Preparation and epitope mapping of broad-spectrum neutralizing monoclonal antibodies against economically important pestiviruses

Pestiviruses are pathogens with broad host ranges, infecting domestic animals and wildlife, with some of them exhibiting antigenic similarity and cross-neutralization capability. However, the underlying molecular mechanism remains elusive. In this study, BALB/c mice were immunized with purified E2 protein of classical swine fever virus (CSFV) LPC strain to generate monoclonal antibodies (mAbs) using hybridoma technology. Immunofluorescence assay (IFA) and Western blot were employed to determine the reactivity of mAbs with pestiviruses and E2 proteins. As a result, 31 mAbs against CSFV E2 protein were generated, with two mAbs TCH034 and TCH052 exhibiting broad-spectrum reactivity against different genotypes of CSFV, BVDV1 and BVDV2, as well as E2 proteins from 8 pestivirus species (CSFV, BVDV1, BVDV2, HobiPeV, BDV, AydinPeV, TSV and ovIT PeV). The pestivirus broad-spectrum mAbs demonstrated neutralizing activity against the available strains of CSFV, BVDV1, and BVDV2. Epitope mapping with truncated and point-mutated E2 proteins showed that TCH034 and TCH052 recognize a highly conserved antigenic epitope in E2 proteins among 8 pestivirus species, comprising residues G ¹¹³ , K ¹¹⁴ , W ¹³³ and G ¹³⁵ , which was further confirmed by the inability of TCH034 and TCH052 to react with or neutralize the rescued CSFV strain JL23 containing mutation K ¹¹⁴ G. Taken together, the findings of this study elucidate the molecular basis underlying antigenic conservation and cross-neutralization among pestiviruses providing valuable support for the development of diagnostic assays targeting economically important pestiviruses.