Recombinant dengue vaccine

Cloning and expression of recombinant dengue
envelope protein (domain-III) and its functional
and molecular characterization
J. PRADEE BABU
SRF, Virology
Defence R&D Establishment

Dengue Burden
 2.5 to 3 billion people (40% of world population ) at risk of infection
 An estimated 50 to 100 million cases of dengue fever occur annually
 2,50,000 and 500, 000 people develop DHF and DSS each year
 20,000 deaths every year

DHF/DSS
VACCINE
THERAPY
x
DENGUE FEVER

Do you know…
 Dengue fever and dengue haemorrhagic fever are the most common
mosquito-borne viral disease in the world
 Only the female mosquito feeds on blood. This is because they need
the protein found in blood to produce eggs. Male mosquitoes feed
only on plant nectar
 The mosquito is attracted by the body odours, carbon dioxide and
heat emitted from the animal or humans
 Aedes are day-biters, most active during dawn and dusk.

DENGUE FEVER
• Fever
• Headache
• Nausea, vomiting
• Retro-orbital pain
• Myalgia and/or arthralgia
• Rash
• Hemorrhage
• Leukopenia
• Normal or decreased platelet count
Secondary infection DHF DSS

Constraints in making a dengue vaccine
Dengue – Four serotypes
Secondary infection with heterologous serotype
Antibody dependant enhancement
DHF/DSS

Primary infection of Dengue virus (D1)
Virus neutralization
(no infection)
Life time immunity
Secondary infection of Dengue virus (D2)
No neutralization
Infection Cured DF
Fc receptor
Human
monocyte/macrophage
Antibody
Dependent
Enhancement
？
DHF
Possible mechanism of DHF
Vascular permiability
Plasma leakage
(Pleural effusion, ascites)
Hemo-concentration（Hct↑）
DIC
(Bleeding)
Death
Activated
T-Cell ?
IL-2
IL-6-
TNFα
IFNγ

Problems with existing vaccines
Whole virus vaccines
Interference
DNA vaccines

A Dengue Vaccine
 There is no licensed vaccine at present
 An efficient vaccine has to be tetravalent
 Several vaccines are in the pipeline
 Effective, safe, low-cost vaccine

E
M
C
RNA
5’ C prM E NS1 NS3 NS5 3’
NC
NC
NC
ns4b
ns4a
ns2b
ns2a
Dengue Virus structure
Genome

Domain II (A)
Domain I (C)
Domain III (B)
Envelope Structure

Domain III of envelope protein
Receptor binding domain

 j
h
Domain III a potential vaccine candidate

Outline of the work
 Cloning, expression and purification of envelope protein
(domain-III) of dengue viruses (DEN 3 & 4 serotypes)
 In vitro refolding of recombinant envelope protein and
characterization
 Studies on immuno-modulatory potential of recombinant
envelope (domain-III) protein.

Map of expression vector containing Den-DIII gene

Restriction digestion of recombinant pET30a+
Plasmid containing D4-DIII
1 - 1kb ladder
2 - Uncut (Clone-1)
3 - Clone-1 digested with Nde1& Sal1
250bp
500bp
750bp
1000bp
1500bp
330bp
1 2 3

NdeI
SalI
D4EIII
f1 Origin
KanR
Lac I
Ori
NdeI
SalI
D4EIII
NdeI
SalI
D4EIII
f1 Origin
KanR
Lac I
Ori
D3-DIII
200
10
15
20
25
40
50
60
12 KDa
1 2 3
30
1 - Marker
2 - Uninduced
3 - Induced
D3-DIII cloned in pET30a+ showing expression
Sal I
Nde I

0.1 mM IPTG
0.5 mM IPTG 1.0 mM IPTG
Expression optimisation of D4-DIII with IPTG concentration and time
200
10
15
20
25
30
40
M UI 1hr 2hr 3hr 4hr
0.2 mM IPTG
M UI 1hr 2hr 3hr 4hr
200
10
15
20
25
30
40
M UI 1hr 2hr 3hr 4hr M UI 1hr 2hr 3hr 4hr

Ni-NTA Purification of D3-DIII
Westernblotting with D3 specific
Monoclonal ab
Westernblotting with 6x His ab
175
6.5
32.5
47.5
25.0
16.5
1 2 3 4 5 6 7 8 9 10
1.0 g 0.5 g 1.0 g 0.5 g

0
10
20
30
40
50
60
70
80
90
100
Percentage
inhibition
(%)
0 5 10 15 20 25 30
Protein concentartion (mg/ml)
D3EIII
BSA
Protein concentration (g/ml)
Competitive inhibition of virus by D3EIII

