A recombinant adenovirus-vectored PEDV vaccine co-expressing S1 and N proteins enhances mucosal immunity and confers protection in piglets.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that causes severe diarrhea and high mortality in neonatal piglets. Effective control requires systemic, mucosal, and T cell-mediated immune responses, highlighting the need for a safe, effective, and versatile vaccine platform. In this study, a recombinant adenovirus type 5-based vaccine co-expressing the PEDV S1 and N proteins (rAd5-S1-N) was developed, and its immunogenicity and protective efficacy were evaluated in mice and piglets. Intranasal immunization of BALB/c mice with rAd5-S1-N induced strong and sustained mucosal and systemic responses, with S1-specific IgA detected in both mucosal tissues and serum up to 12 weeks post-immunization. Systemic IgG responses were also robust via intramuscular, intranasal, and intraperitoneal routes, with geometric mean titers reaching ∼10 by week 4 and remaining stable through week 12. In piglets, immunization via the houhai acupoint elicited significantly stronger humoral and cellular responses than the intramuscular route, as evidenced by a 3.6- to 4.1-fold increase in S1- and N-specific IFN-γ-secreting T cells. Both immunization routes induced durable S1-specific IgG responses that remained stable for at least 11 weeks. Importantly, rAd5-S1-N conferred protection in actively immunized piglets against high-dose oral PEDV challenge (2 × 10TCID), and provided passive protection to neonatal piglets via colostrum antibodies after sow immunization, as indicated by reduced viral shedding, shortened diarrhea duration, milder intestinal lesions, and improved weight gain. These findings demonstrate the potential of rAd5-S1-N as a promising vaccine candidate for effective PEDV prevention in swine.