purwati, s. soegijanto, soetjipto, k. sudiana, e. hendrianto, h susilowati, fedik a. rantam analysis...
TRANSCRIPT
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
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
• 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
Dengue Situation In Indonesia
(MOH, 2012)
(Rantam et.al., 2010)
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
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
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
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
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
RESULTSRESULTS
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
KLONING NS1M D1 D2 D4
600 bp
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
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
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
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
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
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
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).
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
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.
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
CONCLUSION CONCLUSION The recombinant NS1 protein expressed in
a baculovirus system can induce humoral immune response with dominantly IgG1a
Chimeric dengue virus inoculated in cell C6/36 or Vero
Cell