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 establish a mouse model of infection with the minimum lethal dose of Vibrio vulnificus (V. vulnificus) and to evaluate the protective efficacy of inactivated whole-cell (IWC) vaccine using this model.

A mouse model of lethal-dose infection was established by intraperitoneal injection of different doses of V. vulnificus. Bacterial colonization in the organs was detected with tissue homogenate plating, and pathological changes in the organs were observed after tissue section staining. Flow cytometry was used to detect immune cell responses after liver tissues were digested into single-cell suspension. IWC vaccine of V. vulnificus was prepared, and the mice were immunized through different routes to observe the protective efficacy of the vaccine.

A mouse model of infection with the minimum lethal dose at 1×10⁶ CFU of V. vulnificus was successfully established. After infection, the bacteria were mainly colonized in the liver of mice and caused severe pathological damages. Compared with the uninfected mice, the proportion of neutrophils in the liver was significantly increased in the infected mice, whereas the proportions of B cells and T cells were correspondingly decreased (P<0. 05). A single intramuscular or intraperitoneal injection of the IWC vaccine could protect the mice effectively against lethal infection of V. vulnificus in 7 d later (P<0. 01), although the level of serum IgG having no significant increase.

A mouse model of lethal-dose infection with V. vulnificus is successfully established, with histopathological characteristics. The IWC vaccine of V. vulnificus rapidly mediates immune protection in this model probably independent of IgG.

To investigate the distribution of B cells in both tumor and non-tumor tissues of gastric cancer patients, analyze their phenotypic characteristics and explore the impact on T cell proliferation.

Immunohistochemical staining was utilized to detect the expression of B cell surface marker CD19 in tumor and non-tumor tissues from 33 gastric cancer patients. The expression levels of chemokine receptors and immunoglobulin molecules on B cells in both tumor and non-tumor tissues were measured using flow cytometry. Chemotaxis experiments were conducted to examine the role of the CXCL12-CXCR4 axis in B cell chemotaxis. B cells isolated and purified from both tissue types were co-cultured with autologous peripheral T cells to assess their effect on T cell proliferation.

There were significantly more B cells infiltrated in tumor tissues than those infitrated in the non-tumor tissues of gastric cancer patients (P<0.01), and CXCR4 was highly expressed on tumor-infiltrating B cells compared with B cells derived from non-tumor tissues (P<0.05). The Cancer Genome Atlas (TCGA) analysis indicated that the expression level of CXCL12 in tumor tissues was positively correlated with the expression level of CD19 in gastric cancer patients (r=0.15, P<0.01). And the expression level of CXCL12 in tumor tissues of the gastric cancer patients was also positively correlated with the number of B cells infiltrated in tumor tissues. Chemotaxis experiments confirmed that the CXCL12-CXCR4 axis was involved in promoting B cell chemotaxis (P<0.05). Although B cells in tumor and non-tumor tissues had similar levels of IgM, IgG, and IgA expression, tumor-infiltrating B cells significantly inhibited the proliferation of T cells when compared with B cells derived from non-tumor tissues (P<0.01).

There are more B cells infiltrated in gastric cancer tissues, which may be recruited to tumor tissues through the CXCL12-CXCR4 axis, and then inhibit T cell proliferation to promote the progression of gastric cancer.