NdeI
SalI
D4EIII
f1 Origin
KanR
Lac I
Ori
NdeI
SalI
D4EIII
NdeI
SalI
D4EIII
f1 Origin
KanR
Lac I
Ori
1 - Marker
2 - Uninduced
3 - Induced
D4-DIII cloned in pET30a+ showing expression
1 2 3
11
72
55
43
34
26
17
170
11.68 KDa

Expression optimisation of D4-DIII with IPTG concentration and time
0.1 mM IPTG 0.2 mM IPTG
170
11
72
43
34
26
17
0.5 mM IPTG
170
11
72
43
34
26
17
1.0 mM IPTG

Localization and affinity purification of D4-DIII
1 - Marker
2 - Sonicated supernatant
3 - Sonicated pellet in native conditions
4 - Sonicated pellet
5 - Ni-NTA purified D4-DIII
11
72
43
34
26
17
55
170
1 2 3 4 5
D4-DIII

170
72
43
34
26
17
11
1 2
170
72
43
34
26
17
11
55
1 2
11.68 KDa
Western blot analysis of D4-DIII protein
Lane 1 : Marker
Lane 2 : D4DIII Protein
Anti His DEN4 mab

In-vitro refolding of the recombinant proteins
Refolding Method : Rapid Dilution
Oxido-redux shuffling agent : Cystine/Cystamine
Amount of protein refolded : 20 mg
Dilution Factor : 50 fold
Refolding set volume : 1000ml
Total protein after Iex : 1.8 mg
Protein
Concentration
Denaturant
Concentration
Refolding
Solvent
Final
Concentration
Addition of protein with denaturant

-2.00
0.00
50.00
100.00
150.00
200.00
250.00
300.00
Absorbance
at
280
[mAU]
0.00
20.00
40.00
60.00
80.00
100.00
120.00
%
NaCl
280 nm
% NaCl
10
0 70 80
Retention Time/volume (Minutes/ ml)
A
B
Ion-Exchange profile of refolded D4-DIII

SDS-PAGE of elution fractions collected from
ion-exchange chromatography
M R NR
Peak No.A
Marker with b-ME without bME
43
29
20.1
14.3
6.5
3.0
Peak No.B

Analytical Gel filtration profile of refolded D4-DIII
0
10
20
30
40
50
60
70
80
90
5 10 20 25 30
15
Volume (ml)
Absorbance
(mAU)
-4
-2
0
2
4
6
8
10
12
14
16
18
5 10 20 25 30
15
Volume (ml)
Absorbance
(mAU)
Ribonuclease A
13.7kDa
Albumin
67kDa
Ovalbumin
43kDa
Chymotrypsinogen A
25kDa
Den4-DIII
11.68kDa

RP- HPLC profile showing purity and refolding of D4-DIII
C-8 Column, Absorbance at 214 nm, H2O/Acetonitrile
Before Reduction After Reduction
Time (Min)
0
2
4
6
8
10
12
Acetonitrile
(%)
Time (Min)
Acetonitrile
(%)
0
2
4
6
8
10
12

Ellman’s Test (Free thiol assay)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Concentration of free thiol groups
OD
at
412
nm
Reduced D4EIII
Cysteine Standard
Refolded
31.25 M 62.5 M 125 M 250 M 375 M

Functional bioassay demonstrating binding
of D4-DIII to heparan sulfate
0
0.4
0.8
1.2
1.6
2
Control 0.125 0.25 0.5 1 2
D4EIII Concentration (g/ml)
Absorbance
at
490
nm
Refolded
Denatured

Production of rD4-DIII by fed batch Fermentation process
&
Evaluation of its diagnostic potential

Comparative yield of D4EIII protein in shake flask culture
and bioreactor
Media Type of Culture OD600 Dry weight (gl-1) Protein (mgl-1)
Luria Bertani broth Shake flask 2.43 1.57 25.24
Super Broth Shake flask 3.30 2.25 36.35
Terrific Broth Shake flask 3.56 2.37 42.68
Terrific Broth Batch Culture 7.51 4.56 66.34
Terrific Broth Fed-batch culture 36.12 17.34 196.26

Test n % Agreement a % Sensitivityb % Specificityc
Dipstick ELISA/IC test 72 98% (71/72) 97% (38/39) 100% (33/33)
Dipstick ELISA/capture ELISA 72 96% (69/72) 93% (38/41) 100% (31/31)
Comparative evaluation of in-house dipstick ELISA with reference to rapid IC
test and IgM capture ELISA for detection of dengue antibodies

Test n % agreementa %sensitivityb %specifictyc
Dipstick ELISA/IC test 72 97% (70/72) 100% (35/35) 95% (35/37)
Dipstick ELISA/capture 72 97% (71/72) 97% (37/38) 100% (34/34)
Comparative evaluation of in-house dipstick ELISA with reference to
rapid IC test and IgG capture ELISA for detection of dengue IgG antibodies

Immunomodulatory studies of domain III proteins in
BALB/c mice

Immunization Schedule
Adjuvants used in this study
 FCA
 Montanide
 Alum
 Protein alone
Pre-immune
Bleed
Priming
(Day 0)
Bleed 1
(Day 14)
Boost 1
(Day 21)
Bleed 2
(Day 35)
Boost 2
(Day 42)
Bleed 3
(Day 56)
Bleed 4
(Day 70)

Evaluation of humoral Immune response
 Antibody titer and antibody sub typing by ELISA
 Immunofluorescence assay
 Plaque reduction neutralization assay
Evaluation of cell mediated immune response
 Cytokine Profiling
 Splenocyte proliferation assay

0
0.4
0.8
1.2
1.6
2
FCA Montanide Alum Protein alone
Adjuvants
OD
at
490
nm
14th day
35th day
56th day
70th day
Antibody titer in mice immunized with D4-DIII by ELISA

Antibody endpoint titer by ELISA
0
50000
100000
150000
200000
250000
Adjuvants
Antibody
titer
Day 14
Day 35
Day 56
Day 70

0
0.5
1
1.5
2
2.5
FCA Mon Alum Protein alone
Adjuvants
OD
at
490
nm
IgG1
IgG2a
IgG2b
IgG3
Antibody sub-typing

Immunofluorescence assay
Control
LLC-MK2 cells infected with DEN4 virus
1o antibody - anti rD4EIII Ab
2o antibody - anti mouse IgG FITC conjugate
Test

[A] [B] [C]
[A] Healthy cell control
[B] Virus control
[C] PRNT50 titer showing where the virus pfu is reduced by 50%
Plaque reduction neutralization test (PRNT)

0
20
40
60
80
100
0 2 4 8 16 32 64 128 256 512
Serum Dilution
Inhibition
of
DEN-4
Virus
(%)
FCA
Montanide ISA 720
Alum
Preimmune sera
[A]
0
20
40
60
80
100
0 2 4 8 16 32 64 128 256
Serum Dilution
Inhibition
of
DEN-3
Virus
(%)
FCA
Montanide ISA 720
Alum
Preimmune sera
[B]

0
200
400
600
Control FCA Montanide Alum
Adjuvants
Fluorescence
Splenocyte proliferation assay
Cyquant NF cell proliferation assay (in vitrogen)

Cytkoine profiling by
X-map technology

0
200
400
600
Control ConA FCA Mont Alum
IL-2
0
400
800
1200
IL-4
IL-5 IL-10
0
500
1000
1500
2000
2500
0
400
800
1200
X-axis concentration in pg/ml
Y-axis rD4EIII in combination with different adjuvants

IL-12
IFN-g TNF-a
GM-CSF
0
100
200
300
0
300
600
900
1200
1500
0
100
200
300
400
0
200
400
600
800
X-axis concentration in pg/ml
Y-axis rD4EIII in combination with different adjuvants

0
0.5
1
1.5
2
2.5
Adjuvants
OD
at
495
nm
14th day
35th day
56th day
70th day
Antibody titer in mice immunized with D3-DIII by ELISA

0
50000
100000
150000
200000
250000
Adjuvants
Antibody
titer
Day 14
Day 35
Day 56
Day 70
Endpoint ELISA titers

0
0.5
1
1.5
2
2.5
3
Adjuvants
OD
at
495
IgG1
IgG2a
IgG2b
IgG3
Antibody Sub typing

0
20
40
60
80
100
0 2 4 8 16 32 64 128 256 512
Serum Dilution
Inhibition
of
DEN-3
Virus
(%)
FCA
Montanide ISA 720
Alum
Preimmune sera
[A]
0
20
40
60
80
100
0 2 4 8 16 32 64 128 256
Serum Dilution
Inhibition
of
DEN-4
Virus
(%)
FCA
Montanide ISA 720
Alum
Preimmune sera
[B]

0
100
200
300
400
500
600
700
Control FCA Montanide Alum
D3EIII in combination with different adjuvants
Fluorescence
Lymphocyte proliferation assay

