274 Evaluation of recombinant <i>Capripoxvirus</i> antigens in the development of indirect enzyme linked immunosorbent assays

Abstract Capripoxvirus is a genus of DNA viruses that includes sheep pox virus, goat pox virus and lumpy skin disease virus (LSDV). These viruses are considered high consequence viruses due to the severe clinical disease leading to economic losses to production and impact on trade. The recent spread of LSDV in Europe and Asia has demonstrated the transboundary nature of LSDV. The vaccines used to control Capripoxvirus infections currently do not have the ability to differentiate infected from vaccinated animals (DIVA) and the enzyme linked immunosorbent assays (ELISA) available for detection of antibodies to Capripoxvirus infections are not ideal. Previous research has been done to evaluate different Capripoxvirus antigens using E. coli expressed antigens; however, there has been no prior evaluation of baculovirus expressed Capripoxvirus antigens for use in ELISA. The objective of the current research was to produce 6 recombinant Capripoxvirus antigens using a baculovirus expression vector system (BEVS) and to evaluate these antigens in ELISAs to test bovine, sheep, and goat serum. The sequences used to produce the recombinant antigens were based on a consensus obtained using reference genomes consisting of all three viruses within the genus. All six antigens were produced and purified in-house using BEVS followed by benchtop or FPLC purification (Figure 1). To date, the antigens have been fully validated for performance in indirect ELISA using sheep sera (Figure 2) and partially using cattle sera, with goat sera validation to follow. The ELISAs were developed and optimized using known reference sera as controls. The performance of the ELISAs developed was assessed in comparison with the only commercially available Capripoxvirus serological test. The results demonstrate that some of the antigens used for the ELISAs completely outperformed the commercial test. Several antigens were found to be useful for ELISAs to detect responses in sheep and cattle sera. The sensitivities and specificities of the different ELISAs for sheep sera ranged from 99.54% to 53.57% sensitivity and 99.54 to 94.95% specificity with likelihood ratios above 10 using receiver operating characteristic curve analysis, while the commercial kit had a 42.85% sensitivity using the same positive sheep serum panel evaluated. The development of these serological assays allows for the simultaneous evaluation of immunogenicity of Capripoxvirus antigens through the testing of serum from experimentally infected animals. This study demonstrates the proof of principle, that sensitive and specific ELISAs can be developed for Capripoxviruses.