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ANALYSIS OF RECOMBINANT, POLYVALENT DENGUE VIRUS CONTAINING
NONSTRUCTURAL PROTEINS (NS1) FROM SEROTYPES -1, -2 AND -4 AND
EXPRESSED IN BACULOVIRUS
Purwati, S. Soegijanto, Soetjipto, K. Sudiana,
E. Hendrianto, H Susilowati, Fedik A. Rantam
Dr. Soetomo Teaching Hospital – Faculty of Med. – Institute of Tropical Diseases
Airlangga University – Surabaya – Indonesia
INTRODUCTION
 Dengue cases is estimated to be between 528 and 621 per million




population
Not specific antiviral therapy
Potential 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
Dengue Virus
• 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 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 VIRUS
5’
C
PrM
M E
NS1
NS 2A-2B
NS3
NS 4A-4B
NS5
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 METHODS
 Virus isolation
 RNA extraction, RT-PCR amplification, cloning and
transfection
 Production of recombinant NS1 protein
 Immunotyping - 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
NS-Protein
DEN-1,2,3,4
E-Protein
PrM--Protein
DENV-4 DENV-3 DENV-2 DENV-1
RNA-VIRUS
WHOLE GENOME DEN-VIRUS
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
SEROTYPE
E-Protein
PrM-Protein
NS1-Protein
PrM & E-Protein coded humoral respons immune, NS1 coded cellular response immune
RESULTS
Virus Isolation
123 4 M 5 67
DEN-1
DEN-4
DEN-3
Den-3
Den-2
Den-2
A
Purwati, Rantam et al.2010
B
DEN-2
Virus production in vero
cell A. vero cell 80%
confluent, dan B vero cell
innoculation with virus
Dengue pasage 3
KLONING NS1
M
D1
D2
D4
600 bp
GENE
Frg. E Gen
Frg. NS1 Gen
Clone
Clone
NS-Protein
DEN-3
E-Protein
PrM--Protein
DENV-4 DENV-3 DENV-2 DENV-1
Frg. prM Gen
Clone
C
PrM
M
E
NS-1
Den-1 Gen Clone
PrM
M
E
NS-1
Den-2NS3
Gen Clone
PrM
PrM
M
M
E
E
NS-1 NS2a,NS2b NS3
NS-1
NS-4a, NS-4b NS-5
Den-4 Gen Clone
Backbone Chimera Den-1,2,4 Gen Clone
MODIFIED DEN-3 VIRUS (CHIMERA)
C
PrM
NS-Protein
DEN-124
M
E
NS2a,NS2b
NS-2a,NS-2b
NS-Protein
NS3
NS-3
NS-4a, NS-4b NS-5
NS-Protein
DEN-124
DEN-124
DEN-3
DEN-3
DEN-3
PrM--Protein
E-Protein
NS-1
PrM--Protein
E-Protein
PrM--Protein
E-Protein
prMD124
ED124
F666
R1228
NS1D124
3’
F668
F601
R1222
R1229
Cloning & Fusion gene
Gene Frag. Prot prMENS1.
AUG-nt 892 SSEΔ nt 2092
Stop Sac1
Smal - Sac1
pVL , 9,8 kb
pVL
DprMENS1Δ
HIS 11,00 kb
PrPH
PrPH
Sac 1
Smal
Insertion
EΔH6
DENV-3
Infected Vero cells (Prototype of Chimera Vaccine)
Ligation
5’
Kinetic growing of chimera dengue vaccine
in Vero cell
Log10-PFU/ml
Day post infection
Growingchimera vaccine compare
to wild dengue virus base on
plaque forming unit (log10PFU/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
Day post infection
Kinetic growth of chimera dengue virus was compared with
wild dengue virus. Supernatant samples of infected Vero cell
were then analyzed using indirect ELISA.
Kinetic growing of chimera dengue vaccine
in Vero cell
Day post infection
Day post infection
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.
Reciprocal of antigen titer log10
Immunotyping
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
1010
EDS
109
NDV
Serum
Netralisasi Ab
Fever
108
DEN
Pituity Ab
NDL
107
30
20
10
10-5
105
10-4
104
10-3
10-2
10-1
ND+AI
103
Serum Netralisasi Ab
102
101
ND+IB+IBD
100
0
Purwati, Rantam et al.2012
2
4
6
8 10 14
21
Days After Immunization
28
42
Antibody Titer
Interferon Value
40
Jumlah Virus pada serum
106
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
 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