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J D Clements - One of the best experts on this subject based on the ideXlab platform.
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development of mucosal protection against the heat stable enterotoxin st of escherichia coli by oral immunization with a genetic fusion delivered by a Bacterial Vector
Infection and Immunity, 1993Co-Authors: Lucia Cardenas, J D ClementsAbstract:An LT-B-ST (LT-B/ST) fusion peptide was constructed by genetically joining the 5' terminus of a synthetic gene coding for the heat-stable enterotoxin (ST) of Escherichia coli to the 3' terminus of the gene coding for the binding subunit of the heat-labile enterotoxin (LT-B) of E. coli. An eight-amino-acid, proline-containing linker was included between the LT-B and ST moieties. An aroA mutant of Salmonella dublin transformed with a plasmid carrying this genetic construct was shown to express a fusion peptide with antigenic determinants of both LT-B and ST. Mice were immunized orally with this strain or with a control strain expressing just LT-B from the same plasmid. Sera and mucosal secretions were obtained and analyzed for the presence of serum immunoglobulin G and mucosal immunoglobulin A that were able to recognize LT-B and ST by enzyme-linked immunosorbent assay (ELISA) and, more importantly, were able to neutralize native ST in the suckling mouse assay. Sera and mucosal secretions from animals immunized with the strain expressing the LT-B/ST fusion exhibited detectable ELISA reactivity against LT-B but not against native ST. However, even in the absence of detectable ELISA reactivity, both sera and mucosal secretions from these animals were able to neutralize the biological activity of native ST in the suckling mouse assay. These findings are important because they demonstrate the development of mucosal protection against ST by oral immunization with a genetic fusion delivered by a Bacterial Vector.
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Development of mucosal protection against the heat-stable enterotoxin (ST) of Escherichia coli by oral immunization with a genetic fusion delivered by a Bacterial Vector.
Infection and immunity, 1993Co-Authors: Lucia Cardenas, J D ClementsAbstract:An LT-B-ST (LT-B/ST) fusion peptide was constructed by genetically joining the 5' terminus of a synthetic gene coding for the heat-stable enterotoxin (ST) of Escherichia coli to the 3' terminus of the gene coding for the binding subunit of the heat-labile enterotoxin (LT-B) of E. coli. An eight-amino-acid, proline-containing linker was included between the LT-B and ST moieties. An aroA mutant of Salmonella dublin transformed with a plasmid carrying this genetic construct was shown to express a fusion peptide with antigenic determinants of both LT-B and ST. Mice were immunized orally with this strain or with a control strain expressing just LT-B from the same plasmid. Sera and mucosal secretions were obtained and analyzed for the presence of serum immunoglobulin G and mucosal immunoglobulin A that were able to recognize LT-B and ST by enzyme-linked immunosorbent assay (ELISA) and, more importantly, were able to neutralize native ST in the suckling mouse assay. Sera and mucosal secretions from animals immunized with the strain expressing the LT-B/ST fusion exhibited detectable ELISA reactivity against LT-B but not against native ST. However, even in the absence of detectable ELISA reactivity, both sera and mucosal secretions from these animals were able to neutralize the biological activity of native ST in the suckling mouse assay. These findings are important because they demonstrate the development of mucosal protection against ST by oral immunization with a genetic fusion delivered by a Bacterial Vector.
Lucia Cardenas - One of the best experts on this subject based on the ideXlab platform.
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development of mucosal protection against the heat stable enterotoxin st of escherichia coli by oral immunization with a genetic fusion delivered by a Bacterial Vector
Infection and Immunity, 1993Co-Authors: Lucia Cardenas, J D ClementsAbstract:An LT-B-ST (LT-B/ST) fusion peptide was constructed by genetically joining the 5' terminus of a synthetic gene coding for the heat-stable enterotoxin (ST) of Escherichia coli to the 3' terminus of the gene coding for the binding subunit of the heat-labile enterotoxin (LT-B) of E. coli. An eight-amino-acid, proline-containing linker was included between the LT-B and ST moieties. An aroA mutant of Salmonella dublin transformed with a plasmid carrying this genetic construct was shown to express a fusion peptide with antigenic determinants of both LT-B and ST. Mice were immunized orally with this strain or with a control strain expressing just LT-B from the same plasmid. Sera and mucosal secretions were obtained and analyzed for the presence of serum immunoglobulin G and mucosal immunoglobulin A that were able to recognize LT-B and ST by enzyme-linked immunosorbent assay (ELISA) and, more importantly, were able to neutralize native ST in the suckling mouse assay. Sera and mucosal secretions from animals immunized with the strain expressing the LT-B/ST fusion exhibited detectable ELISA reactivity against LT-B but not against native ST. However, even in the absence of detectable ELISA reactivity, both sera and mucosal secretions from these animals were able to neutralize the biological activity of native ST in the suckling mouse assay. These findings are important because they demonstrate the development of mucosal protection against ST by oral immunization with a genetic fusion delivered by a Bacterial Vector.
