PLANT-BASED RECOMBINANT DENGUE ENVELOPE DOMAIN III (EDIII) ANTIGENS FOR THE DEVELOPMENT OF SEROTYPE-SPECIFIC RAPID DIAGNOSTIC KIT

National Center for Vector Borne Diseases Control (NCVBDC), India recorded 10,172 morbid cases of dengue and 346 deaths in 2022 (till 31 May 2022) and 193,245 morbid infections and 346 deaths in 2021. Dengue envelope domain III (EDIII) antigen helps in host cell entry through receptor binding and induction of prolonged immunity against distinct serotypes DENV1, DENV2, DENV3 and DENV4. The wider prevalence of infection and severity of illness require differential diagnosis for control and treatment. Codon-optimized sequences of all four serotypes for expression in bacterial and plant hosts were synthesized and cloned in GATEWAY entry vector. All four EDIII gene sequences were cloned into GATEWAY destination vectors (pDEST527 for E. coli and pEAQ-HT-DEST3 for Nicotiana benthamiana) by LR cloning and transformed into E. coli DH5α. The constructs pDEST527-D1EDIII, pDEST527-D2EDIII, pDEST527-D3EDIII and pDEST527-D4EDIII were expressed following IPTG-induction in E. coli BL21(DE3)pLysS and the recombinant octahistidine-tagged proteins are purified using Ni-NTA metal affinity chromatography. The constructs pEAQ-HT-DEST3-D1EDIII, pEAQ-HT-DEST3- D2EDIII, pEAQ-HT-DEST3-D3EDIII and pEAQ-HT-DEST3-D4EDIII will be transformed into Agrobacterium tumefaciens GV3101 and the transiently expressed recombinant hexahistidine-tagged proteins will be used to develop lateral-flow based rapid diagnostic test kit for serotype-specific diagnosis. Crude and purified DENV1, DENV2, DENV3 and DENV4 rEDIII antigens, ∼ 110 amino acids length and ∼12.7 KDa each, from E. coli were analyzed on SDS-PAGE gel and confirmed using Western blot. All four antigens will be expressed in N. benthamiana system, purified and confirmed. Transiently expressed recombinant antigens in plant system will account to 50-100 mg/kg of leaf fresh weight. The sensitivity and specificity of rEDIII antigen of each serotype will be validated using commercial serotype- specific antibodies, patient and healthy serum samples using in-house ELISA. All four rEDIII antigens from plant-based transient expression will be used to develop a highly sensitive differential diagnostic kit that will be rapid, cost-effective and serotype-specific.