Female BALB/c mice 7 to 12 weeks old were purchased from Orient Bio (Korea), maintained in our animal facility, and used for all experiments. Animal experiments in this study were performed in accordance with the institutional guidelines of Jeju National University (Korea) for animal care and use. Two vaccine antigens used in this study, Mycoplasma (M.) hyopneumoniae and Bordetella (B.) bronchiseptica, were obtained from ChoongAng Vaccine Laboratories (Korea) and KBNP (Korea), respectively. The amount of protein in the formalin-inactivated intact bacteria administered as antigen was measured with the Bradford protein assay (Bio-Rad Laboratories, USA).

Fucoidan derived from Fucus vesiculosus was purchased from Sigma (USA) and dissolved in phosphate buffered saline (PBS). Endotoxin levels in the fucoidan solutions were measured with QCL-1000 Chromogenic LAL endpoint assay (Lonza, USA). The endotoxin level in the solution with the highest concentration (50 µg/mL) was below than the detection limit of the kit (0.1 EU/mL) [11].

The spleen cells were harvested as previously described [10]. The cells were acquired by mincing the spleens recovered from the BALB/c mice. Red blood cells were removed from the spleen cells with an ammonium chloride-potassium buffer. After incubating the cells in a T75 flask for 1 h, the floating cells were collected and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, L-glutamine, penicillin/streptomycin, sodium pyruvate, non-essential amino acids, and 2-mercaptoethanol. The cells were maintained at 37℃ in 5% CO₂.

The spleen cells were seeded at a concentration of 2 × 10⁶ cells/mL in 96-well culture plates for a viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma). The cells were treated with fucoidan and B. bronchiseptica antigen for 2 days. Next, 10 µL/well of MTT solution (final concentration of 0.5 mg/mL) was added and the cells were incubated for 4 h. After this, 10% SDS solution (100 µL/well) was added to the wells and the plate was incubated for 2 h to dissolve the crystals generated by viable cells. Optical density of the wells was measured at 570 nm using a microplate reader (Molecular Devices, USA).

A cytokine-specific ELISA kit was used to measure the amount of cytokines produced by the spleen cells. Culture supernatants were collected and used for the ELISA. The amount of tumor necrosis factor (TNF)-α in the supernatants was measured with CytoSet kit (Invitrogen, USA) according to the manufacturer's instruction.

To determine whether fucoidan affects subsets of spleen cells, flow cytometry analysis was performed with surface marker-specific antibodies. The spleen cells were seeded at a concentration of 2 × 10⁶ cells/mL in 6-well culture plates. The staining procedure was performed as previously described in detail [8]. The cells were stained with biotin-labeled anti-B220, anti-CD19, anti-I-A^d, and anti-CD80 antibody followed by streptavidin-fluorescein isothiocyanate (FITC; all from BD Biosciences, USA). To examine activation markers, the spleen cells were stained with anti-CD25 antibody followed by phycoerythrin (PE)-labeled anti-rat IgM or PE-labeled anti-CD69 antibody. In addition, the cells were stained with biotin-labeled anti-B220, streptavidin-FITC, and PE-labeled anti-CD138 antibody to detect plasma cells. To assess the total effects of fucoidan in vivo, we measured the expression levels of fucoidan-treated cells without gating. The stained cells were analyzed with FACSCalibur (BD Biosciences) and WinMDI 2.9 software.

To measure the effect of fucoidan as an adjuvant in vivo, M. hyopneumoniae antigen (10 µg/mouse) and/or fucoidan (100 mg/kg) was injected into mice twice at a 2-week interval. The dose of fucoidan in vivo was determined based on efficacy and toxicity [12]. PBS was injected as a control. Two weeks after the final injection, serum was collected and antigen-specific antibody levels were measured. Maxisorp Nunc-Immuno module (Thermo Scientific, USA) was coated with M. hyopneumoniae antigen, and the plates were sequentially treated with blocking solution, the samples, horseradish peroxidase-conjugated anti-mouse IgG antibody, substrate, and stop solution. Optical density was measured at 405 nm using a microplate reader.

Data in Figs. 4,5,6 are presented as the mean ± standard deviation (SD). Differences were analyzed using ANOVA and Student t-test. A p value < 0.05 was considered significant.

To investigate the effect of fucoidan on the expression of surface markers specific for B lymphocytes, we stained fucoidan-treated spleen cells with anti-B220 or -CD19 antibody. B220 is a pan B lymphocyte marker while CD19 is a mature B lymphocyte marker. The expression levels of both markers on spleen cells treated with 2 or 10 µg/mL fucoidan were similar to those on the control cells (Fig. 1). However, the expression of both markers was decreased on spleen cells treated with 50 µg/mL fucoidan.

We next measured the expression levels of various immune response-related markers, I-A^d, CD80, and CD25 with CD69, that are associated with antigen presentation, co-stimulatory signal transduction, and lymphocyte activation, respectively. Fucoidan consistently increased the expression level of I-A^d while CD80 expression was decreased (Fig. 2). Additionally, fucoidan increased the expression of both lymphocyte activation markers (CD25 and CD69) on spleen cells treated with 10 or 50 µg/mL fucoidan (Fig. 2). Flow cytometry analysis revealed that fucoidan activated the spleen cells as indicated by increased expression of the immune response-related markers.

To determine whether fucoidan may induce the generation of antibody-producing plasma cells, we treated spleen cells with fucoidan and stained for two plasma cell-specific markers: B220 and CD138. The percentage of plasma cells (B220⁺ CD138⁺) was marginally enhanced among spleen cells treated with 10 or 50 µg/mL fucoidan (Fig. 3). However, the population of plasma cells was relatively small in our study.

To determine whether fucoidan can be used as an immunostimulatory adjuvant for vaccines, we administered fucoidan with B. bronchiseptica vaccine antigen. We then performed an MTT assay to assess the toxicity of fucoidan in combination with the antigen. Compared to the control, B. bronchiseptica antigen significantly increased the viability of spleen cells in a concentration-dependent manner. Addition of fucoidan further increased the viability (Fig. 4). Results of the MTT assay revealed that fucoidan does not cause significant toxicity when used in combination with B. bronchiseptica antigen.

Immunostimulatory cytokines are required to enhance the efficacy of vaccines. We measured the production of TNF-α by spleen cells treated with B. bronchiseptica and fucoidan. TNF-α is an inflammatory cytokine that increases immune responses essential for vaccine efficacy. Results of an ELISA demonstrated that B. bronchiseptica antigen significantly induced TNF-α production by spleen cells at 1.0 µg/mL. Addition of 50 µg/mL fucoidan increased TNF-α production at control and B. bronchiseptica antigen 0.2 µg/mL (Fig. 5). These findings seem to indicate that fucoidan may not have synergistic effects when used in combination with B. bronchiseptica antigen.

We evaluated the adjuvant effect of fucoidan on the production of antigen-specific antibodies in mice using an immunoassay. For these in vivo experiments, M. hyopneumoniae was used as the antigen. No antigen-specific antibody production was detected in the control mice injected with PBS alone (5^-3~5^-6 dilution). Fucoidan enhanced the generation of M. hyopneumoniae-specific antibody compared to treatment with antigen alone (5^-6~5^-7 dilution; Fig. 6).