Dehydroevodiamine enhances PEDV vaccine efficacy through immune-gut microbiota modulation

Porcine Epidemic Diarrhea Virus (PEDV) is a major pathogen of severe enteric disease and mortality in neonatal piglets, and currently available vaccines often provide incomplete and inconsistent protective immunity. The limited immunostimulatory capacity and safety concerns associated with conventional vaccine adjuvants necessitate the development of alternative adjuvants with improved efficacy and biocompatibility. This study evaluated the adjuvant potential of Dehydroevodiamine (DHED), a natural quinazoline alkaloid isolated from Euodiae Fructus, in synergistically enhancing PEDV vaccine-induced humoral and cellular immune responses. In vitro, RT-qPCR results demonstrated that 6.25 μg/mL DHED significantly upregulated the mRNA expression of IL-1β, TNF-α, IL-6, and IL-10 in RAW264.7 macrophages, indicating immunomodulatory potential. In vivo, BALB/c mice were intragastrically administered DHED in conjunction with immunization using a commercial PEDV vaccine. DHED significantly elevated vaccine-induced PEDV-specific IgG and intestinal SIgA levels, upregulated serum IFN-γ and IL-4 concentrations, and promoted CD4⁺ T-cell activation in peripheral blood and spleen, along with increased CD3⁺CD4⁺/CD3⁺CD8⁺ ratios and splenic index, thus enhancing the vaccine's immunoefficacy. Furthermore, 16S rRNA sequencing revealed that DHED-mediated immune enhancement was accompanied by significant alterations in intestinal microbiota composition. Co-administration with PEDV vaccine increased gut microbiota richness and diversity, regulated the relative abundance of beneficial bacteria, and enriched immune-related taxa (e.g., Lachnospiraceae, Lachnospiraceae_NK4A136_group, unclassified_f_Lachnospiraceae) to potentiate immune responses. Collectively, DHED emerges as a promising adjuvant candidate, inducing robust cellular and humoral immunity while overcoming the limitations of conventional adjuvants. These findings suggest that DHED represents a promising natural adjuvant candidate for improving PEDV vaccine efficacy through coordinated modulation of immune responses and intestinal microbial communities. Together, our study provides a safe and innovative adjuvant strategy for PEDV control in swine herds, and offer robust experimental evidence supporting the clinical application of DHED.