Gut adhesive Bacillus subtilis spores as a platform for mucosal delivery of antigens.

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.

Bacillus subtilis endospores at high purity and recovery yields: optimization of growth conditions and purification method.

Bacillus subtilis endospores have applications in different fields including their use as probiotics and antigen delivery vectors. Such specialized applications frequently require highly purified spore preparations. Nonetheless, quantitative data regarding both yields and purity of B. subtilis endospores after application of different growth conditions and purification methods are scarce or poorly reported. In the present study, we conducted several quantitative and qualitative analyses of growth conditions and purification procedures aiming generation of purified B. subtilis spores. Based on two growth media and different incubations conditions, sporulation frequencies up to 74.2 % and spore concentrations up to 7 × 10(9) spores/ml were achieved. Application of a simplified spore isolation method, in which samples were incubated with lysozyme and a detergent, resulted in preparations with highly purified spores at the highest yields. The present study represents, therefore, an important contribution for those working with B. subtilis endospores for different biotechnological purposes.

Refolded dengue virus type 2 NS1 protein expressed in Escherichia coli preserves structural and immunological properties of the native protein.

The dengue virus NS1 protein has been shown to be a protective antigen under different experimental conditions but the recombinant protein produced in bacterial expression systems is usually not soluble and loses structural and immunological features of the native viral protein. In the present study, experimental conditions leading to purification and refolding of the recombinant dengue virus type 2 (DENV-2) NS1 protein expressed in Escherichia coli are described. The refolded recombinant protein was recovered as heat-stable soluble dimers with preserved structural features, as demonstrated by spectroscopic methods. In addition, antibodies against epitopes of the NS1 protein expressed in eukaryotic cells recognized the refolded protein expressed in E. coli but not the denatured form or the same protein submitted to a different refolding condition. Collectively, the results demonstrate that the recombinant NS1 protein preserved important conformation and antigenic determinants of the native virus protein and represents a valuable reagent either for the development of vaccines or for diagnostic methods.

Induction of neutralizing antibodies in mice immunized with an amino-terminal polypeptide of Streptococcus mutans P1 protein produced by a recombinant Bacillus subtilis strain.

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.

Boosting systemic and secreted antibody responses in mice orally immunized with recombinant Bacillus subtilis strains following parenteral priming with a DNA vaccine encoding the enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae B subunit.

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.