Establishing improved platforms for dengue diagnosis and hybridoma development using dengue virus envelope domain III antigen a

Development and characterization of dengue virus serotypes 1 to 4 recombinant envelope Domain III proteins and antibodies as diagnostic reagents for a biotin-streptavidin enhanced indir

ESTABLISHING IMPROVED PLATFORMS FOR

DENGUE DIAGNOSIS AND HYBRIDOMA DEVELOPMENT USING DENGUE ENVELOPE DOMAIN III ANTIGEN

MELVIN TAN LIK CHERN

NATIONAL UNIVERSITY OF SINGAPORE

2010

Melvin Tan Lik Chern

B.Sc (Hons), Monash University, Australia

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF MICROBIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE

2010

ESTABLISHING IMPROVED PLATFORMS FOR

DENGUE DIAGNOSIS AND HYBRIDOMA DEVELOPMENT USING DENGUE ENVELOPE DOMAIN III ANTIGEN

PUBLICATIONS AND PRESENTATIONS GENERATED DURING THE COURSE OF STUDY

Publications:

Tan, L.C.M., Chua, A.J.S., Goh, L.S.L., Pua, S.M., Cheong, Y.K and Ng, M.L (2010) A membrane chromatography method for the rapid purification

of recombinant flavivirus proteins Protein Expr Purif 74:129-37

- Provided at the back of thesis

Tan, L.C.M & Ng, M.L High throughput detection of dengue envelope

domain III-specific antibodies by monovalent and tetravalent streptavidin indirect ELISA Manuscript in preparation

biotin-Book Chapter:

Tan, L.C.M and Ng M.L Dengue envelope domain III protein: properties,

production and potential applications in dengue diagnosis In: Dengue virus: detection, diagnosis and control Chapter 3 Editor: Basak Ganim and Adam Reis ISBN: 978-1-60876-398-6 In press

- Provided at the back of thesis

International Conference Presentations (Oral):

Tan, L.C.M and Ng M.L (2008) Development and characterization of

dengue virus serotypes 1 to 4 recombinant envelope Domain III proteins and antibodies as diagnostic reagents for a biotin-streptavidin enhanced indirect

ELISA Wilbio 5 th International Meeting Bioprocess Technology: Asia Pacific, Singapore

Choi, J.H., Tan L.C.M., Ng M.L., Grotenbreg, G.M., Love, J.C and Ploegh, H.L (2009) Recent advancement of single cell assay as a platform

for infectious disease research International Conference on Materials for Advanced Technologies: GEM4/SMART Symposium on Infectious Diseases, Suntec City, Singapore

International Conference Presentations (Poster):

Tan L.C.M and Ng M.L (2009) Immunogenicity and protective efficacy of

dengue virus serotypes 1 to 4 recombinant envelope domain III proteins 8th Asia Pacific Congress of Medical Virology, Hong Kong SAR, China

Tan L.C.M and Ng M.L (2009) Improved detection of dengue envelope

domain III-specific antibodies by indirect biotin streptavidin ELISA

Emerging Infectious Diseases 2009, 8 - 11 Dec 2009, Duke-NUS, Singapore

Choi J.H., Tan L.C.M., Lee Y.H., Gijsbert M.J., Love J.C., Ng M.L and Ploegh, H.L (2009) Single cell assay for hybridomas and primary B

lymphocytes secreting antibodies against dengue envelope domain III

proteins Emerging Infectious Diseases 2009, 8 - 11 Dec 2009, Duke-NUS,

Singapore (Selected for colloquium)

Goh L.S.L., Tan L.C.M., Chua A.J.S., Pua S.M., Cheong Y.K and Ng M.L (2009) A membrane chromatography method for the rapid purification

of recombinant flavivirus proteins Emerging Infectious Diseases 2009, 8 - 11 Dec 2009, Duke-NUS, Singapore

Tan L.C.M., Choi J.H., Lee Y.H., Gijsbert M.J., Love J.C., Ng M.L and Ploegh, H.L (2010) Protein production and purification of dengue domain III

proteins for all four serotypes and subsequent sortase labelling for single cell

assay 10 th Nagasaki-Singapore Medical Symposium on Infectious Diseases, 15-16 April 2010, National University of Singapore, Singapore

Choi J.H., Tan L.C.M., Lee Y.H., Gijsbert M.J., Love J.C., Ng M.L and Ploegh, H.L (2010) High-throughput single cell assay: Screening by

microengraving and isolating by automatic micromanipulator for hybridomas

secreting antibodies against dengue envelope domain III proteins 10 th Nagasaki-Singapore Medical Symposium on Infectious Diseases, 15-16 April

2010, National University of Singapore, Singapore

Local Conference Presentations (Oral):

Choi, J.H., Tan L.C.M., Ng M.L., Grotenbreg, G.M., Love, J.C & Ploegh, H.L (2009) Progress on single cell assay for B lymphocytes and hybridoma

specific to dengue envelope domain III Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease - Interdisciplinary Research Group) Workshop-11 Jan 2010, Singapore

Local Conference Presentations (Poster):

Choi J.H., Tan L.C.M., Lee, Y.H., Lai, Y.Q., Rafiq, N.B.M., Grotenbreg, G.M., Ng M.L and Ploegh, H.L (2009) Protein purification for

immunization and labelling Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease - Interdisciplinary Research Group) Workshop-2009, Singapore

Choi J.H., Tan L.C.M., Lee Y.H., Ng, M.L., Love, J.C and Ploegh, H.L (2009) Recent advancement of single cell assay for infectious disease

research Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease - Interdisciplinary Research Group) Workshop-2009, Singapore

Choi J.H., Tan L.C.M., Lee Y.H., Gijsbert M.J., Love J.C., Ng M.L and Ploegh, H.L (2010) Advanced single cell assay (1) For hybridomas secreting

antibodies against dengue envelope domain III protein serotype 1 Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease - Interdisciplinary Research Group) Workshop-11 Jan

2010, Singapore

Choi J.H., Tan L.C.M., Lee Y.H., Gijsbert M.J., Love J.C., Ng M.L and Ploegh, H.L (2010) Advanced single cell assay (2) For hybridoma and

primary lymphocyte secreting multi-specific antibodies against dengue

envelope domain III proteins Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease - Interdisciplinary Research Group) Workshop-11 Jan 2010, Singapore

Choi J.H., Tan L.C.M., Lee Y.H., Love J.C., Ng M.L and Ploegh, H.L (2010) Pre-selective single cell assay for monoclonal antibodies specific to

dengue envelope domain III: Selection of single cells specific to only one

serotype or multi-serotypes Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease - Interdisciplinary Research Group) 3 rd Annual Workshop- 7-8 July 2010, Singapore

Choi J.H., Tan L.C.M., Love J.C., Ng M.L and Ploegh, H.L (2010)

Single cell assay for primary B lymphocytes specific to dengue envelope

domain III Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease - Interdisciplinary Research Group) 3 rd Annual Workshop- 7-8 July 2010, Singapore

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to Professor Ng Mah Lee for the

immeasurable amount of support and guidance she has provided me

throughout this study Professor Ng‟s insights into this project and patience towards me have been a true blessing

I sincerely thank the members of the Flavivirus Laboratory: Boon, Bhuvana,

Mun Keat, Han Yap, Xiao Ling, Li Shan, Shu Min, Samuel, Vincent, Adrian,

Edwin, Terence, Kim Long, Anthony and Audrey for their friendship, expert

technical advice on different techniques and constructive criticism I would

also like to thank the members of SMART-Single Cell Assay Group:

Professor Hidde L Ploegh, Dr Choi Jae Hyeok and Lee Ying Hui, for

providing their state of the art technology and expert advice

I am grateful to my parents who never ceased loving and supporting me

This work is dedicated to my wife, Lydia, and my son, Benjamin (whose name

means „Son of my right hand‟) Your presence alone is a constant joy and support for me I thank Jesus for his great abundance of love, wisdom and

grace My heart is truly warmed

Thank you

TABLE OF CONTENTS

Page Number

Publications and presentations generated during the course of study …… i

Acknowledgements ……… ……… iv

Table of Contents ……….……… v

Summary ……… ……… xi

List of Tables ……… ……….……….………… xiii

List of Figures ……… ……….……… xv

Abbreviations ………….……… ……… xx

CHAPTER 1 1.0 LITERATURE REVIEW 1.1 Introduction ……….……… … 1

1.2 Structural studies on the flavivirus envelope and DIII protein……… 4

1.3 Antagonistic activity of DIII protein ……… ………… … 5

1.4 Neutralizing epitopes on DIII protein ………….……… … 6

1.5 Production of DENV rDIII protein ………… ……… 8

1.5.1 rDIII protein expression ……… 8

1.5.2 rDIII protein purification ……… ……… … 11

1.5.2.1 Immobilized metal affinity chromatography (IMAC) …… ……… … 11

1.5.2.2 Membrane adsorber-based IMAC ….……… 13

1.6 DENV rDIII protein as a potential protein subunit vaccine 14

1.7 Potential applications of rDIII protein in dengue diagnosis 21

1.8 Microengraving as a potential tool for antibody generation 24

2.0 MATERIALS AND METHODS

2.1 Tissue culture and propagation of viruses ……… 29

2.1.1 Cell culture ……… ……… 29

2.1.2 Virus source and propagation ……… …… 29

2.1.3 Plaque assay…… ………… ……… …… 30

2.2 Bioinformatics and biostatistical analyses ……… 31

2.3 Construction of DIII-expression plasmids ……… 32

2.4 Transformation of E coli competent cells for protein expression ……….……… 37

2.5 Construction of Series A and B DIII-expression plasmids 37

2.6 Protein expression and purification ……….……… 43

2.6.1 Pilot expression of rDIII protein …… ……… 43

2.6.2 Batch production of rDIII protein ……… 43

2.6.3 Refolding of rDIII protein by dialysis ……… 44

2.6.4 rDIII protein purification ……… 44

2.6.5 Isolation of E coli inclusion bodies enclosing rDIII-protein ……… ……… 45

2.6.6 Metal affinity membrane chromatography … …… 46

2.6.7 Packed-bed chromatography …… ……… 49

2.6.7.1 Purification under native conditions ….…… 49

2.6.7.2 Purification under denaturing conditions… 49

2.6.8 Fast protein liquid chromatography (FPLC) … 49

2.7 Protein storage and product analysis ……….…… 50

2.8 Enzyme linked immunosorbent assay (ELISA) ……… 53

2.9 Immunization procedures ……….…… 56

2.9.1 Immunization of mice with DENV rDIII proteins

(serotypes 1-4) ……… 57

2.9.2 Determining the effects of immunization of mice with

different rDIII-protein-adjuvant formulations over a week period ……… 57

ten-2.9.3 Immunization of mice with rDIII proteins for

hybridoma development (collaboration with SMART) ……… 58 2.10 Plaque reduction neutralization test (PRNT) … ………… 58

2.11 Microengraving and micromanipulation of cells ……… 61

2.11.1 In vitro treatment of splenocytes obtained from mice

immunized with rDIII protein ……… 61

2.11.2 Treatment of splenocytes for microengraving studies

performed on primary B cells ……… 62

2.11.3 Development of DIII-specific hybridomas …… … 63

2.11.4 Preparation of cells for flow cytometry analyses … 64

2.11.5 Microengraving analysis of cells ……… … 64

2.11.6 Robotic retrieval of cells by micromanipulation … 68

3.0 RESULTS DEVELOPMENT OF FUNCTIONAL rDIII

PROTEINS

3.1 Determination of growth kinetics of DENV in C6/36 and Huh7

cells ……… 70 3.2 Bioinformatics analysis of envelope DIII proteins ……… 72

3.3 Construction of DIII-expression vectors ……… 80

3.4 Production of purified DENV rDIII proteins … ………… 80

3.4.1 Pilot protein expression ……… 80

3.4.2 Batch expression and purification of rDIII proteins 87

3.5 Characterization of purified DENV rDIII proteins …… … 93

3.5.1 Determining the homologous and heterologous

neutralizing potential of rDIII protein-specific antibodies ……… ……… 97

3.5.2 Removal of N-terminal hexahistidine tag by thrombin

cleavage reaction ……… ……… 101

4.0 RESULTS DENV rDIII PROTEIN-BASED DIAGNOSTIC

ASSAYS

4.1 DENV rDIII proteins as diagnostic reagents ……… 104

4.2 Development of monovalent biotin-streptavidin enhanced

indirect ELISA [iELISA-(BS)] ……….……… 104

4.2.1 Preliminary test ………… ……… 104

4.2.2 Optimization of monovalent DENV rDIII

iELISA-(BS)……… ……… 107 4.2.2.1 Exploring the suitability of various immuno-

plates for iELISA-(BS) ……….……… 108

4.2.2.2 Determining the duration required for optimal

coating of antigen on the immuno-plate … 111

4.2.2.3 Determining the minimal concentration of

antigen required for effective coating of immuno-plates ……… ………….113

4.2.2.4 Determining the optimal volume of antigen

required for coating of immuno-plate …… 116

4.2.2.5 Determining the optimal time required for assay

blocking ………….………119

4.2.2.6 Determining the time required for optimal

development of TMB substrate via a study comparing two different brands of TMB solutions ……… ……….… 121

