RESUMEN
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.
Asunto(s)
Animales , Vacunas contra Escherichia coli/clasificación , Bacillus subtilis , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/inmunologíaRESUMEN
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 constructedB. 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 developedhigher anti-LTB serum IgG and fecal IgA responses with regard to vaccine strains transformed with plasmids encoding the antigen undercontrol of IPTG-inducible (Pspac) or constitutive (PlepA) promoters. Moreover, surface expression of the vaccine antigen under the controlof the PgsiB promoter enhanced the immunogenicity of vegetative cells, while intracellular accumulation of LTB led to higher antibodyresponses in mice orally immunized with recombinant B. subtilis spores. Specific anti-LTB antibodies raised in vaccinated mice recognizedand neutralized in vitro the native toxin produced by ETEC strains. Nonetheless, only mice orally immunized with recombinant B. subtilisstrains, either as vegetative cells or spores, expressing intracellular LTB under the control of the gsiB promoter conferred partial protectionto lethal challenges with purified LT. The present report further demonstrates that B. subtilis plasmid-based heterologous protein expressionsystems are adequate for antigen delivery via the oral route.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/inmunología , Vacunas/clasificación , Vacunas/administración & dosificación , Vacunas/análisis , Vacunas/antagonistas & inhibidores , Vacunas/efectos adversos , Vacunas/inmunología , Vacunas/provisión & distribuciónRESUMEN
Bacillus subtilis has been successfully engineered to express heterologous antigens genetically fused to surface-exposed spore coat proteins asa vaccine vehicle endowed with remarkable heat resistance and probiotic effects for both humans and animals. Nonetheless, the immunogenicityof passenger antigens expressed by B. subtilis spores is low particularly following oral delivery. In this work, we describe a new episomalexpression system promoting enhanced immunogenicity of heterologous antigens carried by B. subtilis strains, either in the form of sporesor vegetative cells, following oral or parenteral delivery to mice. Based on a bi-directional replicating multicopy plasmid, the gene encodingthe B subunit of the heat-labile toxin (LTB), produced by enterotoxigenic Escherichia coli (ETEC) strains, was cloned under the controlof 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 newepisomal expression system may improve the performance of B. subtilis as a live orally-delivered vaccine carrier.