrkA-EDIII-S1 111
Figure 3.16: Schematics of ptxS1-ediii cloning 113
Figure 3.17: Production and secretion of S1-EDIII protein was detected
by western blot 114
Figure 3.18: Growth impairment was not observed in BPSE3 116
Figure 3.19: Serum IgG response against DENV2 was not detected in
mice immunised with BPSE3 117
Figure 3.20: Schematic representation of gfp and ediii-gfp expressed
under the control of P
nisA
121
Figure 3.21: Effect of nisin concentration on the yield of EDIII-GFP 122
Figure 3.22: Assessment of protein production after semi-aerobic or
aerobic induction 124
Figure 3.23: Significant improvement in fluorescent subpopulation of
LL-EDIII-GFP after aerobic induction 126
Figure 3.24: Schematic representations of ediii(Sec) and ediii(CWA)
expressed under the control of P
nisA

129
Figure 3.25: Production of EDIII(Sec) and EDIII(CWA) proteins were
confirmed by western blot 131
Figure 3.26: Presence of EDIII in culture supernatant of LL-EDIII(Sec)
was further confirmed with ELISA 132
Figure 3.27: Yields of EDIII were improved with the use of bicarbonate-
buffered media 134
Figure 3.28: Surface exposure of EDIII was not detected by flow
cytometry 137
Figure 3.29: Impact of subcellular location on stability of EDIII during
erythromycin and nisin deprivation 139
Figure 3.30: Immunising with LL-EDIII-GFP, LL-EDIII(Sec) or
LL-EDIII(CWA) failed to elicit a functional
anti-DENV2 IgG response 141
xvii

Figure 3.31: Intratracheal administration of LL-EDIII-GFP appeared
to have successfully primed the immune system 145

xviii

LIST OF ABBREVIATIONS

ADCC Antibody-dependent cell-mediated
cytotoxicity
ADE Antibody dependent enhancement
ATP Adenosine triphosphate
BALF Bronchoalveolar lavage fluid
BG Bordet-Gengou medium

BHK Baby hamster kidney cell
BSA Bovine serum albumin
C Capsid
CFU Colony-forming unit
CMC Carboxymethyl cellulose
CPE Cytopathic effect
CWA Cell wall anchor
CWA
M6
Cell wall anchor of S. pyogenes M6
protein
DENV Dengue virus
DF Dengue fever
DHF Dengue haemorrhagic fever
DNA Deoxyribonucleic acid
DNT Dermonecrotic toxin
DSS Dengue shock syndrome
Dsb Disulphide bond forming enzyme
E Envelope
EDIII Domain III of envelope protein
EDTA Ethylenediaminetetraacetic acid
EGFP Enhanced green fluorescent protein
ER Endoplasmic reticulum
EV71 Enterovirus-71
FBS Foetal bovine serum
FDA Food and Drug Administration
FHA Filamentous haemagglutinin
GFP Green fluorescent protein
xix

GRAS Generally recognised as safe
GSK GlaxoSmithKline
HPV Human papilloma virus
IC Intracranial
IFN-α/β/γ Interferon-alpha/beta/gamma
IgA Immunoglobulin A
IgG Immunoglobulin G
IgM Immunoglobulin M
IL-2/4/6/12 Interleukin-2/4/6/12
IN Intranasal
IP Intraperitoneal
IPTG Isopropyl β-D-1-thiogalactopyranoside
IT Intratracheal
IV Intravenous
IWZ Inner wall zone
LAB Lactic acid bacteria
LATV Live attenuated tetravalent vaccine
LB Luria-Bertani
LPS Lipopolysaccharide
M Membrane
M2e Ectodomain of influenza M2 protein
mAb Monoclonal antibody
MADCAM1 Mucosal vascular addressin cell adhesion
molecule 1
MBP Mannose binding protein
MCS Multiple cloning site
MFI Mean fluorescence intensity
NGC New Guinea C strain
NHP Non-human primate
NIAID National Institute of Allergy and

Infectious Diseases
NICE Nisin-controlled expression
NIH National Institutes of Health
NOD/SCID Non-obese diabetic/
xx

severely compromised immunodeficient
NS Non-structural
OPD O-phenylenediamine dihydrochloride
ORF Open reading frame
PAMP Pathogen-associated molecular pattern
PBS Phosphate-buffered saline
PCR Polymerase chain reaction
PDK Primary dog kidney cell
PEG Polyethylene glycol
PFU Plaque-forming unit
POTRA Polypeptide-transport-associated
prM Pre-membrane
PRNT Plaque reduction neutralisation test
PRR Pattern recognition receptor
PTX Pertussis toxin
RNA Ribonucleic acid
SC Subcutaneous
SDS-PAGE Sodium dodecyl sulphate-
polyacrylamide gel electrophoresis
SP Signal peptide
SP
Usp45
Signal peptide of Usp45 protein
SS Stainer-Scholte medium

TBS Tris-buffered saline
TCT Tracheal cytotoxin
Th T helper cell
TLR Toll-like receptor
TNF-α Tumour necrosis factor-alpha
TSA Trypic soy agar
TTFC Tetanus toxin fragment C
UTR Untranslated region
VLP Virus-like particle
WRAIR Walter Reed Army Institute of Research
WT Wild type
YFV Yellow fever vaccine












CHAPTER 1 INTRODUCTION
Chapter 1: Introduction
1

CHAPTER 1 INTRODUCTION

1.1. DENGUE: VIROLOGY, DISEASE AND EPIDEMIOLOGY

1.1.1. Virion structure and assembly

Dengue virus (DENV) is an enveloped, positive stranded RNA virus that
exists as four antigenically distinct serotypes (DENV1-4). The single-stranded
viral RNA genome is 10.7 kb in length and contains a 5’ methyl guanosine cap,
5’ untranslated region (UTR), single open reading frame, and a 3’ UTR
(Figure 1.1, A) [1]. Viral RNA is translated to a polyprotein carrying three
structural proteins (capsid, envelope and membrane) that form the virus
particle and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A,
NS4B and NS5) required for viral replication.

The structure of DENV has been solved using a combination of cryoelectron
microscopy and image reconstruction techniques (Figure 1.1, B) [2, 3]. In the
culture supernatant of infected cells, the virus exists as a mix of mature and
immature forms with diameters of 50 nm and 60 nm, respectively [4]. Both
particles possess a surface glycoprotein shell and, beneath that, a host-derived
lipid bilayer. At the centre of the virion is the nucleocapsid core consisting of
genomic RNA and capsid (C) protein. The glycoprotein shell is composed of
envelope (E) and membrane (prM/M) proteins. Immature virions are non-
infectious and have spiky projections of 60 trimers of prM-E heterodimers.
Mature virions, on the other hand, are infectious and possess a smooth surface
composed of 90 E homodimers arranged to form an icosahedral scaffold
(Figure 1.1, C).

Cleavage of prM is necessary for DENV maturation and infectivity. This
cleavage is mediated by the host protease furin and generates the M protein,
which associates with the mature virion as a transmembrane protein beneath
the E protein shell [2-5]. The E protein is involved in receptor binding and

fusion with the host membrane. Structurally, it consists of three domains (DI,
DII and DIII) with DIII being identified as the putative host receptor binding