Development of structure-guided bivalent vaccine antigens for lumpy skin disease virus in Nicotiana benthamiana

Lumpy skin disease virus (LSDV) poses a significant threat to the global cattle industry. While current live-attenuated vaccines carry safety risks, subunit vaccines—though safer—face limitations due to high production costs. The Nicotiana benthamiana expression system offers a scalable and cost-effective alternative, yet its potential for developing LSDV subunit vaccines remains underexplored. In this study, we designed three chimeric immunogen variants by strategically fusing the tandem LSDV060 and LSDV122 antigens. Structural modeling with AlphaFold3 indicated that all three constructs adopt a stable "butterfly-like" architecture. These antigens were successfully expressed and purified from N. benthamiana, exhibiting high thermal stability and favorable biophysical properties. In mouse immunization studies, the plant-produced antigens elicited potent neutralizing antibodies against LSDV, with efficacy comparable to Expi293F-cell-derived counterparts. This work establishes the N. benthamiana platform as a promising system for producing effective LSDV subunit vaccine