purwati, s. soegijanto, soetjipto, k. sudiana, e. hendrianto, h susilowati, fedik a. rantam analysis...

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Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING NONSTRUCTURAL PROTEINS (NS1) FROM SEROTYPES -1, -2 AND -4 AND EXPRESSED IN BACULOVIRUS Dr. Soetomo Teaching Hospital – Faculty of Med. – Institute of Tropical Diseases Airlangga University – Surabaya – Indonesia

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Page 1: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Purwati, S. Soegijanto, Soetjipto, K. Sudiana,

E. Hendrianto, H Susilowati, Fedik A. Rantam

ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING NONSTRUCTURAL PROTEINS (NS1)

FROM SEROTYPES -1, -2 AND -4 AND EXPRESSED IN BACULOVIRUS

Dr. Soetomo Teaching Hospital – Faculty of Med. – Institute of Tropical Diseases

Airlangga University – Surabaya – Indonesia

Page 2: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

INTRODUCTIONINTRODUCTIONDengue cases is estimated to be between 528 and

621 per million populationNot specific antiviral therapyPotential dengue virus vaccines have been

developed, but they may not protect against all serotypes.

Recombinant protein vaccines with many epitopes have been produced for yellow fever virus and baculovirus (lobigs et al., 1987; Satropoli et al., 1996), but it is not clear which antigen determines the induction of neutralizing antibodies

In this research : vaccine used a baculovirus system to express a multivalent, recombinant NS1 protein for vaccine subunit development

Page 3: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

• Causes dengue and dengue hemorrhagic fever • Transmitted by mosquitoes • Has 4 serotypes (DEN-1, 2, 3, 4) Each

serotype provides specific lifetime immunity, and short-term cross-immunity

• All serotypes can cause severe and fatal disease

• Genetic variation within serotypes • Some genetic variants within each serotype • appear to be more virulent or have greater • epidemic potential

Dengue Virus

Page 4: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING
Page 5: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Dengue Situation In Indonesia

(MOH, 2012)

Page 6: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

(Rantam et.al., 2010)

Page 7: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING
Page 8: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING
Page 9: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Where are we today ?

• No vaccine licensed• High levels of country interest • Robust vaccine pipeline• Candidates in different stages of

evaluation• Large-scale clinical trials – underway

Page 10: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

GENOME STRUCTUR OF DENGUE GENOME STRUCTUR OF DENGUE VIRUSVIRUS

C PrM M E NS1 NS 2A-2B

NS3 NS 4A-4B

NS5

5’ 3’

11000 bp

Structural protein

Nonstructural protein

C: capsid, : New virion assembling

PrM; Premembran,M: matrix, Virion release

E: Envelope, attachment, Ab netralization

NS1-4: Ag HI, CF, Ag net., NS5: Polymerase

Page 11: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

MATERIALS and METHODSMATERIALS and METHODS

Virus isolation Virus isolation RNA extraction, RT-PCR amplification, RNA extraction, RT-PCR amplification,

cloning and transfectioncloning and transfectionProduction of recombinant NS1 proteinProduction of recombinant NS1 proteinImmunotyping - ELISAImmunotyping - ELISA

Page 12: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

The NS1 Protein genes from three isolates of dengue virus (Den V -1, V-2 and V-4) were isolated, clone into E. coli and than sub clone into a baculovirus vector. The last transfection into Sf9 cells. The recombinant NS1 genes were inserted between Smal and Sacl sites of the plasmid

Page 13: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

PROTEIN CODING

C PrM M E NS-1 NS2a,NS2b NS3 NS-4a, NS-4b NS-5

C PrM M E NS-1 NS2a,NS2b NS3 NS-4a, NS-4b NS-5

C PrM M E NS-1 NS2a,NS2b NS3 NS-4a, NS-4b NS-5

C PrM M E NS-1 NS2a,NS2b NS3 NS-4a, NS-4b NS-5

DEN

V-1

DEN

V-2

DEN

V-3

DEN

V-4

E-ProteinPrM--Protein

NS-Protein

DEN-1,2,3,4

WHOLE GENOME DEN-VIRUS

RNA-VIRUS

SEROTYPEE-Protein

PrM-Protein NS1-Protein

PrM & E-Protein coded humoral respons immune, NS1 coded cellular response immune

Page 14: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

RESULTSRESULTS

Page 15: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Virus Isolation

Den-2Den-3

Den-2

1 2 3 4 M 5 6 7

AB Virus production in vero

cell A. vero cell 80% confluent, dan B vero cell innoculation with virus Dengue pasage 3

