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Bacillus subtilis spores have been used as safe and heat-resistant antigen delivery vectors. Nonetheless, the oral administration of spores typically induces weak immune responses to the passenger antigens, which may be attributed to the fast transit through the gastrointestinal tract. To overcome this limitation, we have developed B. subtilis spores capable of binding to the gut epithelium by means of expressing bacterial adhesins on the spore surface. The resulting spores bound to in vitro intestinal cells, showed a longer transit through the mouse intestinal tract, and interacted with Peyer's patch cells. The adhesive spores increased the systemic and secreted antibody responses to the Streptococcus mutans P1 protein, used as a model antigen, following oral, intranasal, and sublingual administration. Additionally, P1-specific antibodies efficiently inhibited the adhesion of the oral pathogen Streptococcus mutans to abiotic surfaces. These results support the use of gut-colonizing B. subtilis spores as a new platform for the mucosal delivery of vaccine antigens.

The oral pathogen Streptococcus mutans expresses a surface protein, P1, which interacts with the salivary pellicle on the tooth surface or with fluid-phase saliva, resulting in bacterial adhesion or aggregation, respectively. P1 is a target of protective immunity. Its N-terminal region has been associated with adhesion and aggregation functions and contains epitopes recognized by efficacious antibodies. In this study, we used Bacillus subtilis, a gram-positive expression host, to produce a recombinant N-terminal polypeptide of P1 (P1(39-512)) derived from the S. mutans strain UA159. Purified P1(39-512) reacted with an anti-full-length P1 antiserum as well as one raised against intact S. mutans cells, indicating preserved antigenicity. Immunization of mice with soluble and heat-denatured P1(39-512) induced antibodies that reacted specifically with native P1 on the surface of S. mutans cells. The anti-P1(39-512) antiserum was as effective at blocking saliva-mediated aggregation of S. mutans cells and better at blocking bacterial adhesion to saliva-coated plastic surfaces compared with the anti-full-length P1 antiserum. In addition, adsorption of the anti-P1 antiserum with P1(39-512) eliminated its ability to block the adhesion of S. mutans cells to abiotic surfaces. The present results indicate that P1(39-512), expressed and purified from a recombinant B. subtilis strain, maintains important immunological features of the native protein and represents an additional tool for the development of anticaries vaccines.

Recombinant Bacillus subtilis strains, either spores or vegetative cells, may be employed as safe and low cost orally delivered live vaccine vehicles. In this study, we report the use of an orally delivered B. subtilis vaccine strain to boost systemic and secreted antibody responses in mice i.m. primed with a DNA vaccine encoding the structural subunit (CfaB) of the CFA/I fimbriae encoded by enterotoxigenic Escherichia coli (ETEC), an important etiological agent of diarrhea among travelers and children living in endemic regions. DBA/2 female mice submitted to the prime-boost immunization regimen developed synergic serum (IgG) and mucosal (IgA) antibody responses to the target CfaB antigen. Moreover, in contrast to mice immunized only with one vaccine formulation, sera harvested from prime-boosted vaccinated individuals inhibited adhesion of ETEC cells to human red blood cells. Additionally, vaccinated dams conferred full passive protection to suckling newborn mice challenged with a virulent ETEC strain. Taken together the present results further demonstrate the potential use of recombinant B. subtilis strains as an alternative live vaccine vehicle.

Recombinant Bacillus subtilis strains, either in the form of spores or vegetative cells, may be employed as safe and low-cost vaccine vehicles. In this study, we studied the role of promoter sequences and antigen-sorting signals on the immunogenicity based on previously constructed B. subtilis episomal expression systems. Mice orally immunized with spores or cells encoding the B subunit of the heat labile toxin (LTB), originally expressed by some enterotoxigenic Escherichia coli (ETEC) strains, under control of the stress-inducible gsiB promoter developed higher anti-LTB serum IgG and fecal IgA responses with regard to vaccine strains transformed with plasmids encoding the antigen under control of IPTG-inducible (Pspac) or constitutive (PlepA) promoters. Moreover, surface expression of the vaccine antigen under the control of the PgsiB promoter enhanced the immunogenicity of vegetative cells, while intracellular accumulation of LTB led to higher antibody responses in mice orally immunized with recombinant B. subtilis spores. Specific anti-LTB antibodies raised in vaccinated mice recognized and neutralized in vitro the native toxin produced by ETEC strains. Nonetheless, only mice orally immunized with recombinant B. subtilis strains, either as vegetative cells or spores, expressing intracellular LTB under the control of the gsiB promoter conferred partial protection to lethal challenges with purified LT. The present report further demonstrates that B. subtilis plasmid-based heterologous protein expression systems are adequate for antigen delivery via the oral route.

Bacillus subtilis has been successfully engineered to express heterologous antigens genetically fused to surface-exposed spore coat proteins as a vaccine vehicle endowed with remarkable heat resistance and probiotic effects for both humans and animals. Nonetheless, the immunogenicity of passenger antigens expressed by B. subtilis spores is low particularly following oral delivery. In this work, we describe a new episomal expression system promoting enhanced immunogenicity of heterologous antigens carried by B. subtilis strains, either in the form of spores or vegetative cells, following oral or parenteral delivery to mice. Based on a bi-directional replicating multicopy plasmid, the gene encoding the B subunit of the heat-labile toxin (LTB), produced by enterotoxigenic Escherichia coli (ETEC) strains, was cloned under the control of the B. subtilis glucose starvation inducible (gsiB) gene promoter, active in vegetative cells submitted to heat and other stress conditions. The recombinant plasmid proved to be structurally and segregationally stable in both cells and spores under in vitro and in vivo conditions. Moreover, BALB/c mice orally immunized with B. subtilis cells or spores elicited enhanced anti-LTB systemic (serum IgG) and secreted (fecal IgA) antibody responses, thus, suggesting that antigen expression occurred during in vivo transit. These results indicate that the new episomal expression system may improve the performance of B. subtilis as a live orally-delivered vaccine carrier.