4.2.2.7 Determining the optimal concentration of

streptavidin-HRP conjugate for DENV rDIII iELISA-(BS) ……….… 124

4.2.2.8 Determining the optimal concentration of

secondary antibody-biotin conjugate for DENV rDIII iELISA-(BS) ……….……… 127

4.3 Development of monovalent normal indirect ELISA

[iELISA-(N)]……… ……… 130

4.4 Sensitivity differences between iELISA-(BS) and

iELISA-(N)……… ……… 134 4.5 Assessing the specificity of DENV rDIII iELISA-(BS)

platforms for detection of homotypic and heterotypic DENV rDIII protein-specific antibodies ……… ….……… 136

4.6 Development of a tetravalent iELISA-(BS) platform … … 139

4.7 Establishing the suitability of monovalent and tetravalent

DENV rDIII iELISA-(BS) for high-throughput screening 140

4.8 Establishing the suitability of monovalent DENV rDIII

iELISA-(BS) for detection of seroconversion in mice ….… 144

4.9 Determining the relative avidities of rDIII protein-specific

antibodies ……….……….… 146

4.10 Application of iELISA-(BS) to rDIII-protein subunit vaccine

research ……… ……… 149

4.11 Detection of DIII-specific antibodies in human sera using

human IgM and IgG tetravalent rDIII protein-based (BS) ……….……… … 153

iELISA-4.12 Reducing background signal for human IgG tetravalent DENV

rDIII iELISA-(BS) platform ……… …… … 160

5.0 RESULTS ESTABLISING IMPROVED PLATFORMS FOR

HYBRIDOMA DEVELOPMENT

development……… … 163

5.2 Determining the feasibility of Sortase labelling of DENV rDIII

proteins by homology modeling analysis ………….……… 164

5.3 Construction of DENV rDIII Series A and B expression

vectors……… 166

5.4 Production and purification of Series A and B DENV rDIII

proteins……… ……….……… 168 5.5 Pushing the boundaries of microengraving ……… ……… 176

5.5.2 Refining processes to obtain stimulated primary B-cells

for microengraving ……….………… 181

5.5.3 Microengraving analysis of primary B cells ……… 182

5.6 Development of hybridomas secreting DENV rDIII serotype-specific monoclonal antibodies ……….……… 188

5.7 Further characterization of clones 1B2, 1B3, 3B4 and 3B22……… ……… 203

6.0 DISCUSSION ……….……… … ……… 214

REFERENCES ……… 229

APPENDICES Appendix 1 Reagents of cell culture and virus infection ……… 242

Appendix 2 Reagents for molecular cloning ……… 244

Appendix 3 Reagents for protein research ……… 246

Appendix 4 Reagents for ELISA ……… 254

Appendix 5 Reagents for microengraving ……… … 255

Appendix 6 Optimization of protocols and reagents for DENV iELISA-(BS) ……… 257

Appendix 7 Validation of DENV rDIII iELISA-(BS) platforms 272

Appendix 8 A membrane chromatography method for the rapid purification of recombinant flavivirus proteins Protein Expr Purif 74:129-37……… 285

Appendix 9 Book Chapter: Dengue envelope domain III protein: properties, production and potential applications in dengue diagnosis In: Dengue virus: detection, diagnosis and control Chapter 3 Uncorrected Proof ……… 295

Summary

Dengue is a mosquito-transmitted viral disease of global importance By

exploiting the immunogenic properties of dengue virus (DENV) envelope

recombinant Domain III (rDIII) proteins, several innovative platforms were

established for research and diagnosis of dengue infection In this study, a

metal affinity membrane chromatography method for rapid purification of

DENV rDIII proteins (serotypes 1 - 4) was established for the first time for

flavivirus research This method of purification is superior over traditional

packed-bed chromatography because it is faster, more consistent and more

cost-effective to perform Purified DENV rDIII proteins were subsequently

characterized and used for functional studies

Biotin-streptavidin enhanced indirect ELISA platforms [iELISA-(BS)] for

detection of DIII-specific antibodies in mice and human sera were also

established When compared against the normal indirect ELISA, the former

demonstrated three times improvement in signal-to-noise ratio and at least one

logarithmic improvement in the limit of detection Further to having improved

sensitivity, these monovalent DENV rDIII iELISA-(BS) platforms (serotypes

1 - 4) exhibited excellent high-throughput screening potential (z-factor >

0.75) These assays were used for detection of seroconversion in mice after

DENV rDIII immunization, as well as characterization of monoclonal

antibodies Additionally, the monovalent DENV rDIII iELISA-(BS) platforms

were modified to detect for relative avidity rDIII protein-specific antibodies to

its corresponding proteins Significantly, human IgM and IgG tetravalent

DENV rDIII protein-based ELISAs developed in this study were able to

effectively detect DIII protein-specific antibodies in DENV patient sera The

assays were able to assist in differentiation between primary and secondary

infection in patient sera These results supported that DIII protein-based

ELISAs are potential alternatives for commercially available diagnostic kits

A further study was performed to establish an integrated platform (using

state-of-the-art technologies such as sortagging, microengraving and

micromanipulation) to dengue research The sortagging protein labelling

method allowed for site-directed labelling of DENV rDIII proteins (serotypes

1 - 4) without masking its epitopes Microengraving method was improved for

examination of individual primary B cells (obtained from mice immunized

with DENV rDIII protein) for its antibody secreting capability It was also

used for rapid screening of hybridomas which secreted DIII protein-specific

monoclonal antibodies Each hybridoma clone was identified and retrieved

using a specialized robot, via the process of micromanipulation Monoclonal

antibodies produced by these clones were subsequently characterized via

monovalent DENV rDIII-protein iELISA-(BS) and plaque reduction

neutralization test Through this process, four commercially viable clones of

hybridomas secreting mono-specific neutralizing monoclonal antibodies (1B2,

1B3, 3B4 and 3B22) were developed

Table 2.1 Primers used for PCR amplification of DIII DNA of DENV

(serotypes 1 - 4) with Nhe I and Xho I RE recognition sites

engineered at the 5‟ and 3‟ ends, respectively ……… …… 34

Table 2.2 Primers used for PCR amplification of DIII DNA of DENV

(serotypes 1 - 4) with Nco I and Xho I RE recognition sites

engineered at the 5‟ and 3‟ ends, respectively ……… …… 40

Table 2.3 Immunization of mice with DENV3 and 4 rDIII proteins over a

ten-week period ……… ……… … 59

Table 3.1 Composition of DENV DIII protein (serotypes 1 - 4) ….… 75

Table 3.2 Study to optimize protein purification via MA-based

chromatography ……… 89

Table 3.3 Mass spectrometry analyses of DENV rDIII proteins (serotypes

1 - 4) ……… ……… 96

Table 3.4 PRNT50 results of DENV DIII-specific antisera against DENV

(serotypes 1 - 4), Wengler or Kunjin strains of WNV…… 100

Table 4.1 Suitability of various immuno-plates for iELISA-(BS)

