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Recombinant dengue vaccine

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Pradeep B J Reddy

This document describes research on cloning and expressing the domain III region of the dengue virus envelope protein (DENV EDIII) from serotypes 3 and 4. Key findings include: 1) DENV EDIII from serotypes 3 and 4 were successfully cloned and expressed in E. coli, and the recombinant proteins were purified using nickel affinity and size exclusion chromatography. 2) The purified recombinant EDIII proteins were able to fold correctly and form stable monomers, as shown through analytical methods like SDS-PAGE and gel filtration chromatography. 3) In mouse studies, immunization with the recombinant EDIII proteins, especially when combined with adjuvants, induced strong humoral and cellular immune responses as measured by

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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
PRESENT STUDY
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 M UI 1hr 2hr 3hr 4hr M UI 1hr 2hr 3hr 4hr 0.5 mM IPTG 170 11 72 43 34 26 17 M UI 1hr 2hr 3hr 4hr M UI 1hr 2hr 3hr 4hr 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 FCA Montanide Alum Protein alone 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] Plaque reduction neutralization test (PRNT)
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 Control ConA FCA Mont Alum IL-4 IL-5 IL-10 0 500 1000 1500 2000 2500 Control ConA FCA Mont Alum 0 400 800 1200 Control ConA FCA Mont Alum 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 Control ConA FCA Mont Alum 0 300 600 900 1200 1500 Control ConA FCA Mont Alum 0 100 200 300 400 Control ConA FCA Mont Alum 0 200 400 600 800 Control ConA FCA Mont Alum X-axis concentration in pg/ml Y-axis rD4EIII in combination with different adjuvants
0 0.5 1 1.5 2 2.5 FCA Montanide Alum Protein alone 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 FCA Montanide Alum Protein alone Adjuvants Antibody titer Day 14 Day 35 Day 56 Day 70 Endpoint ELISA titers
0 0.5 1 1.5 2 2.5 3 FCA Montanide Alum Protein alone 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] Plaque reduction neutralization test (PRNT)
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 Control ConA FCA Mont Alum D3EIII in combination with adjuvants pg/ml IL -2 0 100 200 300 400 500 Control ConA FCA Mont Alum D3EIII in combination wit adjuvants pg/ml IL-4 0 100 200 300 400 500 Control ConA FCA Mont Alum D3EIII in combination with adjuvants pg/ml IL -10 0 100 200 300 Control ConA FCA Mont Alum D3EIII in combination with adjuvants 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)
Thank you

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Recombinant dengue vaccine

  • 1. 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
  • 2. 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
  • 4. 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.
  • 5. 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
  • 6. Constraints in making a dengue vaccine Dengue – Four serotypes Secondary infection with heterologous serotype Antibody dependant enhancement DHF/DSS
  • 7. 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
  • 8. Problems with existing vaccines Whole virus vaccines Interference DNA vaccines
  • 9. 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
  • 11. E M C RNA 5’ C prM E NS1 NS3 NS5 3’ NC NC NC ns4b ns4a ns2b ns2a Dengue Virus structure Genome
  • 12. Domain II (A) Domain I (C) Domain III (B) Envelope Structure
  • 13. Domain III of envelope protein Receptor binding domain   j h Domain III a potential vaccine candidate
  • 14. 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.
  • 15. Map of expression vector containing Den-DIII gene
  • 16. 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
  • 17. 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
  • 18. 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
  • 19. 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
  • 20. 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
  • 21. 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
  • 22. 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 M UI 1hr 2hr 3hr 4hr M UI 1hr 2hr 3hr 4hr 0.5 mM IPTG 170 11 72 43 34 26 17 M UI 1hr 2hr 3hr 4hr M UI 1hr 2hr 3hr 4hr 1.0 mM IPTG
  • 23. 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
  • 24. 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
  • 25. 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
  • 27. 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
  • 28. 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
  • 29. 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
  • 30. 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
  • 31. 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
  • 32. Production of rD4-DIII by fed batch Fermentation process & Evaluation of its diagnostic potential
  • 33. 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
  • 34. 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
  • 35. 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
  • 36. Immunomodulatory studies of domain III proteins in BALB/c mice
  • 37. 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)
  • 38. 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
  • 39. 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
  • 40. Antibody endpoint titer by ELISA 0 50000 100000 150000 200000 250000 FCA Montanide Alum Protein alone Adjuvants Antibody titer Day 14 Day 35 Day 56 Day 70
  • 41. 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
  • 42. Immunofluorescence assay Control LLC-MK2 cells infected with DEN4 virus 1o antibody - anti rD4EIII Ab 2o antibody - anti mouse IgG FITC conjugate Test
  • 43. [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)
  • 44. 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] Plaque reduction neutralization test (PRNT)
  • 45. 0 200 400 600 Control FCA Montanide Alum Adjuvants Fluorescence Splenocyte proliferation assay Cyquant NF cell proliferation assay (in vitrogen)
  • 47. 0 200 400 600 Control ConA FCA Mont Alum IL-2 0 400 800 1200 Control ConA FCA Mont Alum IL-4 IL-5 IL-10 0 500 1000 1500 2000 2500 Control ConA FCA Mont Alum 0 400 800 1200 Control ConA FCA Mont Alum X-axis concentration in pg/ml Y-axis rD4EIII in combination with different adjuvants
  • 48. IL-12 IFN-g TNF-a GM-CSF 0 100 200 300 Control ConA FCA Mont Alum 0 300 600 900 1200 1500 Control ConA FCA Mont Alum 0 100 200 300 400 Control ConA FCA Mont Alum 0 200 400 600 800 Control ConA FCA Mont Alum X-axis concentration in pg/ml Y-axis rD4EIII in combination with different adjuvants
  • 49. 0 0.5 1 1.5 2 2.5 FCA Montanide Alum Protein alone Adjuvants OD at 495 nm 14th day 35th day 56th day 70th day Antibody titer in mice immunized with D3-DIII by ELISA
  • 50. 0 50000 100000 150000 200000 250000 FCA Montanide Alum Protein alone Adjuvants Antibody titer Day 14 Day 35 Day 56 Day 70 Endpoint ELISA titers
  • 51. 0 0.5 1 1.5 2 2.5 3 FCA Montanide Alum Protein alone Adjuvants OD at 495 IgG1 IgG2a IgG2b IgG3 Antibody Sub typing
  • 52. 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] Plaque reduction neutralization test (PRNT)
  • 53. 0 100 200 300 400 500 600 700 Control FCA Montanide Alum D3EIII in combination with different adjuvants Fluorescence Lymphocyte proliferation assay
  • 54. 0 100 200 300 400 500 Control ConA FCA Mont Alum D3EIII in combination with adjuvants pg/ml IL -2 0 100 200 300 400 500 Control ConA FCA Mont Alum D3EIII in combination wit adjuvants pg/ml IL-4 0 100 200 300 400 500 Control ConA FCA Mont Alum D3EIII in combination with adjuvants pg/ml IL -10 0 100 200 300 Control ConA FCA Mont Alum D3EIII in combination with adjuvants pg/ml IFN-g Cytokine profiling of mice immunized with D3EIII
  • 55. 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
  • 56. 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
  • 57.  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
  • 58.  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
  • 59. 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
  • 60. 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)