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Development of mucosal protection against the heat-stable enterotoxin (ST) of Escherichia coli by oral immunization with a genetic fusion delivered by a Bacterial Vector.
Infection and immunity, 1993Co-Authors: Lucia Cardenas, J D ClementsAbstract:An LT-B-ST (LT-B/ST) fusion peptide was constructed by genetically joining the 5' terminus of a synthetic gene coding for the heat-stable enterotoxin (ST) of Escherichia coli to the 3' terminus of the gene coding for the binding subunit of the heat-labile enterotoxin (LT-B) of E. coli. An eight-amino-acid, proline-containing linker was included between the LT-B and ST moieties. An aroA mutant of Salmonella dublin transformed with a plasmid carrying this genetic construct was shown to express a fusion peptide with antigenic determinants of both LT-B and ST. Mice were immunized orally with this strain or with a control strain expressing just LT-B from the same plasmid. Sera and mucosal secretions were obtained and analyzed for the presence of serum immunoglobulin G and mucosal immunoglobulin A that were able to recognize LT-B and ST by enzyme-linked immunosorbent assay (ELISA) and, more importantly, were able to neutralize native ST in the suckling mouse assay. Sera and mucosal secretions from animals immunized with the strain expressing the LT-B/ST fusion exhibited detectable ELISA reactivity against LT-B but not against native ST. However, even in the absence of detectable ELISA reactivity, both sera and mucosal secretions from these animals were able to neutralize the biological activity of native ST in the suckling mouse assay. These findings are important because they demonstrate the development of mucosal protection against ST by oral immunization with a genetic fusion delivered by a Bacterial Vector.
Yuanxing Zhang - One of the best experts on this subject based on the ideXlab platform.
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Cell-penetrating peptides mediated protein cross-membrane delivery and its use in Bacterial Vector vaccine.
Fish & shellfish immunology, 2014Co-Authors: Lingyu Guan, Qin Liu, Jingfan Xiao, Yuanxing ZhangAbstract:It is an attractive strategy to develop a recombinant Bacterial Vector vaccine by expressing exogenous protective antigen to induce the immune response, and the main concern is how to enhance the cellular internalization of antigen produced by Bacterial Vector. Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides which facilitate cellular uptake of various molecular cargoes and therefore have great potentials in Vector vaccine design. In this work, eleven different CPPs were fused to the C-terminus of EGFP respectively, and the resultant EGFP-CPP fusion proteins were expressed and purified to assay their cross-membrane transport in macrophage J774 A.1 cells. Among the tested CPPs, TAT showed an excellent capability to deliver the cargo protein EGFP into cytoplasm. In order to establish an efficient antigen delivery system in Escherichia coli, the EGFP-TAT synthesis circuit was combined with an in vivo inducible lysis circuit PviuA-E in E. coli to form an integrated antigen delivery system, the resultant E. coli was proved to be able to lyse upon the induction of a mimic in vivo signal and thus release intracellular EGFP-TAT intensively, which were assumed to undergo a more efficient intracellular delivery by CPP to evoke protective immune responses. Based on the established antigen delivery system, the protective antigen gene flgD from an invasive intracellular fish pathogen Edwardsiella tarda EIB202, was applied to establish an E. coli recombinant Vector vaccine. This E. coli Vector vaccine presented superior immune protection (RPS = 63%) under the challenge with E. tarda EIB202, suggesting that the novel antigen delivery system had great potential in Bacterial Vector vaccine applications.
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FERMENTATION PREPARATION OF RECOMBINANT Vibrio anguillarum VACCINE WITH HETEROGENEOUS ANTIGEN DISPLAY
Preparative biochemistry & biotechnology, 2013Co-Authors: Sanying Wang, Qin Liu, Menghao Cai, Qiyao Wang, Yuanxing ZhangAbstract:In the design of recombinant Bacterial Vector vaccine, heterogeneous antigen is displayed on the outer membrane of the Vector strain to evoke polyvalent immunological protection. Thus, the expression of heterogeneous antigen in cells and its display on the outer membrane are of great concern for vaccine preparation. In our previous work, a multivalent Bacterial Vector vaccine MVAV6203A-1 was constructed by displaying the protective antigen GAPDH from Aeromonas hydrophila on the surface of an attenuated Vibrio anguillarum MVAV6203. In this work, a new fermentation medium was designed by a four-step method to improve the cell growth and antigen display of V. anguillarum MVAV6203A-1. First, suitable carbon and nitrogen sources were selected by a component swapping method. Second, the initial concentrations of carbon and nitrogen sources were determined by orthogonal design. Then three main factors to significantly affect cell growth and antigen expression were screened by a Plackett–Burman design. Finally, t...
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A novel in vivo inducible expression system in Edwardsiella tarda for potential application in Bacterial polyvalence vaccine
Fish & shellfish immunology, 2011Co-Authors: Lingyu Guan, Qin Liu, Yijian Yan, Yuanxing ZhangAbstract:Abstract Recombinant Bacterial Vector vaccine is an attractive vaccination strategy to induce the immune response to a carried protective antigen, and the main concern of Bacterial Vector vaccine is to establish a stable antigen expression system in Vector bacteria. Edwardsiella tarda is an important facultative intracellular pathogen of both animals and humans, and its attenuated derivates are excellent Bacterial Vectors for use in recombinant vaccine design. In this study, we design an in vivo inducible expression system in E. tarda and establish potential recombinant E. tarda Vector vaccines. With wild type strain E. tarda EIB202 as a Vector, 53 different bacteria-originated promoters were examined for iron-responsive transcription in vitro , and the promoters P dps and P yncE showed high transcription activity. The transcription profiles in vivo of two promoters were further assayed, and P dps revealed an enhanced in vivo inducible transcription in macrophage, larvae and adult zebra fish. The gapA 34 gene, encoding the protective antigen GAPDH from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the P dps -based protein expression system, and transformed into attenuated E. tarda strains. The resultant recombinant Vector vaccine WED/pUTDgap was evaluated in turbot ( Scophtalmus maximus ). Over 60% of the vaccinated fish survived under the challenge with A. hydrophila LSA34 and E. tarda EIB202, suggesting that the P dps -based antigen delivery system had great potential in Bacterial Vector vaccine application.
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Iron-regulated lysis of recombinant Escherichia coli in host releases protective antigen and confers biological containment.
Infection and immunity, 2011Co-Authors: Lingyu Guan, Jingfan Xiao, Yuanxing Zhang, Qiyao Wang, Jonathan Champeimont, Werner Lubitz, Qin LiuAbstract:The use of a recombinant Bacterial Vector vaccine is an attractive vaccination strategy to induce an immune response to a carried protective antigen. The superiorities of live Bacterial Vectors include mimicry of a natural infection, intrinsic adjuvant properties, and the potential for administration by mucosal routes. Escherichia coli is a simple and efficient Vector system for production of exogenous proteins. In addition, many strains are nonpathogenic and avirulent, making it a good candidate for use in recombinant vaccine design. In this study, we screened 23 different iron-regulated promoters in an E. coli BL21(DE3) Vector and found one, PviuB, with characteristics suitable for our use. We fused PviuB with lysis gene E, establishing an in vivo inducible lysis circuit. The resulting in vivo lysis circuit was introduced into a strain also carrying an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible PT7-controlled protein synthesis circuit, forming a novel E. coli-based protein delivery system. The recombinant E. coli produced a large amount of antigen in vitro and could deliver the antigen into zebrafish after vaccination via injection. The strain subsequently lysed in response to the iron-limiting signal in vivo, implementing antigen release and biological containment. The gapA gene, encoding the protective antigen GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the E. coli-based protein delivery system, and the resultant recombinant Vector vaccine was evaluated in turbot (Scophtalmus maximus). Over 80% of the vaccinated fish survived challenge with A. hydrophila LSA34, suggesting that the E. coli-based antigen delivery system has great potential in Bacterial Vector vaccine applications.
Adilson José Silva - One of the best experts on this subject based on the ideXlab platform.
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Vacinas recombinantes contra erisipela suína: desenvolvimento integrado de bioprocesso, da biologia molecular ao biorreator
Programa de Pós-graduação em Biotecnologia, 2011Co-Authors: Adilson José SilvaAbstract:Swine erysipelas is among the diseases that causes great economic losses in swine cultures worldwide. The disease is caused by the bacterium Erysipelothrix rhusiopathiae, and the surface protein SpaA is one of its main antigens. Herein, we report studies concerning the development of recombinant vaccines against swine erysipelas based on the SpaA antigen. Protein production for a subunit vaccine formulation was studied in shaken flasks and 5.0 L bioreactors. For this propose, a 1026 bp fragment of the spaA gene was cloned in Escherichia coli cells under the lac promoter control. The recombinant organism (E. coli BL21(DE3) pET28a_spaA) was cultivated in fed batch using complex medium with glycerol as carbon source. Nonconventional induction strategies were evaluated and high protein yield (198 mgprot/gDCW) and productivity values (0.4 gprot/L.h) were reached. The same antigen was cloned for expression and secretion in attenuated Salmonella typhimurium cells to obtain a live Bacterial Vector for the SpaA antigen. The recombinant lineage was able to express and secrete the SpaA fragment fused to the alpha-hemolysin secretion signal both in vitro and in vivo. High plasmid maintenance was observed in both conditions. The vaccinal vehicle showed to be able to colonize the Peyer patches and to invade the gut epithelial barrier in the inoculated animals. Immunization tests in murine model showed that the recombinant antigen delivered by Salmonella cells inoculated by oral route induced the production of seric IgG antibodies anti-SpaA. According to the literature, these antibodies must be able to promote pathogen opsonization in case of infection, contributing to confer a protective immunity against swine erysipelas to the vaccinated animals. In summary, this work presents contributions to development of subunit vaccines against swine erysipelas, in the form of recombinant protein formulations, or SpaA antigen delivery by attenuated S. typhimurium cells.Valée S.A.A erisipela suína é uma das enfermidades que causam grandes prejuízos na suinocultura em todo o mundo. A doença é causada pela bactéria Erysipelothrix rhusiopathiae, e a proteína de superfície SpaA desse microrganismo é um de seus principais antígenos. Neste trabalho, estudou-se o desenvolvimento de vacinas recombinantes contra a erisipela suína a partir do antígeno SpaA. Avaliou-se a produção de uma vacina de subunidade composta pelo antígeno recombinante, a qual foi estudada em frascos agitados e em biorretores de bancada de 5,0 L. Para isso, um fragmento de 1026 pb do gene spaA foi clonado em células de Escherichia coli sob controle do promotor lac e o organismo recombinante (E. coli BL21(DE3) pET28a_spaA) foi cultivado em batelada alimentada, utilizando-se meio complexo contendo glicerol como fonte de carbono. Estratégias não convencionais de indução foram avaliadas e altos valores de rendimento (198 mgprot/gDCW) e produtividade (0,4 gprot/L.h) da proteína recombinante foram alcançados. O mesmo antígeno foi clonado em um plasmídeo que possibilita a expressão e secreção da proteína recombinante em Salmonella typhimurium atenuada, a fim de se obter um vetor bacteriano vivo para o antígeno em questão. A linhagem recombinante foi capaz de expressar e secretar o fragmento da proteína SpaA fusionado ao sinal de secreção da alfa-hemolisina tanto in vitro quanto in vivo, apresentando alta taxa de manutenção plasmidial nas duas condições. Além disso, o veículo vacinal se mostrou capaz de colonizar as placas de Peyer e de invadir a barreira epitelial do intestino dos animais inoculados. Ensaios de imunização em modelo murino mostraram que a veiculação do antígeno pelas células de Salmonella inoculadas por via oral induziu a produção de anticorpos IgG séricos anti-SpaA, que de acordo com a literatura, devem ser capazes de promover a opsonização do patógeno em caso de infecção, contribuindo para conferir uma imunidade protetora contra a erisipela suína aos animais vacinados. Em suma, este trabalho apresenta contribuições para o desenvolvimento de vacinas de subunidade contra a erisipela suína na forma de uma vacina de proteína recombinante, ou por veiculação do antígeno SpaA por linhagens atenuadas de S. typhimurium
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Vacinas recombinantes contra erisipela suína: desenvolvimento integrado de bioprocesso, da biologia molecular ao biorreator
Universidade Federal de São Carlos, 2011Co-Authors: Adilson José SilvaAbstract:A erisipela suína é uma das enfermidades que causam grandes prejuízos na suinocultura em todo o mundo. A doença é causada pela bactéria Erysipelothrix rhusiopathiae, e a proteína de superfície SpaA desse microrganismo é um de seus principais antígenos. Neste trabalho, estudou-se o desenvolvimento de vacinas recombinantes contra a erisipela suína a partir do antígeno SpaA. Avaliou-se a produção de uma vacina de subunidade composta pelo antígeno recombinante, a qual foi estudada em frascos agitados e em biorretores de bancada de 5,0 L. Para isso, um fragmento de 1026 pb do gene spaA foi clonado em células de Escherichia coli sob controle do promotor lac e o organismo recombinante (E. coli BL21(DE3) pET28a_spaA) foi cultivado em batelada alimentada, utilizando-se meio complexo contendo glicerol como fonte de carbono. Estratégias não convencionais de indução foram avaliadas e altos valores de rendimento (198 mgprot/gDCW) e produtividade (0,4 gprot/L.h) da proteína recombinante foram alcançados. O mesmo antígeno foi clonado em um plasmídeo que possibilita a expressão e secreção da proteína recombinante em Salmonella typhimurium atenuada, a fim de se obter um vetor bacteriano vivo para o antígeno em questão. A linhagem recombinante foi capaz de expressar e secretar o fragmento da proteína SpaA fusionado ao sinal de secreção da alfa-hemolisina tanto in vitro quanto in vivo, apresentando alta taxa de manutenção plasmidial nas duas condições. Além disso, o veículo vacinal se mostrou capaz de colonizar as placas de Peyer e de invadir a barreira epitelial do intestino dos animais inoculados. Ensaios de imunização em modelo murino mostraram que a veiculação do antígeno pelas células de Salmonella inoculadas por via oral induziu a produção de anticorpos IgG séricos anti-SpaA, que de acordo com a literatura, devem ser capazes de promover a opsonização do patógeno em caso de infecção, contribuindo para conferir uma imunidade protetora contra a erisipela suína aos animais vacinados. Em suma, este trabalho apresenta contribuições para o desenvolvimento de vacinas de subunidade contra a erisipela suína na forma de uma vacina de proteína recombinante, ou por veiculação do antígeno SpaA por linhagens atenuadas de S. typhimurium.Swine erysipelas is among the diseases that causes great economic losses in swine cultures worldwide. The disease is caused by the bacterium Erysipelothrix rhusiopathiae, and the surface protein SpaA is one of its main antigens. Herein, we report studies concerning the development of recombinant vaccines against swine erysipelas based on the SpaA antigen. Protein production for a subunit vaccine formulation was studied in shaken flasks and 5.0 L bioreactors. For this propose, a 1026 bp fragment of the spaA gene was cloned in Escherichia coli cells under the lac promoter control. The recombinant organism (E. coli BL21(DE3) pET28a_spaA) was cultivated in fed batch using complex medium with glycerol as carbon source. Nonconventional induction strategies were evaluated and high protein yield (198 mgprot/gDCW) and productivity values (0.4 gprot/L.h) were reached. The same antigen was cloned for expression and secretion in attenuated Salmonella typhimurium cells to obtain a live Bacterial Vector for the SpaA antigen. The recombinant lineage was able to express and secrete the SpaA fragment fused to the alpha-hemolysin secretion signal both in vitro and in vivo. High plasmid maintenance was observed in both conditions. The vaccinal vehicle showed to be able to colonize the Peyer patches and to invade the gut epithelial barrier in the inoculated animals. Immunization tests in murine model showed that the recombinant antigen delivered by Salmonella cells inoculated by oral route induced the production of seric IgG antibodies anti-SpaA. According to the literature, these antibodies must be able to promote pathogen opsonization in case of infection, contributing to confer a protective immunity against swine erysipelas to the vaccinated animals. In summary, this work presents contributions to development of subunit vaccines against swine erysipelas, in the form of recombinant protein formulations, or SpaA antigen delivery by attenuated S. typhimurium cells
Lingyu Guan - One of the best experts on this subject based on the ideXlab platform.
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Cell-penetrating peptides mediated protein cross-membrane delivery and its use in Bacterial Vector vaccine.
Fish & shellfish immunology, 2014Co-Authors: Lingyu Guan, Qin Liu, Jingfan Xiao, Yuanxing ZhangAbstract:It is an attractive strategy to develop a recombinant Bacterial Vector vaccine by expressing exogenous protective antigen to induce the immune response, and the main concern is how to enhance the cellular internalization of antigen produced by Bacterial Vector. Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides which facilitate cellular uptake of various molecular cargoes and therefore have great potentials in Vector vaccine design. In this work, eleven different CPPs were fused to the C-terminus of EGFP respectively, and the resultant EGFP-CPP fusion proteins were expressed and purified to assay their cross-membrane transport in macrophage J774 A.1 cells. Among the tested CPPs, TAT showed an excellent capability to deliver the cargo protein EGFP into cytoplasm. In order to establish an efficient antigen delivery system in Escherichia coli, the EGFP-TAT synthesis circuit was combined with an in vivo inducible lysis circuit PviuA-E in E. coli to form an integrated antigen delivery system, the resultant E. coli was proved to be able to lyse upon the induction of a mimic in vivo signal and thus release intracellular EGFP-TAT intensively, which were assumed to undergo a more efficient intracellular delivery by CPP to evoke protective immune responses. Based on the established antigen delivery system, the protective antigen gene flgD from an invasive intracellular fish pathogen Edwardsiella tarda EIB202, was applied to establish an E. coli recombinant Vector vaccine. This E. coli Vector vaccine presented superior immune protection (RPS = 63%) under the challenge with E. tarda EIB202, suggesting that the novel antigen delivery system had great potential in Bacterial Vector vaccine applications.
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A novel in vivo inducible expression system in Edwardsiella tarda for potential application in Bacterial polyvalence vaccine
Fish & shellfish immunology, 2011Co-Authors: Lingyu Guan, Qin Liu, Yijian Yan, Yuanxing ZhangAbstract:Abstract Recombinant Bacterial Vector vaccine is an attractive vaccination strategy to induce the immune response to a carried protective antigen, and the main concern of Bacterial Vector vaccine is to establish a stable antigen expression system in Vector bacteria. Edwardsiella tarda is an important facultative intracellular pathogen of both animals and humans, and its attenuated derivates are excellent Bacterial Vectors for use in recombinant vaccine design. In this study, we design an in vivo inducible expression system in E. tarda and establish potential recombinant E. tarda Vector vaccines. With wild type strain E. tarda EIB202 as a Vector, 53 different bacteria-originated promoters were examined for iron-responsive transcription in vitro , and the promoters P dps and P yncE showed high transcription activity. The transcription profiles in vivo of two promoters were further assayed, and P dps revealed an enhanced in vivo inducible transcription in macrophage, larvae and adult zebra fish. The gapA 34 gene, encoding the protective antigen GAPDH from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the P dps -based protein expression system, and transformed into attenuated E. tarda strains. The resultant recombinant Vector vaccine WED/pUTDgap was evaluated in turbot ( Scophtalmus maximus ). Over 60% of the vaccinated fish survived under the challenge with A. hydrophila LSA34 and E. tarda EIB202, suggesting that the P dps -based antigen delivery system had great potential in Bacterial Vector vaccine application.
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Iron-regulated lysis of recombinant Escherichia coli in host releases protective antigen and confers biological containment.
Infection and immunity, 2011Co-Authors: Lingyu Guan, Jingfan Xiao, Yuanxing Zhang, Qiyao Wang, Jonathan Champeimont, Werner Lubitz, Qin LiuAbstract:The use of a recombinant Bacterial Vector vaccine is an attractive vaccination strategy to induce an immune response to a carried protective antigen. The superiorities of live Bacterial Vectors include mimicry of a natural infection, intrinsic adjuvant properties, and the potential for administration by mucosal routes. Escherichia coli is a simple and efficient Vector system for production of exogenous proteins. In addition, many strains are nonpathogenic and avirulent, making it a good candidate for use in recombinant vaccine design. In this study, we screened 23 different iron-regulated promoters in an E. coli BL21(DE3) Vector and found one, PviuB, with characteristics suitable for our use. We fused PviuB with lysis gene E, establishing an in vivo inducible lysis circuit. The resulting in vivo lysis circuit was introduced into a strain also carrying an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible PT7-controlled protein synthesis circuit, forming a novel E. coli-based protein delivery system. The recombinant E. coli produced a large amount of antigen in vitro and could deliver the antigen into zebrafish after vaccination via injection. The strain subsequently lysed in response to the iron-limiting signal in vivo, implementing antigen release and biological containment. The gapA gene, encoding the protective antigen GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the E. coli-based protein delivery system, and the resultant recombinant Vector vaccine was evaluated in turbot (Scophtalmus maximus). Over 80% of the vaccinated fish survived challenge with A. hydrophila LSA34, suggesting that the E. coli-based antigen delivery system has great potential in Bacterial Vector vaccine applications.
