ABSTRACT
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.
Subject(s)
Bacillus subtilis/immunology , Bacterial Toxins/biosynthesis , Bacterial Toxins/metabolism , Bacterial Vaccines/immunology , Enterotoxins/biosynthesis , Enterotoxins/metabolism , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/metabolism , Promoter Regions, Genetic , Protein Sorting Signals/genetics , Administration, Oral , Animals , Antibodies, Bacterial/analysis , Antibodies, Bacterial/blood , Antigens, Bacterial/biosynthesis , Antigens, Bacterial/immunology , Antigens, Bacterial/metabolism , Antitoxins/analysis , Antitoxins/blood , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Toxins/immunology , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/genetics , Disease Models, Animal , Enterotoxins/immunology , Escherichia coli/genetics , Escherichia coli Proteins/immunology , Female , Immunoglobulin A/analysis , Immunoglobulin G/blood , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neutralization Tests , Plasmids/genetics , Poisoning/immunology , Protein Subunits/biosynthesis , Protein Subunits/immunology , Protein Subunits/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Spores, Bacterial/immunology , Survival AnalysisABSTRACT
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.