serotypes 1 - 4 (examining P/N ratios) ……….……… 110

Table 4.2 Optimal duration required for effective antigen coating

(examining P/N ratios) ……… ……… 112

Table 4.3 Determining the minimal concentration of antigen required for

effective coating (examining P/N ratios) …… ……… 115

Table 4.4 Optimal volume of antigen required for coating (examining P/N

ratios) ……… 118

Table 4.5 Optimal time required for assay blocking (examining P/N

ratios) ……… …….……… 120

Table 4.6 Optimal concentration of streptavidin-HRP required for

iELISA-(BS) (examining P/N ratios) ………126

Table 4.7 Optimal concentration of secondary antibody-biotin conjugate

for iELISA-(BS) (examining P/N ratios) ………… ……… 129

Table 4.8 Optimal concentration of secondary antibody-HRP conjugate

for iELISA-(N) (examining P/N ratios) ……… … ……… 133

Table 4.9 Difference in sensitivity between iELISA-(BS) and iELISA-(N)

(examining P/N ratios) ……… ……… 135

Table 4.10 Sero-crossreactivity of rDIII protein-specific homotypic or

heterotypic antibodies with DIII proteins Results were normalized in the form of SP ratios ……… ……… 138

Table 4.11 Z-factor calculations for monovalent DIII iELISA-(BS) and

tetravalent DIII iELISA-(BS) ……… … 143

Table 4.12 PRNT50 results obtained for DENV3 or 4 DIII-specific

antibodies against respective viruses ……… 152

Table 4.13 Comparison of tetravalent DENV rDIII iELISA-(BS) IgM and

IgG platforms against Commercial Panbio IgM and IgG serological assays ……… ……… 158

Table 4.14 Summary of serological test results ………… ……… 159

Table 5.1 Calibration of HiLoad 16/60 (Superdex 75) an AKTA

FPLCTM liquid chromatography system ………….……… 171

Table 5.2 Summary of PRNT50 results performed using MAbs 1B2, 1B3,

3B4 and 3B22 ……… ………… 213

LIST OF FIGURES

Page Number

Figure 1.1 Translation of viral RNA yields a single polypeptide that is

further processed by cellular and viral proteases, generating three structural and seven non-structural proteins Structure of the envelope protein dimer of a mature dengue virus particle……… 3

Figure 1.2 Model for antibody-dependent enhancement of virus

infection……… …….……… 16

Figure 2.1 DNA map of amplicons after PCR amplification using primers

described in Table 2.1 Nhe I and Xho I RE recognition sites

Plasmid map of pET28a (Novagen) expression vector ….… 35

Figure 2.2 DNA map of amplicons for Series A and B, respectively, after

PCR amplification using primers described in Table 2.2 Nco I

RE and Xho I recognition sites ……… ……… 39

Figure 2.3 Set-up of MA-based chromatography for purification of rDIII

proteins ……… ……… 47

Figure 2.4 Schematic diagram representing the different components of

iELISA-(BS) ……….……… 54

Figure 2.5 Schematic diagram depicting the workflow for microengraving

of a heterogenous culture of cells ……….……… 65

Figure 2.6 Automated robotic retrieval of single cell of interest using a

micromanipulator ……… ……… 69

Figure 3.1 Growth kinetics of DENV (serotypes 1 – 4) in C6/36 mosquito

and Huh7 human hepatoma cells ……… ………… 71

Figure 3.2 Alignment of the amino acid sequences of DENV envelope

DIII proteins (serotypes 1 - 4) ……… …… 73

Figure 3.3 Antigenicity and hydrophilicity plots of individual DIII

proteins (serotypes 1 - 4) ………… ……… 76

Figure 3.4 An overall comparison of DENV1 - 4 protein antigenic indexes

and hydrophilicity plots ……… 78

Figure 3.5 PCR amplification of DIII cDNA ……….……… 81

Figure 3.6 Plasmid maps of pET28a comprising DENV DIII inserts

(serotypes 1 - 4) ……… 82

Figure 3.7 SDS-PAGE analyses of time course protein expression of

DENV rDIII proteins ……… 84

Figure 3.8 SDS-PAGE analysis of rDIII protein expression levels of

selected E coli strain Rosetta(DE3) colonies after IPTG

induction ……… …… 85

Figure 3.9 SDS-PAGE analyses of the effect of IPTG concentration on

DENV rDIII protein expression ……… 86

Figure 3.10 SDS-PAGE analyses of the purification profiles for MA-based

purification of DENV3 rDIII protein ……… 88

Figure 3.11 SDS-PAGE analyses of the purification profiles for MA-based

purification of rDIII proteins of DENV (serotypes 1 - 4).… 91

Figure 3.12 Quantitative densitometry analyses of protein eluates (E1, E3,

E5, E7, E9 and E11) of DENV (serotypes 1 - 4) …… …… 92

Figure 3.13 SDS-PAGE analysis of the purification profile for DENV3

rDIII protein purification via packed-bed metal affinity chromatography ……… 94

Figure 3.14 Characterization of DENV rDIII proteins (serotypes 1 - 4) 95 Figure 3.15 Neutralizing activity of DENV DIII-specific anti-sera against

DENV (serotypes 1 - 4), respectively ……… …… 98

Figure 3.16 Thrombin cleavage of DENV3 rDIII protein carried out at 2 hr

and overnight (O/N) durations ……… ……… 102

Figure 4.1 Preliminary test on DENV rDIII-protein monovalent

iELISA-(BS) serotypes 1 - 4 ……… ……… 106

Figure 4.2 Examining the suitability of various immuno-plates for

iELISA-(BS) serotypes 1 - 4 ……….…… 110

Figure 4.3 Determining the optimal duration required for effective coating

of antigen on Multisorp plate ……… 112

Figure 4.4 Determining the minimal concentration of antigen required for

effective coating of immuno-plates ……… ………… 114

Figure 4.5 Determining the optimal volume of antigen required for coating

of immuno-plate ……… 118

Figure 4.6 Determining the optimal time required for assay blocking…120

Figure 4.7 Determining the optimal time required for TMB substrate

development ……… … 122

Figure 4.8 Determining the optimal concentration of streptavidin-HRP for

DENV rDIII iELISA-(BS) ……… 125

Figure 4.9 Determining the optimal concentration of secondary

antibody-biotin conjugate for DENV rDIII iELISA-(BS) ………… 128

Figure 4.10 Determining the optimal concentration of secondary

antibody-HRP conjugate for DENV rDIII iELISA-(N) ……… …… 132

Figure 4.11 Comparison of the P/N ratios for (BS) and

iELISA-(N)……….………… 135

Figure 4.12 Comparison of the LOD for iELISA-(BS) and iELISA-(N) 137 Figure 4.13 Development of tetravalent iELISA-(BS) for detection of

