Mapping neutralizing epitopes and developing protective chimeric antibodies against porcine epidemic diarrhea virus...

Porcine epidemic diarrhea virus (PEDV) causes serious economic losses to the global pig industry. Although commercial vaccines remain the main preventive tool, their effectiveness is often reduced due to the emergence of variant strains and insufficient immune protection transferred from sows to piglets through milk. In this study, we developed three neutralizing monoclonal antibodies (mAbs) that target conserved parts of the virus as a potential treatment option. Using hybridoma technology, we immunized BALB/c mice with purified PEDV particles and obtained four neutralizing mAbs-1H9, 1E7, 3A9, and 1B12. Among these, mAbs 1H9, 1E7, and 3A9 showed strong neutralizing ability against both G1 and G2 types of PEDV. We found that mAb 1H9 recognizes a. epitope on the S1 part of the spike protein, which depends on three key amino acids: T516, N537, and N556. In comparison, mAbs 1E7 and 3A9 bind to two separate linear sequences, ¹⁸⁶KNDWSRVATK¹⁹⁵ and ⁷⁵⁵GVCKSGSIGY⁷⁶⁴, respectively. And all the four mAbs inhibit the viral entry by blocking a post-attachment step in viral entry process. To make these antibodies more suitable for use in pigs, we engineered three mouse-porcine chimeric antibodies, r1H9, r1E7 and r3A9. Among them, r1H9 exhibited the strongest protective efficacy, characterized by significant reductions in small intestinal viral loads (4-5-log decrease), fecal virus shedding (6-log decrease), and histopathological lesions compared to the control group. This finding provides critical insights for the development of mAbs-based therapies and informs the rational design of next-generation subunit vaccines against PEDV.