Bacterial spores as vaccine vehicles

Abstract

For the first time, bacterial spores have been evaluated as vaccine vehicles. Bacillus subtilis spores displaying the tetanus toxin fragment C (TTFC) antigen were used for oral and intranasal immunization and were shown to generate mucosal and systemic responses in a murine model. TTFC-specific immunoglobulin G titers in serum (determined by enzyme-linked immunosorbent assay) reached significant levels 33 days after oral dosing, while responses against the spore coat proteins were relatively low. Tetanus antitoxin levels were sufficient to protect against an otherwise lethal challenge of tetanus toxin (20 50% lethal doses). The robustness and long-term storage properties of bacterial spores, coupled with simplified genetic manipulation and cost-effective manufacturing, make them particularly attractive vehicles for oral and intranasal vaccination.

FIG. 1.

Detection of presence of CotB and TTFC by immunofluorescence. Sporulation of B. subtilis strains was induced by the resuspension method (17), and samples were taken 5 h after the onset of sporulation. (A and B) PY79 (wild type). (C and D) RH103 (CotB-TTFC-expressing strain). Samples were labeled with rabbit anti-CotB and mouse anti-TTFC antisera, followed by anti-rabbit IgG-fluorescein isothiocyanate (green) and anti-mouse IgG-tetramethyl rhodamine isothiocyanate (red) conjugates.

FIG. 2.

Systemic responses after mucosal immunizations. Serum anti-TTFC specific IgG responses after oral (A) or intranasal (B) immunization with recombinant B. subtilis spores expressing CotB-TTFC. Groups of seven mice were immunized (↑) orally or intranasally with spores expressing CotB-TTFC (RH103) (•) or nonrecombinant spores (PY79) (○). Doses of 1.67 × 10¹⁰ spores were used for the oral route and of 1.1 × 10⁹ spores were used for the intranasal route, and individual serum samples from groups were tested by ELISA for TTFC-specific IgG. Sera from a naïve control group (▵) and a group orally immunized with 4 μg of purified TTFC protein per dose (◊) were also assayed. Data are presented as arithmetic means and error bars are standard deviations.

FIG. 3.

Antibody isotype profiles. Anti-TTFC antibody isotype profiles on day 54 post-oral immunization or day 48 post-intranasal (i.n.) immunization with recombinant spores expressing CotB-TTFC (RH103) or nonrecombinant B. subtilis spores (PY79) as described in the legend to Fig. 2. TTFC-specific IgG1, IG2a, IgG2b, IgG3, IgM, and IgA isotypes were determined by indirect ELISA. Sera from a naïve control group were also assayed. The end-point titer was calculated as the dilution of serum producing the same optical density as a 1/40 dilution of a pooled preimmune serum. Data are presented as arithmetic means and error bars are standard deviations.

FIG. 4.

TTFC-specific fecal IgA responses. Groups of seven mice were immunized orally (A) or intranasally (B) with recombinant spores expressing CotB-TTFC (RH103) or nonrecombinant spores (PY79) as described in the legend to Fig. 2. Fresh fecal pellets were collected from these immunized mice as well as from a naïve group and were tested for the presence of TTFC-specific IgA as described in Materials and Methods. The end-point titer was calculated as the dilution of the fecal extract producing the same optical density as the undiluted preimmune fecal extract. Data are presented as arithmetic means and error bars are standard deviations.

FIG. 5.

Antispore serum IgG and mucosal IgA responses. Groups of seven mice were immunized (↑) by the oral (A and B) or intranasal (C and D) route with recombinant spores expressing CotB-TTFC (•) or nonrecombinant spores (○) as described in the legend to Fig. 2. Individual samples were tested by indirect ELISA for B. subtilis spore coat-specific serum IgG (A and C) or spore coat-specific fecal IgA (B and D). Sera from a naïve group (▵) were also assayed. The end-point IgG titer was calculated as the dilution of serum producing the same optical density as a 1/40 dilution of a pooled preimmune serum. The end-point IgA titer was calculated as the dilution of the fecal extract producing the same optical density as the undiluted preimmune fecal extract. Data are presented as arithmetic means and error bars are standard deviations.