DENV rDIII protein-specific anti-sera (serotypes 1 - 4)… 141

Figure 4.14 Detection of seroconversion in mice after rDIII protein

immunization (serotypes 1 - 4) ……… 145

Figure 4.15 Mean relative avidity of DENV rDIII specific anti-sera,

serotypes 1 - 4, against its corresponding proteins … …… 148

Figure 4.16 Sero-conversion of mice immunized with DENV3 or 4 rDIII

proteins ……….… 150

Figure 4.17 Relative avidity of anti-sera obtained from mice immunized

with DENV3 or 4 rDIII proteins ……… ……… 152

Figure 4.18 DENV rDIII protein-based tetravalent iELISA-(BS) IgM and

IgG platform detection of DIII-specific antibodies in patient sera Results for representative samples from patients B, K and P……….……… 155

Figure 4.19 DENV rDIII protein-based tetravalent iELISA-(BS) IgM and

IgG platform detection of DIII-specific antibodies in patient sera Results for representative samples from patients D, O and Q……….…… 156

Figure 4.20 Effect of E coli extract on reduction of background for human

Figure 5.1 Ribbon diagram of dengue (serotypes 1-4) DIII protein

structures ……… ………… 165

Figure 5.2 PCR amplification of DENV DIII DNA ………… ……… 167

Figure 5.3 SDS-PAGE analyses of the purification profiles for Series A

DENV rDIII proteins (serotypes 1 - 4) ……….……… 169

Figure 5.4 SDS-PAGE analyses of the purification profiles for Series B

DENV rDIII proteins (serotypes 1 - 4) …… … ………… 170

Figure 5.5 Purification of Series A DENV rDIII proteins (serotypes 1 - 4)

Figure 5.8 Investigating the optimal time required for effective sortase

enzymatic reaction on Series A DENV1 rDIII proteins… 179

Figure 5.9 Investigating the effect of increasing reagent (Sortase and

nucleophile) concentrations on sortase enzymatic reaction on Series A DENV1 rDIII proteins ……… 180

Figure 5.10 Flow cytometry analysis of mice splenocytes cultured in

RPMI-10/HI media enriched with CPG-ODN 1826 and LPS… … 183

Figure 5.11 Examination of specific-antibody secretion qualities of primary

B cells via microengraving technique ……… … 185

Figure 5.12 Photograph showing the improved micromanipulation

technique for depositing 1 µl buffer containing a single cell into

a well of a PCR tube ……… ……… … 187

Figure 5.13 Microarray analysis of slides processed via microengraving

method ……… 189

Figure 5.14 Images representing the complete process for programmed

retrieval of cell from a single microwell, using CellCelector micromanipulator ……… ……… 191

Figure 5.15 Brightfield images of hybridomas - 1B1, 1B2, 1B3, 1B4, 1B4,

1B6 and 1B12, respectively ……… ……… 194

Figure 5.16 Brightfield images of hybridomas - 3B4, 3B5, 3B6, 3B7, 3B16,

3B22, 3B23, 3B33, 3B35 and 3B36, respectively ….… … 195

Figure 5.17 Indirect ELISA detection of rDIII protein-specific antibodies in

tissue culture fluid (TCF) obtained from 1B1, 1B2, 1B3, 1B4, 1B6 and 1B12 ……… ………… 198

Figure 5.18 Indirect ELISA detection of rDIII protein-specific antibodies in

tissue culture fluid (TCF) obtained from 3B4, 3B5, 3B6, 3B7, 3B16, 3B22, 3B23, 3B33, 3B35 and 3B36 ……… 200

Figure 5.19 Affinity purification of antibodies using HiTrapTM Protein G

Columns (GE Healthcare, USA) coupled with AKTA FPLC system ……… ……… 204

Figure 5.20 Qualitative analysis of specificity of Mabs 1B2, 1B3, 3B4 and

3B22 to DENV1, 2, 3 or 4 using DENV-coated indirect ELISA……… 206

Figure 5.21 Neutralizing activity of MAbs against DENV1 and 3 … 209 Figure 5.22 Neutralizing activities of 1B2 and 1B3 MAbs against DENV1

isolated from Patients 4172 and 3908 ……… …… 211

Figure 5.23 Neutralizing activities of 3B4 and 3B22 MAbs against DENV3

isolated from Patients 4176 and 2392 ……… … 212

Figure 6.1 Establishing improved platforms for dengue diagnosis and

hybridoma development using dengue envelope Domain III antigen ……… ……… 228

BALB/C Bagg Albino Strain C

BCIP/NBT 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium

cDNA Complementary deoxyribonucleic acid

DAPI 4',6-diamidino-2-phenylindole

EDTA Ethylenediaminetetraacetic acid

ELISA Enzyme linked immunosorbent assay

FPLC Fast protein liquid chromatography

H2O Water

HPLC High performance liquid chromatography

His-tag Histidine tag

HRP Horse raddish peroxidase

HAT Hypoxanthine thymidine aminopterin

iELISA-(BS) Indirect ELISA enhanced with a biotin-streptavidin system iELISA-(N) Indirect ELISA normal (without signal enhancement using a

biotin-streptavidin system IMAC Immobilized metal-ion affinity chromatography

IPTG Isopropyl β-D-1-thiogalactopyranoside

MALDI-TOF Matrix-assisted laser desorption/ionization - Time of flight

MOI Multiplicity of infection

PBST Phosphate buffered saline/Tween 20

PCR Polymerase chain reaction

PRNT Plaque reduction neutralization test

PVDF Polyvinylidene fluoride

rDIII Recombinant Domain III

RT-PCR Reverse transcription polymerase chain reaction

SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SMART Singapore - Massachusetts Institute of Technology Alliance for

Research and Technology

CHAPTER 1 Literature Review

Dengue virus (DENV) is a positive-sense, single-stranded RNA virus

belonging to the genus Flavivirus within the Flaviviridae family It causes

disease ranging from mild dengue fever (DF) to severe dengue hemorrhagic

fever and dengue shock syndrome (DHF/DSS) (Alvarez et al., 2006; Henchal

& Putnak, 1990; Kyle & Harris, 2008; Malavige et al., 2004) It has been

estimated that more than 2.5 billion people in over 100 countries are at risk of

dengue infection, with several hundred thousand cases of life threatening

DHF/DSS occurring every year (Gubler, 1998, 2002) Other members of the

Flaviviridae family include Japanese encephalitis virus (JEV), tick-borne

encephalitis virus (TBEV) and West Nile virus (WNV)

Dengue fever is categorized by rapid onset of fever, severe headache,

retro-orbital pain, gastrointestinal discomfort and rashes (Martina et al., 2009)

