Characterization of two monoclonal antibodies that capture and neutralize LSDV via distinct novel epitopes

Functional monoclonal antibodies (mAbs) are crucial for the development of effective control and therapeutic strategies against lumpy skin disease (LSD), a cattle disease caused by Lumpy skin disease virus (LSDV) that results in significant economic losses. In this study, the soluble recombinant ORF117 protein of LSDV was expressed in 293F cells. It was then employed as an immunogen to generate two mAbs, designated 11C4 and 28B5. Bio-layer interferometry measurements indicated high binding affinities, with equilibrium dissociation constants (Kd) values of 0.05 nM for 11C4 and 0.8 nM for 28B5. Indirect ELISA revealed half-maximal effective concentration (EC₅₀) value of 6.69 × 10^-3μg/mL for 11C4 and 11.22 × 10^-3μg/mL for 28B5. Immunofluorescence assays confirmed that both mAbs recognized the native viral ORF117. Competition ELISA demonstrated that the mAbs preferentially binded to viral particles rather than to recombinant ORF117 in a co-incubation system. Neutralization assays showed that 11C4 achieved 45.1 % neutralization of LSDV infection, while 28B5 exhibited comparable activity. Epitope mapping indicated both mAbs recognized novel distinct epitopes. 11C4 targeted the epitope (IFPGDDDETNERNINHREKT) with loop structure, whereas 28B5 bound to a novel distinct region (KKIINERYSNYISIDDDEISDILKDSFISNEEMQI) with helix structure. Structural docking simulations revealed that 11C4 recognized a conserved segment IFPGDDDET among Chordopoxvirinae subfamily, with Asp12 and Glu14 identified as critical residues forantibody binding. This study provided valuable tools to advance the development of diagnostic and therapeutic measures against LSDV and related poxviruses.