0
100
200
300
400
500
D3EIII in combination with adjuvants
pg/ml
IL -2
0
100
200
300
400
500
D3EIII in combination wit adjuvants
pg/ml
IL-4
0
100
200
300
400
500
pg/ml
IL -10
0
100
200
300
pg/ml
IFN-g
Cytokine profiling of mice immunized with D3EIII

Real-time kinetics
Amplification Plots
-0.02
-0.01
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
1 11 21 31
Cycles
Fluorescence
(dRn)
A1, GAPDH Control, SYBR
A2, GAPDH Control, SYBR
A3, GAPDH FCA, SYBR
A4, GAPDH FCA, SYBR
A5, GAPDH Mont, SYBR
A6, GAPDH Mont, SYBR
A7, GAPDH Alum, SYBR
A8, GAPDH Alum, SYBR
GAPDH Amplification Plots
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
1 11 21 31
Cycles
Fluorescence
(dRn)
C1, IFN-g Controll, SYBR
C2, IFN-g Control, SYBR
C3, IFN-g FCA, SYBR
C4, IFN-g FCA, SYBR
C5, IFN-g Mont, SYBR
C6, IFN-g Mont, SYBR
C7, IFN-g Alum, SYBR
C8, IFN-g Alum, SYBR
IFN-g
Amplification Plots
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
1 11 21 31
Cycles
Fluorescence
(dRn)
D1, IL-10 Control, SYBR
D2, IL-10 Control, SYBR
D3, IL-10 FCA, SYBR
D4, IL-10 FCA, SYBR
D5, IL-10 Mont, SYBR
D6, IL-10 Mont, SYBR
D7, IL-10 Alum, SYBR
D8, IL-10 Alum, SYBR
IL-10

Cytokine profiling of mice immunized with
D3EIII by real-time PCR
0
0.5
1
1.5
2
2.5
3
3.5
FCA Montanide Alum
D3EIII with different adjuvants
Fold
up
regulation
IFN-g
0
0.5
1
1.5
2
2.5
FCA Montanide Alum
D3EIII with different adjuvants
Fold
up
regulation
IL-10

 Cloning, expression and purification of biologically functional D3DIII and
D4DIII with high yields
 The fed-batch fermentation strategy employed in this work is probably
one of the cost effective means to enhance cell mass and protein
production
 D4EIII protein as an antigen in dipstick ELISA resulted in excellent
agreement with the findings of commercial rapid IC test and capture
ELISA
 Proteins expressed in E.coli were successfully refolded under in vitro
conditions. Biophysical and biochemical characterization of refolded
proteins was carried out to show the refolding of the protein
 Functional characterization was carried out to demonstrate the biological
activity of the protein that can bind to heparan sulfate
Conclusions

 The recombinant domain III protein deserves further study as a
potential subunit DEN vaccine candidate
 FCA and montanide ISA 720 yielding high titers of neutralizing
antibodies and mixed Th1/Th2 response
 Correlation between the cytokine gene expression and cytokine
secretion in cell culture supernatant
 Since FCA is not approved by FDA for human use, montanide ISA
720 could be an efficient adjuvant for further studies
Conclusions

J. Pradeep Babu, Priyabrata Pattnaik, Nimesh Gupta, Ambuj Shrivastava, Mohsin
Khan, P.V. Lakshmana Rao. Immunogenicity of a recombinant envelope domain III
protein of Dengue virus type-4 with various adjuvants in mice. Vaccine. 26 (2008)
4655-4663.
Nagesh K. Tripathi, J. Pradeep Babu, Ambuj Shrivastva, Manmohan Parida, Asha M.
Jana, P.V. Lakshmana Rao. Production and characterization of recombinant dengue
virus type 4 envelope domain III protein. Journal of Biotechnology; J Biotechnol 134
(2008) 278–286.
Pattnaik P, Babu JP, Verma SK, Tak V, Rao PV. Bacterially expressed and refolded
envelope protein (domain III) of dengue virus type-4 binds heparan sulfate. J
chromatogr B; 2006, 846(1-2):184-94.
J. Pradeep Babu, Priyabrata Pattnaik, Nimesh Gupta, K. Sathyaseelan, G. B. K. S
Prasad, P.V. Lakshmana Rao. 2008. High-level expression, in vitro refolding and
characterization of dengue virus type-3 envelope domain III protein. Biotechnology
Progress, Communicated.
List of Publications

Acknowledgements
Dr. PVL Rao (Supervisor)
Dr. G.B.K.S Prasad (co-guide)
Dr. R. Vijayaraghavan (Director, DRDE, Gwalior)
Dr. M. M. Parida, Dr. P. K Dash, Dr. R. Barghava
Dr. Priyabratha Pattnaik (Head, Bioprocess, Millipore)

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