Engineering a recombination-resistant live attenuated vaccine candidate with suppressed interferon antagonists for...

Porcine epidemic diarrhea virus (PEDV) is a deadly coronavirus (CoV) for neonatal piglets, and no effective vaccine is available. Live attenuated vaccines (LAVs) show promise, but risks of recombination and reversion to virulence hinder their application. For LAV development, we previously engineered a recombination-resistant PEDV mutant RMT by rewiring the transcriptional regulatory sequence (TRS)-core sequences (TRS-CSs). Because TRS is critical for CoV replication, the incompatibility between wild-type TRS-CS and the rewired TRS-CS should disrupt the structural and accessory protein-encoding mRNA transcription from a recombinant PEDV genome, preventing the production of progeny infectious viruses. However, the RMT contained a 189-nt insertion upstream of the E gene TRS-CS and a missing guanine in the N gene TRS-CS. Here, we generated RMTv1 via correcting these mutations and removing the EGFP gene in RMT. Using the RMTv1 as a backbone, we generated a series of PEDV mutants carrying one or two attenuating mutations of non-structural protein 1 (nsp1), nsp15, and nsp16. Their replication efficiency, sensitivity to interferons (IFNs), induction of IFNs, and genetic stability were tested in Vero and/or LLC-PK1 cells. We selected RMTv1 and RMTv1-nsp1 + nsp15 for the pathogenesis studies in neonatal gnotobiotic pigs and tested the immunogenicity of RMTv1-nsp1 + nsp15. The RMTv1-nsp1 + nsp15 was further attenuated, causing no pig mortality, compared with RMTv1. All the pigs infected with RMTv1-nsp1 + nsp15 were protected from severe diarrhea and death post-challenge with virulent PEDV at 21 days post-inoculation, whereas 50% of mock-challenged piglets died. These findings establish RMTv1-nsp1 + nsp15 as a promising PEDV LAV candidate and can be further evaluated.IMPORTANCEPEDV continues to cause devastating economic losses in the global swine industry. Exposing pregnant sows to feedback materials from infected pigs is still one of the main methods used to control PEDV outbreaks in U.S. swine farms but carries the risk of transmitting other pathogens. Effective and safe vaccines are desperately needed to replace the feedback materials but are still not available. We revised the recombination-resistant vaccine backbone and combined it with targeted attenuation mutations in viral IFN antagonists to generate six PEDV mutants. Among them, the RMTv1-nsp1 + nsp15 had significant advancements in safety and protective efficacy in neonatal piglets, demonstrating its vaccine potential to control PEDV outbreaks and improve swine health globally. By addressing key challenges in LAV development, including risks of reversion to virulence and generation of new variants via recombination, this work establishes a robust foundation for PEDV vaccine strategies and potentially inspires the development of vaccines against other emerging coronaviruses.