Porcine epidemic diarrhea virus (PEDV) continues to be a major infectious threat in swine, especially endangering piglets. The COE and S1D domains have been identified as crucial antigens suitable for designing subunit vaccines. Nanodiamonds (NDs), owing to their biocompatibility, large surface area, and modifiable surfaces, have gained interest as novel carriers to improve recombinant protein vaccines. In this study, we transiently expressed a COE-S1D fusion protein containing the GCN4pII motif (COE-S1D-pII) in Nicotiana benthamiana. The recombinant protein was subsequently mixed with nanodiamonds at various mass ratios to form COE-S1D:ND complexes. SDS-PAGE and Western blot analyses identified the optimal ratio as 1:24 (w/w). Additional size, zeta and morphology characterization of these complexes was carried out. We then assessed the immune response of the COE-S1D:ND complex (1:24, w/w) in pregnant sows and their piglets, comparing it to the response induced by the free COE-S1D-pII protein. After administering a booster dose, the COE-S1D:ND mixture significantly enhanced PEDV-specific IgG and COE-S1D-specific IgA levels, as well as neutralizing antibody titers, as measured by ELISA and virus neutralization assays in their piglets. Overall, the results highlight that ND nanoparticles can strengthen both systemic and mucosal immunity, supporting the potential of using plant-produced COE-S1D-pII protein in combination with nanodiamonds as a next-generation subunit vaccine candidate against PEDV.

Kynurenic acid (KYNA), a small molecule derived from the tryptophan–kynurenine pathway, can readily diffuse across biological membranes and act as an endogenous ligand for receptors such as the aryl hydrocarbon receptor (Ahr). While KYNA dysregulation is implicated in neurodegenerative disorders, the role of the KYNA–Ahr-IL-6 axis in MSC proliferation and differentiation remains poorly defined. We investigated the impact of KYNA on murine bone marrow-derived MSCs (BM-MSCs) at various concentrations (10–200 μM) and time points (8–48 h). The BM-MSC phenotype was assessed via flow cytometry; proliferation, via cell counting; and the gene expression of Ahr, Cyp1a1, Cyp1b1, and Il-6, via quantitative real-time PCR. Multipotency was evaluated through adipogenic, osteogenic, and chondrogenic differentiation assays with histochemical confirmation. KYNA significantly upregulated Ahr mRNA expression. Among the tested concentrations, 100 μM KYNA induced the highest Ahr expression (~19.1 ± 1.5-fold greater than that of the untreated controls, p < 0.005). Notably, 10 and 50 μM KYNA caused moderate induction, whereas compared with 100 μM KYNA, 200 μM did not further increase expression. In addition, KYN treatment increased Cyp1a1, Cyp1b1, and Il-6 expression, with increases of ~64.6 ± 4.5-fold, ~43.6 ± 2.3-fold, and ~41.6 ± 1.2-fold, respectively. Compared with no treatment, 100 µM KYNA enhanced BM-MSC proliferation by 1.210 ± 0.02, 1.189 ± 0.03, and 1.242 ± 0.02-fold across passages P3, P4, and P5, respectively (p < 0.05), without altering Sca-1, CD90, or CD45 expression or impairing trilineage differentiation potential. KYNA may activate the AHR–IL-6 signaling axis to promote BM-MSC expansion. This controlled proliferative effect, without loss of phenotypic or functional integrity, highlights the pharmacological potential of KYNA as a small-molecule modulator for stem cell-based therapies.