Immune-enhancing effect and mechanism of natural plant-derived immunostimulatory molecule ophiopogonin

To explore the effect and preliminary mechanism of the plant-derived immunostimulatory molecule, ophiopogonin, on enhancing the immune response of a subunit vaccine with the receptor-binding domain（RBD） of coronavirus spike protein as the antigen.

CCK-8 assay was used to determine the cytotoxicity of ophiopogonin D’（OPD’） on bone marrow-derived dendritic cells（BMDCs）. Female Balb/c mice were randomly divided into RBD, RBD/OPD’, RBD/Alum, and control groups.The immunization dose was 5 μg of antigen per mouse and 100 μg of adjuvant per mouse, and immunization was carried out according to the intramuscular injection immunization procedure on days 0, 21, and 42. The titers of specific IgG and its subtype antibodies were detected by ELISA. The cytokine levels in the supernatant of splenocytes were detected using ELISA. The number of splenocytes secreting IFN-γ was detected by ELISpot. Laser confocal microscopy was employed to observe the uptake of antigen by BMDCs.The phagocytic ability of BMDCs for antigen was quantitatively analyzed by flow cytometry. The mechanism of its enhanced immune effect was preliminarily explored using transcriptomics technology combined with bioinformatics research.

When the concentration of OPD’ was less than 5 μg/mL, the survival rate of BMDCs was 100%. After a single intramuscular injection in mice, except for a slight decrease in body weight, the other biochemical indicators were within corresponding normal ranges. After intramuscular injection immunization of the vaccine, the titers of serum-specific IgG, IgG1, and IgG2a in the RBD/OPD’group were significantly higher than those in the RBD group（P＜0. 05）. Compared with the RBD group, the RBD/OPD’ group induced a high-level Th1 cell immune response of IL-1β, TNF-α, and IFN-γ（P＜0. 01） and had more lymphocytes secreting IFN-γ（P＜0. 001）. Laser confocal microscopy displayed that BMDCs took up more antigens after OPD’ treatment, which was further confirmed with flow cytometry in quantitative analysis on antigen uptake rate（P＜0. 01）. Transcriptomics results indicated that there was more significant enrichment of the PPAR signaling pathway in the RBD/OPD’ group than the RBD group, suggesting that OPD’ may activate the PPAR signaling pathway to exert its adjuvant effect.

OPD’ effectively enhances the immune response of the RBD subunit vaccine, and its action mechanism may be related to the activation of the PPAR signaling pathway.