DEN-1

DEN-4

DEN-3

DEN-2

Purwati, Rantam et al.2010

Page 16: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

KLONING NS1M D1 D2 D4

600 bp

Page 17: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

GENE

PrM M E NS-1

PrM M E NS-1 NS3

C PrM M E NS-1 NS2a,NS2b NS3 NS-4a, NS-4b NS-5

PrM M E NS-1

DEN

V-1

DEN

V-2

DEN

V-3

DEN

V-4

E-ProteinPrM--Protein

NS-Protein

DEN-3

Den-1 Gen Clone

Den-2 Gen Clone

Den-4 Gen Clone

Backbone Chimera Den-1,2,4 Gen Clone

Frg. NS1 Gen Clone

Frg. prM Gen Clone

Frg. E Gen Clone

Page 18: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

MODIFIED DEN-3 VIRUS (CHIMERA)

E-ProteinPrM--Protein

NS-Protein

DEN-3

NS2a,NS2b NS3C PrM M E NS-1 NS-4a, NS-4b NS-5

DEN-124

E-ProteinPrM--Protein

NS-Protein

DEN-3

DEN-124

E-ProteinPrM--Protein

NS-Protein

DEN-3

DEN-124

NS-3NS-2a,NS-2b

Page 19: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

F666 F601

R1229

F668

R1222R1228

Lig

atio

n

prMD124 ED124 NS1D1245’

3’

Cloning & Fusion gene

pVL , 9,8 kb

SmalSac 1

pVL DprMENS1Δ

HIS 11,00 kb

PrPHPrPH

EΔH6

Smal - Sac1

Gene Frag. Prot prMENS1. AUG-nt 892 SSEΔ nt 2092 Stop Sac1

DENV-3

Infected Vero cells (Prototype of Chimera Vaccine)

Insertion

Page 20: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Log10-PFU/ml

Growingchimera vaccine compare to wild dengue virus base on plaque forming unit (log10-PFU/ml) in 1 moi. Showed that chimera virus was still low than others.

Kinetic growing of chimera dengue vaccine in Vero cell

Day post infection

Page 21: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Kinetic growing of chimera dengue vaccine in Vero cell

Kinetic growth of chimera dengue virus was compared with wild dengue virus. Supernatant samples of infected Vero cell were then analyzed using indirect ELISA.

Day post infectionDay post infection

Page 22: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Kinetic growing of chimera dengue vaccine in Vero cell

Antigen expression intracelular in infected sel vero was compared with wild dengue virus A. virus was 3th passage, B. virus was 6th passage. The results showed chimera dengue vaccine indicated relative stabil growing in vero cell.

Day post infectionDay post infection

Page 23: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Immunotyping

Rec

ipro

cal o

f an

tigen

tite

r lo

g10

Rec

ipro

cal o

f an

tigen

tite

r lo

g10

MAb types

Analysis of chimera dengue vaccine antigenicity. Indirect ELISA was used to confirm that chimera dengue vaccine protein induced an immune response. Chimera dengue vaccine proteins were reacted with various isotypes of mAb (IgM, IgG, IgG1a, IgG2a, IgG2b).

Page 24: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Neutralization Test

0 2 4 6 8 10 14 21 28 42

10-1

10-2

10-3

10-4

10-5

Jum

lah

Viru

s p

ad

a s

eru

m

10

20

30

40

Inte

rfe

ron

Va

lue

100

101

105

107

Serum Netralisasi Ab Pituity Ab

Serum Netralisasi Ab

An

tibo

dy

Tite

r

Days After Immunization

Fever

1010

EDS

NDL

NDV

102

104

103

106

108

109

ND+IB+IBD

ND+AI

DEN

Purwati, Rantam et al.2012

Page 25: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Discussion NS1 is part of immunogenic of dengue virus for all

serotype (DENV 1, 2,3,4), but in this research we explore for DENV 1, 2, and 4 because of mostly of dengue infection at Surabaya is DENV 2 , DENV 1 and then DENV 4.

So this research was design by chimera models vaccine using by expression of baculovirus system. To purified DNA of restriction product were used by clone, was showed in figure 2. After ligation between product restriction of DENV 1, 2 and 4 insert into plasmid and than transfection into baculovirus.

After 1 week incubation the protein product was purified and than immunized by bulb c and finally characterized using immunotyping , was showed in figure 4.

This protein has immunogenic properties and can induced many kinds of subtype og immunoglobulin .

So that why we called that protein with polyvalent protein.

Page 26: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Immunogenicity : more higher antibody titer more good their immunogenicity, In this research, Ig1a dominantly for this chimera model

Neutralization test: good vaccine has potent of neutralization

Good Candidate vaccine

Page 27: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

CONCLUSION CONCLUSION The recombinant NS1 protein expressed in

a baculovirus system can induce humoral immune response with dominantly IgG1a

Page 28: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING

Chimeric dengue virus inoculated in cell C6/36 or Vero

Cell

Page 29: Purwati, S. Soegijanto, Soetjipto, K. Sudiana, E. Hendrianto, H Susilowati, Fedik A. Rantam ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING