RESUMEN
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.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Enterotoxinas/inmunología , Proteínas de Escherichia coli/inmunología , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos , Regiones Promotoras Genéticas , Administración Oral , Animales , Anticuerpos Antibacterianos/sangre , Bacillus subtilis/inmunología , Toxinas Bacterianas/genética , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Enterotoxinas/genética , Ensayo de Inmunoadsorción Enzimática , Proteínas de Escherichia coli/genética , Heces/química , Femenino , Inmunoglobulina A/análisis , Inmunoglobulina G/sangre , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos C57BL , Plásmidos/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
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.
Asunto(s)
Humanos , Escherichia coli Enterotoxigénica/inmunología , Vacunas/inmunologíaRESUMEN
Bacillus subtilis and some of its close relatives have a long history of industrial and biotechnological applications. Search for antigen expression systems based on recombinant B. subtilis strains sounds attractive both by the extensive genetic knowledge and the lack of an outer membrane, which simplify the secretion and purification of heterologous proteins. More recently, genetically modified B. subtilis spores have been described as indestructible delivery vehicles for vaccine antigens. Nonetheless both production and delivery of antigens by B. subtilis strains face some inherent obstacles, as unstable gene expression and reduced immunogenicity that, otherwise, can be overcome by already available gene technology approaches. In the present review we present the status of B. subtilis-based vaccine research, either as protein factories or delivery vectors, and discuss some alternatives for a better use of genetically modified strains.
Asunto(s)
Antígenos Bacterianos/inmunología , Bacillus subtilis/inmunología , Vacunas Bacterianas , Portadores de Fármacos , Diseño de Fármacos , Antígenos Bacterianos/genética , Antígenos Bacterianos/aislamiento & purificación , Antígenos Heterófilos/genética , Antígenos Heterófilos/inmunología , Antígenos Heterófilos/aislamiento & purificación , Bacillus subtilis/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Bacillus subtilis e alguns de seus parentes mais próximos possuem uma longa história de aplicações industriais e biotecnológicas. A busca de sistemas de expressão de antígenos baseados em linhagens recombinants de B. subtilis mostra-se atrativa em função do conhecimento genético disponível e ausência de uma membrana externa, o que simplifica a secreção e a purificação de proteínas heterólogas. Mais recentemente, esporos geneticamente modificados de B. subtilis foram descritos com veículos indestrutíveis para o transporte de antígenos vacinais. Todavia a produção e o transporte de antígenos por linhagens de B. subtilis encontra obstáculos, como a expressão gênica instável e imunogenicidade reduzida, que podem ser superados com o auxílio de tecnologias genéticas atualmente disponíveis. Apresentamos nesta revisão o estado atual da pesquisa em vacinas baseadas em B. subtilis, empregado tanto como fábrica de proteínas ou veículos, e discute algumas alternativas para o uso mais adequado de linhagens geneticamente modificadas.
Asunto(s)
Humanos , Antígenos Bacterianos , Bacillus subtilis , Vacunas Bacterianas , Portadores de Fármacos , Diseño de Fármacos , Antígenos Heterófilos , Proteínas RecombinantesRESUMEN
Attempts to obtain a recombinant protein using prokaryotic expression systems can go from a rewarding and rather fast procedure to a frustrating time-consuming experience. In most cases production of heterologous proteins in Escherichia coli K12 strains has remained an empirical exercise in which different systems are tested without a careful insight into the various factors affecting adequate expression of the encoded protein. The present review will deal with E. coli as protein factory and will cover some of the aspects related to transcriptional and translational expression signals, factors affecting protein stability and solubility and targeting of proteins to different cell compartments. Based on the knowledge accumulated over the last decade, we believe that the rate of success for those dedicated to expression of recombinant proteins based on the use E. coli strains can still be significantly improved.