Bacillus subtilis spores as vaccine adjuvants: further insights into the mechanisms of action

Abstract

Bacillus subtilis spores have received growing attention regarding potential biotechnological applications, including the use as probiotics and in vaccine formulations. B. subtilis spores have also been shown to behave as particulate vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. In this study, we further evaluated the immune modulatory properties of B. subtilis spores using a recombinant HIV gag p24 protein as a model antigen. The adjuvant effects of B. subtilis spores were not affected by the genetic background of the mouse lineage and did not induce significant inflammatory or deleterious effects after parenteral administration. Our results demonstrated that co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen after subcutaneous administration to BALB/c and C57BL/6 mice. Spores promoted activation of antigen presenting cells as demonstrated by the upregulation of MHC and CD40 molecules and enhanced secretion of pro-inflammatory cytokines by murine dendritic cells. In addition, in vivo studies indicated a direct role of the innate immunity on the immunomodulatory properties of B. subtilis spores, as demonstrated by the lack of adjuvant effects on MyD88 and TLR2 knockout mouse strains.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Adsorption of the p24 protein to spores of the B. subtilis WW02 strain.

(A) Evaluation of protein adsorption to spores at different pHs. Spores (2×109) were suspended in PBS buffer at pH 4, pH 7 or pH 10, mixed with purified p24 protein (2 µg) and kept for 1 h at room temperature. The samples were centrifuged, washed twice with PBS and the spore coat proteins were extracted and submitted to electrophoresis in SDS-PAGE at 15%. The control lane (Con) represents spore samples without incubation with the protein and the control lane (Pr) shows 2 µg of purified p24. Image is representative of four independent experiments. (B) Detection of protein released from spores after adsorption. Spores adsorbed with p24 (PBS at pH 4) were resuspended in PBS at pH 4, pH 7 or pH 10 and incubated for different times. At indicated time points (15, 30, 45, and 60 min) samples were centrifuged and the supernatants and pellet (spores) were loaded onto 15% SDS-PAGE gel. (C) Determination of the saturation curve of p24 protein adsorption to B. subtilis spores. Increasing amounts of protein (0.5 to 4 µg) were added to B. subtilis spores (2×109) following the procedure described before. The amount of protein adsorbed to the spores coat was quantified by dot-blot assay.

Figure 2. Evaluation of antibody responses following subcutaneous immunization with p24 protein adsorbed or mixed with spores.

Mouse groups (n = 5) were immunized (three doses on days 0, 14 and 28) with 2×109 spores (□), 700 ng of p24 only (○), 700 ng of p24 in combination with 2×109 spores (▪) or 700 ng of p24 adsorbed at 2×109 spores (•). Levels of anti-p24 (A and B) and anti-spores (C and D) specific IgG antibodies were detected in serum samples at 13, 27 and 41 days after immunization in BALB/c and C57 BL/6 mice. The results are presented as the mean ± s.e.m. of three independent experiments, and the values obtained from PBS immunized mice were subtracted from each group. *** p<0.001 when compared with mice immunized with p24 only.

Figure 3. Humoral responses after subcutaneous immunization with p24 protein mixed with live or heat-killed spores.

Detection of anti-p24 specific IgG antibodies in serum samples of immunized BALB/c (A) and C57 BL/6 mice (B). Mouse groups (n = 5) were immunized (three doses on days 0, 14 and 28) with viable spores (▵), heat-killed spores (HkSp) (◊), p24 protein (○), p24 protein in combination with Alum (▪), p24 protein in combination with spores (•) or p24 protein in combination with heat-killed spores (▴). *** p<0.001 when compared with group immunized with p24 only or with each other. Specific IgG subclasses elicited in BALB/c (C) and C57BL/6 (D) mice after complete immunization regimen. The IgG2a/IgG1 or IgG2c/IgG1 ratios are indicated at the top of the figure. * p<0.05 or *** p<0.001. All the results are presented as the mean ± s.e.m. of three independent experiments, and the values obtained from PBS immunized mice were subtracted from each group.

Figure 4. Intracellular cell staining and flow cytometry analysis of p24-specific CD4+ and CD8+ T cells.

BALB/c mice were immunized (three doses on days 0, 14 and 28) with p24 protein (700 ng), p24 (10 µg) admixed with LT1 (1 µg), p24 (700 ng) admixed with spores (2×109) or p24 (700 ng) admixed with heat-killed spores (2×109). Spleen cells were collected two weeks after the last dose, and the detection of IL-4 producing p24-specific CD4+ T cells (A) and IFN-γ producing p24-specific CD4+ (B) and CD8+ T cells (C) was carried out after stimulation with MHC class I or II restricted p24 peptide and cell surface staining for CD8 (FITC), CD4 (Cy) and intracellular staining for IL-4 or IFN-γ (PE). The percentage of each population is indicated in the upper right corners and the frequencies in all groups in the absence of stimulus were below 0.70% (data not shown). Measurements were performed in duplicate for each individual sample, and the data are representative of two independent experiments.

Figure 5. Maturation of dendritic cells after stimulation with B. subtilis spores.

(A) Induction of pro-inflammatory cytokines after stimulation with spores. BMDCs from C57BL/6 mice were treated with PBS (control group), LT1 and live or heat-killed spores at a MOI of 10.000. Supernatants were collected after 24 h, and the IL-1β, IL12p70 and TNF-α concentrations were quantified by ELISA. (B) Expression of surface molecules by CD11c+ cells following exposure to spores. BMDCs were treated as described above and the surface expression levels of CD40, MHC-I and MHC-II were measured by flow cytometry. Gray-filled histograms represent samples incubated with PBS and open histograms represent samples incubated with spores. The MFI for both conditions is shown in the upper left in each histogram. (C) Graphical comparison of the expression of CD40, MHC-I and MHC-II. Data represent mean values ± s.e.m. of two independent experiments. *p<0.05, **p<0.01 or *** p<0.001 when compared with control group or with each other. Sp: B. subtilis WW02 spores; HkSp: Heat-killed B. subtilis WW02 spores.

Figure 6. The adjuvant effects of B. subtilis spores require stimulation of TLR2.

MyD88−/− (A) and TLR2−/− (B) C57 BL/6 mice (n = 5) were immunized (three doses on days 0, 14 and 28) with p24 protein only (700 ng) or p24 (700 ng) in combination with live or heat-killed spores (2×109). Levels of anti-p24 specific IgG antibodies were detected in serum samples 13 days after the third dose. The results represent the means ± s.e.m. of two experiments, and the values obtained from PBS immunized mice were subtracted from each group. *** p<0.001.