Non-recombinant display of the B subunit of the heat labile toxin of Escherichia coli on wild type and mutant spores of Bacillus subtilis

Abstract

BackgroundMucosal infections are a major global health problem and it is generally accepted that mucosal vaccination strategies, able to block infection at their entry site, would be preferable with respect to other prevention approaches. However, there are still relatively few mucosal vaccines available, mainly because of the lack of efficient delivery systems and of mucosal adjuvants. Recombinant bacterial spores displaying a heterologous antigen have been shown to induce protective immune responses and, therefore, proposed as a mucosal delivery system. A non-recombinant approach has been recently developed and tested to display antigens and enzymes.ResultsWe report that the binding subunit of the heat-labile toxin (LTB) of Escherichia coli efficiently adsorbed on the surface of Bacillus subtilis spores. When nasally administered to groups of mice, spore-adsorbed LTB was able to induce a specific immune response with the production of serum IgG, fecal sIgA and of IFN-γ in spleen and mesenteric lymph nodes (MLN) of the immunized animals. Dot blotting experiments showed that the non-recombinant approach was more efficient than the recombinant system in displaying LTB and that the efficiency of display could be further increased by using mutant spores with an altered surface. In addition, immunofluorescence microscopy experiments showed that only when displayed on the spore surface by the non-recombinant approach LTB was found in its native, pentameric form.ConclusionOur results indicate that non-recombinant spores displaying LTB pentamers can be administered by the nasal route to induce a Th1-biased, specific immune response. Mutant spores with an altered coat are more efficient than wild type spores in adsorbing the antigen, allowing the use of a reduced number of spores in immunization procedures. Efficiency of display, ability to display the native form of the antigen and to induce a specific immune response propose this non-recombinant delivery system as a powerful mucosal vaccine delivery approach.

The sps Gene Products Affect the Germination, Hydrophobicity, and Protein Adsorption of Bacillus subtilis Spores

“…Spores of B subtilis efficiently adsorb heterologous proteins, and adsorption is not dependent on specific spore surface molecules but rather on the negatively charged and hydrophobic surface of the spore (35,36). We have previously shown that in the adsorption of the ␤-galactosidase of Alicyclobacillus acidocaldaricus, the electrostatic interactions do not play a major role (20).…”

Mucosal vaccine delivery by non-recombinant spores of

“…D) At the end of sporulation the mother cell lyses releasing the mature spore with the antigen stably anchored to its surface. a dose of 2 × 10 9 spores, were displayed by the recombinant approach (CotC-LTB), while the same number of spores was able to display up to 5.5 μg of LTB by the nonrecombinant approach [37]. The 25-fold higher efficiency of display obtained with the non-recombinant strategy than with the recombinant one was further improved using mutant spores with an altered outer coat (cotH mutant spores).…”

“…LTB displayed by both strategies was able to induce a specific immune response in mucosally immunized mice [37,38]. An average of 9.6 × 10 -5 pg of LTB/spore, corresponding to approx.…”

“…2 × 10 9 cotH spores adsorbed approx. 14 μg of LTB, about 70-fold more efficient than the antigen displayed by the recombinant approach [37]. The increased efficiency of display of non-recombinant vs. recombinant approach is particularly relevant since it allows to reduce the number of spores needed to induce an immune response.…”

“…A recent report has compared the efficiency of recombinant and non-recombinant spore display using as a model antigen the binding subunit of the heat-labile toxin (LTB) of Escherichia coli [37]. LTB displayed by both strategies was able to induce a specific immune response in mucosally immunized mice [37,38].…”