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.

To investigate the antibacterial effect and its preliminary mechanism of lavender essential oil on multi-drug resistant Acinetobacter baumannii.

Micro-dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of lavender essential oil against multi-drug resistant Acinetobacter baumannii, and bactericidal kinetic study was employed to determine the onset and maintenance time of lavender essential oil. Meanwhile, the promoting and therapeutic effects of lavender essential oil on wound healing were observed in a mouse model of infection. Subsequently, crystal violet staining was used to determine the inhibition and clearance of multi-drug resistant Acinetobacter baumannii biofilm by lavender essential oil, and laser confocal microscopy was utilized to observe the survival of bacteria in biofilms. NanoDrop instrument was utilized to quantify the leakage of bacterial DNA nucleic acid and protein after intervention with 3 and 6 mg/mL lavender essential oil, and the leakage of bacterial potassium ion was measured by potassium ion test kit. Proteomics technology combined with bio-informatics were applied to explore the action mechanism of lavender essential oil against multi-drug resistant Acinetobacter baumannii.

The MIC and MBC of lavender essential oil were both 6 mg/mL, which could kill almost all multi-drug resistant Acinetobacter baumannii at the time point of 120 min, and showed an obvious dose- and time-dependent manner. The overall animal model evaluation showed that both 3 and 6 mg/mL lavender essential oil could promote wound healing, and the curative effect was obvious. Further studies confirmed that 3 mg/mL lavender essential oil had a certain biofilm inhibitory effect on multi-drug resistant Acinetobacter baumannii, and 6 mg/mL also had a certain biofilm clearance effect under the same conditions. Meanwhile, when incubated at 37 ℃ for 1 h, the dose of 3 mg/mL could increase the leakage of DNA nucleic acid and protein, and significantly promote the efflux of potassium ions. Proteomic analysis suggested that the antibacterial effect of lavender essential oil may be related to affecting the oxidorereductase activity and cell metabolic process of multi-drug resistant Acinetobacter baumannii, and interfering with the biosynthesis of cell wall/membrane/envelope and other structures.

Lavender essential oil at 3 mg/mL can play an antibacterial effect against multi-drug resistant Acinetobacter baumannii, and its mechanism may be related to the destruction of bacterial biofilm and interference with bacterial metabolism.

To evaluate the stability, safety and immune enhancement and anti-tumor effects of Wilms' tumor gene 1 (WT1) peptide combined with AddaVax^TM emulsion vaccine for acute myeloid leukemia.

The stability of WT1 peptide in the adjuvant vaccine was evaluated using MALDI-TOF-MS time-of-flight mass spectrometry. Female C57BL/6 mice were randomly divided into PBS group, WT1 peptide group, and WT1 peptide+AddaVax^TM emulsion adjuvant vaccine group. The immunization was performed at a dose of 50 μg/mouse for antigen and 50 μg/mouse for adjuvant, with intramuscular injection on days 0, 14, and 28. HE staining was used to assess the toxicity of intramuscular vaccination on mouse organ tissues. Cytokine levels were detected by ELISA, and the number of IFN-γ-secreting splenocytes was measured by ELISpot. Flow cytometry was employed to detect the maturation of bone marrow-derived dendritic cells (BMDCs) promoted by the vaccine in vitro and the promotion for lymphocyte activation, and H-2Db WT1 tetramer was utilized to detect the proportion of specific CD8⁺ T cells. After establishing a mouse leukemia tumor model using the C1498-mWT1 stable cell line, the anti-tumor effects of the vaccine for prevention and treatment were evaluated.

The WT1 peptide stably existed in the vaccine without causing significant organ tissue changes in mice after intramuscular injection. Compared to the mice immunized with WT1 aqueous solution, the mice after intramuscular injection of the WT1 peptide emulsion adjuvant vaccine showed stronger immune responses of Th1 cells, including IFN-γ and TNF-α, as well as Th17 cells of IL-17A (P<0.05), and the mice had not only promoted number of IFN-γ secreting splenocytes (P<0.01) but also enhanced maturation of BMDCs, as indicated by an increase in the proportions of CD40⁺/CD11c⁺ and CD86⁺CD80⁺/CD11c⁺ cells (P<0.05). Additionally, there were increases in both the proportion of CD4⁺/CD3⁺ T and CD69⁺/CD8⁺ T cells (P<0.05) and the proportion of specific CD8⁺ T cells (P<0.05). In the anti-tumor effect study using the C1498-mWT1 mouse model, the median survival time of the WT1+AddaVax^TM group was extended by 6 d compared to the WT1 aqueous solution group. At day 50, the survival rate of mice in the WT1+AddaVax^TM group was still 28.5%, while all mice in the other groups had died (P<0.05).

The vaccine with the WT1 peptide and AddaVax^TM emulsion adjuvant exhibits good immunological and anti-tumor effects.