LOW-COST PRODUCTION OF DENGUE VIRUS E-DOMAIN III FOR AN IMMUNOASSAY DEVELOPMENT

MARÍA EMILIA SMITH; ALEXANDRA M. TARGOVNIK; JULIETA CEREZO; MARÍA VICTORIA MIRANDA; JULIAN RODRÍGUES TALOU

Panamá

Encuentro; Pan-American Dengue Research Network Meeting Panama; 2016

Introduction Dengue is a viral disease transmitted to human by Aedesmosquitoes. The incidence of dengue has grown dramatically in the last years and around 390 million infections occur each year. Currently, most of the available serological immunoassays use the whole virus as a source of antigens, what implies high costs, potential biohazard and a high cross reactivitywith others flavivirus. Thus, the aim of this work was to express a recombinant antigen of dengue virus in Rachiplusia nu larvae for the development of a specific immunoassay for dengue diagnosis.Therecombinant antigen consists on the domain III of the envelope protein of dengue virus type-2 fused to hydrophobin (rDomIIIHFB) for its purification by an aqueous two-phase system (ATPS) and for its immobilization in immunoassay plates. Materials and methodsAntigen production: Rachiplusia nularvaewere infected with a recombinant baculoviruscontaining the expression cassette for rDomIIIHFBby intrahemoceleinjection. At 3 day post-infection, larvae were harvested and total proteins were extracted.Purification:was performed by ATPS with Triton X-114 at 2%, 5% and 8% v/v. Samples of each step were analyzed by SDS-PAGE and Western Blot. Protein immobilization:0.1µg/ml to 12.5µg/ml purified rDomIIIHFBsolutions were loaded in multiwell plates (Polystyrene and Polysorp plates, Nunc) and incubated overnight at 4°C.Immunoassay: polystyrene plates coated with rDomIIIHFBwere used for the detection of anti-dengue IgG antibodies in serum samples.ResultsR. nu larvae expressed 4.5 mg of rDomIIIHFB per g of larva, with the expected molecular weight of 22 kDa. We could efficiently purified rDomIIIHFB by ATPS. In a single purification step, when using a 2% of surfactant, we recovered 3.3 mg of rDomIIIHFB per g of infected larvae, with a purity of 80% and a yield of 73%. We found that rDomIIIHFB was efficiently immobilized in polystyrene and Polysorp plates and we selected 6.25µg/ml as the concentration to coat plates for further experiments. In these conditions, with the amount of rDomIIIHFB recovered from a single larva we could coat 22 plates of 96 wells.The immunoassay developed with rDomIIIHFBwas able to detect IgG specifics for dengue virus type-2 in patient samples and was not able to detect IgG for the other serotypes.ConclusionWe developed a low-cost expression and purification platform for the production of a dengue virus antigen. The rDomIIIHFB-based immunoassay shows specificity for dengue virus type-2 IgG.