Minor haemorrhagic manifestations such as petechiae formation may be

observed Patients suffering from DHF generally experience severe

haemorrhage due to increased vascular leakage and thrombocytopenia, which

may lead to life-threatening DSS (or DHF stage IV) At this stage, the blood

pressure decreases, normally resulting in death 12 – 36 hours after shock if

patients are left untreated (Gubler, 1998, 2002; Martina et al., 2009)

DENV comprises four antigenically distinct serotypes (1 to 4) Its viral

genome encodes for three structural proteins: the capsid protein, premembrane

protein and envelope glycoprotein; and seven non-structural (NS) proteins:

NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 (Fig 1.1A) (Clyde et al., 2006;

Mackenzie et al., 2004) The envelope protein comprises 3 regions: Domain I,

Domain II and Domain III (Fig 1.1B) Domain I is the central domain,

Domain II is the dimerization and fusion domain, while Domain III (DIII) is

an immunoglobulin-like receptor binding domain (Mukhopadhyay et al.,

2005; Rey et al., 1995)

Experimental evidences have shown that the DIII protein is a possible receptor

binding domain (Bhardwaj et al., 2001; Chin et al., 2007; Chu et al., 2005;

Zhang et al., 2007b) In addition, it has been demonstrated to be highly

immunogenic and able to elicit production of neutralizing antibodies against

wild-type virus (Gromowski et al., 2008; Guzman et al., 2010) For this

reason, DIII protein is an important immunogen for the development of a

prospective protein subunit vaccine and also a prospective diagnostic reagent

for improved clinical diagnosis of dengue infection Although to date, there is

no DIII protein-based diagnostic assay available commercially, many in-house

tests have demonstrated the possibility of using DIII protein as a reagent to

serologically detect presence of DIII-specific antibodies in dengue patient sera

(Pattnaik et al., 2007; Tripathi et al., 2008)

Fig 1.1 (A) Translation of viral RNA yields a single polypeptide that is

further processed by cellular and viral proteases, generating three structural

and seven non-structural proteins Picture adapted from Whitehead et al.,

2005 (B) Structure of the envelope protein dimer of a mature dengue virus

particle Domain I is illustrated in red, Domain II in yellow and Domain III in blue The envelope protein is modelled from DENV3 and illustrated here according to a two-fold symmetry axis (top and side-view) Picture adapted

from Modis et al., 2005

A

B

1.2 Structural studies on flavivirus envelope and DIII protein

A huge step forward in the field of flavivirus research was made when the

structure of flavivirus major envelope protein was first determined in TBEV

(Rey et al., 1995) Domain I of TBEV envelope protein consists of three

segments which span across residues 1 - 51, 137 - 189 and 285 - 302

(according to the TBEV envelope amino acid sequence) Domain II comprises

2 segments, ranging between residues 52 - 136 and 190 - 284 DIII consists

only 1 segment that span across residues 303 - 395, and is located C-terminal

to Domains I and II Furthermore, DIII consists mainly of β-barrels that project perpendicularly to the viral surface For this reason, accessibility of

DIII protein on flaviviral envelope supports its role as a receptor-binding

protein and an immunogenic protein (Rey et al., 1995)

The study on the envelope glycoprotein of other flaviviruses ensued

Advancement in this field included the determination of the structure of

DENV2 envelope protein (Kuhn et al., 2002; Modis et al., 2003; Zhang et al.,

2003), DENV3 envelope protein (Modis et al., 2005); WNV envelope protein

(Kanai et al., 2006; Mukhopadhyay et al., 2003), DENV4 DIII protein (Volk

et al., 2007); immature WNV (Zhang et al., 2007a) and DENV (Yu et al., 2008), and the precursor membrane-envelope protein complex (Li et al.,

2008) Several of these studies were key to the prediction of the viral envelope

membrane fusion mechanism

It is believed that DENV enter cells via receptor-mediated endocytosis The

association of envelope protein with cell-surface receptor, possibly via DIII

protein, allowed endosomal uptake of virus particle into the cell (Modis et al.,

2004) In a study by Modis and colleagues in 2003, the structure of DENV2

envelope glycoprotein was analyzed in the presence or absence of a detergent,

n-octyl-β-D-glucoside, during crystallization This led to the finding of a

hydrophobic pocket lined by amino acid residues that influence pH threshold

for viral fusion This pocket lay in the “hinge” region of the envelope protein

(Rey et al., 1995), thus supporting the notion that a fusogenic conformational

change may be triggered by acidic environment of endosomes after

flaviviruses enter cells by receptor-mediated endocytosis (Modis et al., 2003;

Rey, 1995; Zhang et al., 2004) Furthermore, the binding of the detergent

denoted the pocket as a potential site for small-molecule fusion inhibitors

(Modis et al., 2004)

By obtaining structural information of viral surface envelope glycoproteins at

the atomic level, these findings can facilitate better understanding of the

molecular interactions that occur between the viral surface proteins and their

receptors (Rey, 2003) The elucidation of the three dimensional structure of

envelope protein can also enhance future development of anti-viral drugs that

potentially bind to specific target sites on the envelope protein (i.e DIII region

or the ligand binding pocket) and lead to neutralization of the virus (Modis et

al., 2004; Rey, 2003)

1.3 Antagonistic activity of DIII protein

The antagonistic activity of flavivirus DIII protein against flavivirus infection

has been demonstrated with recombinant DIII (rDIII) proteins derived from

TBEV (Bhardwaj et al., 2001), WNV (Chu et al., 2005) and DENV (Chin et

al., 2007; Jaiswal et al., 2004; Zhang et al., 2007b) Furthermore, competitive

inhibition was also similarly demonstrated using recombinant DENV2

envelope protein (Chen et al., 1996; Chiu & Yang, 2003) The

bacteria-expressed rDIII or recombinant envelope protein acts as an antagonist,

competitively inhibiting entry of flaviviruses into cells in vitro The binding of

these proteins to corresponding cellular surface receptors reduces the number

of receptors available for binding to the viruses during infection, thus leading

to a reduction of viral infection of these cells Based on these studies, there is

sufficient experimental evidence to suggest that DIII protein is involved in

host cell receptor binding

1.4 Neutralizing epitopes on DIII protein

The flaviviral envelope DIII protein contains critical, virus-specific

neutralization epitopes The DENV DIII protein is highly immunogenic and

able to generate serotype-specific and DENV complex-specific neutralizing

antibodies against the virus (Gromowski et al., 2008; Guzman et al., 2010)

