Identification of two immunodominant and neutralizing linear B-cell epitopes exposed on the surface of the porcine deltacoronavirus spike protein.

Abstract

Porcine deltacoronavirus (PDCoV) is an enteropathogenic virus that causes severe diarrhea in pigs, particularly suckling piglets, and exhibits cross-species transmission with zoonotic potential. The S1 subunit of the viral spike (S) protein mediates cell entry and elicits neutralizing antibodies, making it an ideal target for diagnostics, prophylaxis, and therapeutics. Here, we produced two monoclonal antibodies (mAbs), B8F10 and G10C2, by immunizing BALB/c mice with a recombinant PDCoV S1 protein fused to a human IgG Fc fragment, expressed in an insect baculovirus system and purified using Protein A/G magnetic beads. Both mAbs can specifically recognize native PDCoV S protein in indirect immunofluorescence assays and western blot analyses under denaturing conditions, indicating their binding to linear epitopes. Isotyping classified both as IgG1/κ, and sequence analysis revealed distinct heavy-chain complementarity-determining regions (CDRs) but identical light-chain CDRs. Using truncated S1 proteins coupled with Pepscan ELISA, the B8F10 and G10C2 epitopes were precisely mapped toLETNFMCTandVINNTVVG, respectively. These epitopes can be recognized by both mAbs and swine PDCoV antiserum, confirming their immunodominance. While global PDCoV strain alignments revealed high conservation of these epitopes, a V491A mutation within the G10C2-binding site abolished mAb binding. Structural analysis confirmed both epitopes are surface-exposed on S1. These findings demonstrate the diagnostic potential of these mAbs and the epitopes' suitability as vaccine targets. Their high conservation suggests broad applicability, whereas the V491A mutation may represent an immune escape mechanism. These epitopes could serve as diagnostic markers, immunotherapeutics, or the foundation for epitope-based vaccines to induce protective immunity.