Recombinant protein-based ELISA for serological differentiation of LSD Virus-Infected and Vaccinated Animals (DIVA Assay)

Background:

The effective control of infectious diseases through mass vaccination requires a serological test capable of differentiating infected from vaccinated animals (DIVA). India has recently developed and commercialized a homologous live-attenuated lumpy skin disease (LSD) vaccine, Lumpi-ProVacInd, derived from the local Ranchi strain. The vaccine has been approved by the Central Drugs Standard Control Organization (CDSCO), Govt. of India and is now commercially available as Biolumpivaxin®. The Indian state of Uttar Pradesh has already adopted LSD-specific vaccine to phase out goatpox, and Maharashtra is transitioning. This Ranchi LSD vaccine strain possesses a unique 801-nucleotide deletion in the inverted terminal repeat (ITR) region (ORF154), distinguishing it from field strains of the LSD virus (LSDV). In this invention, we cloned, expressed, and purified the ORF154 protein from a field LSDV strain using the pET28a vector and Ni-NTA affinity chromatography. SDS-PAGE and Western blot confirmed a ~28 kDa His-tagged protein. The recombinant ORF154 protein was employed as a trapping antigen in an ELISA, optimized at 500 ng/well antigen concentration and 1:50 serum dilution. The assay demonstrated high sensitivity (96.12%) and specificity (95.77%) at a positivity cut-off of 22%, reacting exclusively with sera from infected animals and not with those from vaccinated cattle. These results establish the ORF154-based ELISA as a reliable DIVA-compatible diagnostic tool. Lumpi -ProVac Ind, developed by ICAR-NRCE, thus represents the world’s first live-attenuated LSD vaccine with an inherent DIVA capability

Technology Details:

The invention utilizes a recombinant ORF154 protein, uniquely present in field LSDV strains but deleted in the Ranchi vaccine strain. The ORF154 gene was cloned into a pET-28a(+) expression vector and expressed in E. coli, yielding a purified ~28 kDa His-tagged recombinant protein. This protein is used as an antigen in an indirect ELISA capable of distinguishing infected (≥22% positivity) from vaccinated/naïve animals (≤22%). The assay showed 96.12% sensitivity and 95.77% specificity, offering a highly accurate, reproducible, and economical DIVA diagnostic platform.