This is consistent with studies performed on DIII protein of other flaviviruses,

such as WNV, where it was determined to be an important target for

neutralizing antibodies (Beasley & Barrett, 2002) For these reasons, DIII

protein is believed to be a promising candidate as a protein subunit vaccine

Antibody-mediated neutralization of flaviviruses is generally believed to occur

via a “multi-hit” mechanism of neutralization This means multiple antibodies are required to engage individual virus particles (by binding to different

epitopes) in order for neutralization to occur Evidence for a multi-hit

mechanism of neutralization has been demonstrated with DENV2 and WNV

DIII protein-specific monoclonal antibodies (MAbs) (Gromowski & Barrett,

2007; Pierson et al., 2007) The binding of DIII protein-specific MAbs on

these viruses blocked epitopes required for cellular receptor recognition, thus

reducing infectivity of these viruses Lok and colleagues (2008) proposed an

alternative hypothesis in their recent study They believed that the binding of

DIII protein-specific neutralizing antibodies to the virus caused an alteration

of the spatial arrangement between the glycans on the envelope proteins This

caused hidden epitopes to be exposed and further attacked by neutralizing

antibodies, leading to increased viral neutralization (Lok et al., 2008)

DENV envelope-specific MAbs are able to block virus entry into cells (Crill &

Roehrig, 2001) MAbs that are specific against Domains I (IB4C-2, 4A5C-8,

2B3A-1 and 9A4D-1) and II (6B6C-1, 4G2, 4E5, 1A5D-1, 1B7 and 10A1D-2)

have been shown to be capable of blocking virus entry into Vero cells (18 -

46 % blocking) Notably, DIII-specific MAbs (3H5, 9A3D-8, 10A4D-2,

1A1D-2 and 9D12) showed a stronger inhibition in viral entry (36 - 49 %

blocking) as compared to Domains I and II-specific MAbs (Crill & Roehrig,

2001)

In addition, fine mapping of neutralizing epitopes on DENV2 DIII protein

carried out by Gromowski and Barrett (2007) demonstrated (through the use

of seven DIII protein-specific MAbs) that amino acid residues K305 and P384

on the DENV2 envelope protein were critical for binding [Mutation on

adjacent locations S306, K307, T330 or T332 has also resulted in escape

mutants in WNV (Oliphant et al., 2005)] More importantly, they showed that

the level of viral neutralization was associated with the relative occupancy of

MAbs on DIII protein of the virion (i.e degree of viral neutralization increases

as antibody occupancy on the virus increases) Therefore, viral neutralization

is predicted to occur once the threshold for occupancy is reached (Gromowski

& Barrett, 2007) In addition, a study by Gromowski and colleagues (2008)

demonstrated that serotype-specific MAbs were more potent than DENV

complex-specific MAbs Complex-specific MAbs were observed to require a

higher occupancy level on the virion than serotype-specific MAbs, thus

explaining for the observed lowered effectiveness in viral neutralization

Notably, MAbs that are cross-reactive against the four DENV serotypes have

been developed DIII-specific MAbs, 4E11 and 9F12, have been shown to be

able to cross-neutralize all 4 DENV serotypes with varying effectiveness

(Lisova et al., 2007; Rajamanonmani et al., 2009) However, it is also noted

most of the DIII-specific MAbs discussed in literature are still not yet

available commercially

1.5 Production of DENV rDIII protein

1.5.1 rDIII protein expression

Currently, there is a need for the production of cost effective and safe rDIII

protein-related biologics for the development of protein subunit vaccines or

diagnostic reagents For these purposes, the recombinant proteins produced

must maintain their biological activity (i.e generate neutralizing antibodies

against wild-type virus or able to bind to anti-DENV antibodies found in

patient serum) rDIII proteins may be expressed using various hosts, such as

bacteria, yeast and even in the leaves of tobacco plants (Etemed et al., 2008;

Martinez et al., 2010; Saejung et al., 2006, 2007; Tripathi et al., 2008)

Escherichia coli (E coli) is by far the most commonly used host for the

production of rDIII proteins In general, the DIII gene is first cloned into

expression vectors, such as the pET28a or pET30a vectors (Novagen)

Subsequently, E coli is transformed with the recombinant plasmids for protein

expression For high-yield protein production, BL21(DE3) E coli or its

derivatives is the strain of choice It has the advantage of being deficient in

both lon and ompT proteases and it is also highly compatible with the T7 lacO

promoter system (Jeong et al., 2009; Graslund et al., 2008)

Vectors encoding resistance to antibiotics such as kanamycin, as in the case of

the pET28a vector, are widely used to allow for antibiotic selection of

recombinant clones This ensures that majority of the culture consists of E

coli clones that carry the required vector for protein expression Using T7

systems, protein expression can be induced either with the chemical inducer

isopropyl-β-D-thiogalactopyranoside (IPTG) or by manipulating carbon

sources during E coli growth (auto-induction) (Fickert & Muller-Hill, 1992;

Studier, 2005) Protein expression is often induced at mid-log phase of the

growth curve to ensure maximal yield while circumventing problems

associated with cells going into stationary phase (i.e induction of proteases)

(Chin et al., 2007; Graslund et al., 2008)

Small scale pilot expression is widely used as a predictive tool to determine

which of the derivative clones comparatively produce better yield of the

protein of interest It is also generally a platform for optimizing parameters for

large-scale production of recombinant proteins such as the rDIII proteins

(Graslund et al., 2008) Parameters such as the type of culture media to use,

type and duration of induction, incubation temperature and concentration of

the chemical inducers (such as IPTG) are tested

The up-scaling process of rDIII protein production may be performed by

replacing commonly used batch cultivation in Erlenmeyer flasks (Babu et al.,

2008; Chin et al., 2007; Pattnaik et al., 2007; Tripathi et al., 2008; Zhang et

al., 2007b) to fed-batch or continuous batch cultivation in a bioreactor (Tripathi et al., 2008), thereby tremendously increasing the protein yield

Additionally, the enhancement of the culture media used (i.e from Luria

Bertani broth to Terrific broth), experimentally improved DENV4 rDIII

protein yield (Tripathi et al., 2008)

By incorporating the DIII gene of DENV2 into a tobacco mosaic virus-based

vector (TocJ), the DENV2 rDIII protein can be effectively expressed in the

leaves of Nicotiana benthamiana plants (Saejung et al., 2007) The DENV2

rDIII protein after extraction and subsequent purification, was detectable by

enzyme-linked immunosorbent assay (ELISA) and was able to elicit the

generation of neutralizing antibodies in mice against the wild-type virus This

was the first time the DENV rDIII protein expression was reported to have

been successfully performed in plant hosts

In addition, rDIII proteins can also be expressed using the mammalian protein

expression system Taking advantage of the high expression potential of the

methylotrophic yeast, Pichia pastoris, a chimeric tetravalent DIII protein was

successfully expressed at high concentrations (Etemad et al., 2008) The

advantages of using Pichia sp for protein expression are that this yeast has a

high growth rate, able to grow on simple inexpensive media, and is suitable

for small scale pilot expression that can be scaled up to industrial size

1.5.2 rDIII protein purification

1.5.2.1 Immobilized metal affinity chromatography (IMAC)

To facilitate the purification of rDIII proteins, DIII proteins are commonly

produced as fusion proteins that comprise an affinity tag, such as the

hexahistidine tag (Chin et al., 2007; Pattnaik et al., 2007; Uhlen et al., 1992;

