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

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

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 VIRUS CPrMMENS1NS 2A-2BNS3NS 4A-4BNS5 5’ 3’ 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 Virus isolation RNA extraction, RT-PCR amplification, cloning and transfection RNA extraction, RT-PCR amplification, cloning and transfection Production of recombinant NS1 protein Production of recombinant NS1 protein Immunotyping - ELISA 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 CPrMMENS-1NS2a,NS2bNS3 NS-4a, NS-4bNS-5 CPrMMENS-1NS2a,NS2bNS3 NS-4a, NS-4bNS-5 CPrMMENS-1NS2a,NS2bNS3 NS-4a, NS-4bNS-5 CPrMMENS-1NS2a,NS2bNS3 NS-4a, NS-4bNS-5 DENV-1 DENV-2 DENV-3 DENV-4 E-Protein PrM--Protein NS-Protein DEN-1,2,3,4 WHOLE GENOME DEN-VIRUS RNA-VIRUS SEROTYPE E-Protein PrM-Protein NS1-Protein PrM & E-Protein coded humoral respons immune, NS1 coded cellular response immune

RESULTS

Virus Isolation Den-2 Den-3 Den M A B 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 NS1 M D1 D2 D4 600 bp

GENE PrMMENS-1 PrMMENS-1NS3 CPrMME NS-1NS2a,NS2bNS3 NS-4a, NS-4bNS-5 PrMMENS-1 DENV-1 DENV-2 DENV-3 DENV-4 E-Protein PrM--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-Protein PrM--Protein NS-Protein DEN-3 NS2a,NS2bNS3 C PrM M ENS-1 NS-4a, NS-4bNS-5 DEN-124 E-Protein PrM--Protein NS-Protein DEN-3 DEN-124 E-Protein PrM--Protein NS-Protein DEN-3 DEN-124 NS-3NS-2a,NS-2b

F666 F601 R1229 F668 R1222 R1228 Ligation prMD124ED124 NS1D124 5’3’ Cloning & Fusion gene pVL, 9,8 kb Smal Sac 1 pVL DprMENS1Δ HIS 11,00 kb PrPH 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 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 3 th passage, B. virus was 6 th passage. The results showed chimera dengue vaccine indicated relative stabil growing in vero cell. Day post infection

Immunotyping Reciprocal of antigen titer log10 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 Jumlah Virus pada serum Interferon Value Serum Netralisasi Ab Pituity Ab Serum Netralisasi Ab Antibody Titer Days After Immunization Fever 10 EDS NDL NDV 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 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