Zhang et al., 2007b) The advantages of using a hexahistidine tag are

manifold Firstly, hexahistidine-tagged proteins can be purified using

immobilized metal-ion affinity chromatography (IMAC) by the means of a

relatively simple protocol (Arnau et al., 2006; Block et al., 2009) Secondly,

hexahistidine tags rarely affect the characteristics of the protein Lastly, the

hexahistidine tag is relatively small and generally does not alter the solubility

of the protein of interest (Graslund et al., 2008)

In general, IMAC purification procedures are relatively straightforward

(Arnau et al., 2006; Gaberic-Porekar & Menart, 2001) The processed lysate is

first loaded onto the IMAC column and the protein of interest subsequently

binds to the column via its affinity tag Several factors may adversely affect

the binding of the recombinant proteins to the IMAC column Parameters such

as pH of buffer, the presence of chelators such as EDTA, or high

concentration of imidazole or DTT must be considered for IMAC protein

purification to be successful (Graslund et al., 2008) The column is

subsequently washed with a buffer comprising intermediate concentrations of

imidazole This “washing” step removes unwanted proteins from the column Following that, the recombinant protein is eluted with a higher concentration

of imidazole (i.e 200 mM to 500 mM imidazole) (Graslund et al., 2008)

Normally, trace amounts of bacterial proteins co-elute with the recombinant

protein SlyD protein, which comprise multihistidine residues, and other

proteins such as GroES, Fur, CA, RplB, DnaJ, GroEL and DnaK that are

found in E coli, are common contaminants of the IMAC purified proteins

(Bolanos-Garcia & Davis, 2006; Howell et al., 2006) To circumvent these

issues, purity of IMAC-purified rDIII proteins can be further enhanced by gel

filtration via high-performance liquid chromatography (HPLC) or fast protein

liquid chromatography (FPLC) methods In order to determine that the

purified protein is the protein of interest, Western blot using affinity

tag-specific and antigen-tag-specific antibodies may be performed to identify the

affinity tag and also the protein of interest, respectively (Chin et al., 2007; Chu

et al., 2007; Pattnaik et al., 2007; Tripathi et al., 2008) As an additional

confirmation procedure, the identity of the purified protein of interest can be

confirmed by mass spectrometry

1.5.2.2 Membrane adsorber-based IMAC

Chromatography is by far the most widely used technique for high-resolution

separation and analysis of proteins (Ghosh, 2002) Even though traditional

packed-bed chromatography is a popular method for purifying recombinant

proteins, it is faced with several limitations This method requires diffusion of

target molecules to binding sites found within the pores of the resin matrix in

order for binding to occur This increases the process time and consequently

the need for increased recovery liquid (to elute the bound proteins) In

addition, the possibility of channeling (formation of flow passages due to

cracking of the packed-bed) makes quality assurance difficult as purification

quality is not easily maintained, especially on a larger scale For these reasons,

packed-bed chromatography could not be easily and readily up-scaled to

industrial size (Ghosh, 2002)

In contrast, metal affinity membrane chromatography via the use of membrane

adsorbers (MA) is a superior chromatography method and an alternative to

packed-bed chromatography (Ghosh, 2002) Recent improvements in

membrane materials and chemistries have generated renewed interest in

applications of membrane chromatography for bioprocessing (Charcosset,

2006) As the active binding sites for the target proteins are readily available

in convective through-pores in the membrane, the flow rate of biomolecules

can be enhanced by an external pump Thus, MA-based protein separation can

be performed rapidly at higher volumetric rates without compromising its high

yield (Charcosset, 2006)

Membrane absorbers are versatile and can be used for numerous applications

such as purification of proteins (Ghosh, 2002; Shi et al., 2003; Gebauer et al.,

1997), monoclonal antibodies (Knudsen et al., 2001), oligonucleotides

(Deshmukh et al., 2000) and viruses (Karger et al., 1998) In addition, MAs

can also complement the HPLC system for improved purity of the protein of

interest (Boi, 2007) Most importantly, as compared to traditional packed-bed

chromatography columns, MAs have the major advantage of relatively ease of

scale-up from laboratory to industrial scale (Demmer & Nussbaumer, 1999;

Ghosh, 2002; Plate et al., 2006)

For these reasons, metal affinity membrane chromatography presents a more

practical means for consistent and reliable production and purification of rDIII

proteins As MA-based IMAC has not been reported previously for

purification of flavivirus proteins, the establishment of this contemporary

purification technique for rDIII protein purification would thus be a novel

approach for rDIII protein production

1.6 DIII protein as a potential protein subunit vaccine

Currently, there is no commercially available vaccine against DENV infection

Efforts to develop a suitable vaccine against DENV have focused mainly on

live attenuated vaccines, followed by other approaches such as inactivated

virus, protein subunit and DNA vaccines (Barrett, 2001; Guirakhoo et al.,

2004; Whitehead et al., 2007) As the DIII protein is able to elicit the

generation of neutralizing antibodies against wild-type DENV, it is therefore a

prospective candidate as a protein subunit vaccine

One major challenge to DENV vaccine development is the potential

development of antibody-dependent enhancement (ADE) of virus replication,

which is believed to cause DHF and DSS (Halstead, 1988) ADE occurs when

heterotypic non-neutralizing antibodies present in the host bind to the DENV

particle during a subsequent heterotypic infection but cannot neutralize the

virus Instead, this complex attaches to the Fcγ receptors (FcγR) on the

circulating monocytes This therefore facilitates the infection of FcγR cell

types in the body, which are normally not readily infected in the absence of a

non-neutralizing antibody Consequently, this leads to an increase in viral

infection, leading to the potential development of a more severe disease

(Fig 1.2) (Guzman & Kouri, 2002; Whitehead et al., 2007) In addition,

immunization against one dengue serotype induces life-long immunity against

the homologous serotype and short-lived immunity against the other serotypes

For these reasons, it is widely believed that for a DENV vaccine to be

effective, it must comprise neutralizing epitopes from all four serotypes

(tetravalent) (Halstead, 1988; Whitehead et al., 2007)

Presently, rDIII protein immunization in animal models has demonstrated

promising results (Table 1.1) In these studies, a range of parameters affecting

rDIII protein immunogenicity have been evaluated These include: antigen

combination - monovalent, bivalent, or tetravalent rDIII, type of animal model

Fig 1.2 Model for antibody-dependent enhancement of virus infection

(Adapted from Whitehead et al., 2007)