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Heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) is one of the major virulence factors for causing diarrhea in piglets, and LT is a strong immunogen. Thus, LT represents an important target for development of vaccines and diagnostic tests. In this study, bioinformatic tools were used to predict six antigenic B cell epitopes in the B subunit of LT protein (LTB) of ETEC strains. Then, seven antigenic B cell epitopes of LTB were identified by polyclonal antisera (polyclonal antibody (PAb)) using a set of LTB-derived peptides expressed as maltose-binding protein (MBP) fusion protein. In addition, one LTB-specific monoclonal antibody (MAb) was generated and defined its corresponding epitope as mentioned above. This MAb was able to specifically bind with native LT toxin and has no cross-reaction with LT-II (type II heat-labile enterotoxin), Stx1 (Shiga toxin I), Stx2 (Shiga toxin II), STa (heat-stable enterotoxin I), and STb (heat-stable enterotoxin II) toxins. Further, this MAb was able to interrupt LT toxin specific binding to GM1 receptor, indicating that the corresponding epitope is the specific binding region to GM1 receptor. Moreover, in vitro and in vivo assay showed that the MAb was able to neutralize the native LT toxin. Diarrheal suckling pigs challenged with LT-positive ETEC strain recovered when an enema with this purified MAb was administered. This study will provide the foundation for further studies about the interaction between LT toxin and GM1 receptor and about the developing of epitope-based vaccines and specific therapeutic agent.

Previous epidemiological study showed that most of the porcine enterotoxin Escherichia coli (ETEC) strains harbor multiple enterotoxins but lack any of the fimbriae or non-fimbrial adhesion genes. Therefore, effective ETEC vaccines need to aim directly at all the enterotoxin antigens. The objective of this study was to evaluate the simultaneous immune effect of two live attenuated E. coli strains expressing LTR192G-STaA13Q and LTR192G-STb fusion immunogen, respectively. The results showed that both local mucosal and systemic immune responses against LT, STa, STb, and F41 were induced in BALB/c mice immunized orally with the recombinant E. coli strains ER-A and ER-B simultaneously. In addition, results of cellular immune responses showed that stimulation index (SI) values of immunized mice were significantly higher than control mice (P < 0.05) and a marked shift toward type-2 helper T lymphocyte (Th 2) immunity. Moreover, the induced antibodies demonstrated neutralizing effects on LT, STa, and STb producing E. coli infection. These data indicated that the use of recombinant E. coli ER-A and ER-B could be a valuable strategy for future polyvalent vaccine development of ETEC.

Enter toxigenic Escherichia coli (ETEC) is a major pathogen of swine industry that can have a substantial impact on morbidity and mortality. Therefore, it is necessary to develop effective vaccines for the prevention of ETEC infection. Live attenuated bacteria delivery system are effective tools for mucosal immunization. The purpose of this study was to construct a novel delivery system that can present the LTR192G-STb fusion protein as oral vaccine candidate. Firstly, the PRPL-mKate2 fluorescent cassette was inserted into the genome (yaiT pseudogene) of an attenuated E. coli by homologous recombination methods to construct the delivery system O142(yaiT::PRPL-mKate2). Secondly, the oral vaccine O142(yaiT:: LT192-STb) (ER-B) was derived for replacing the PRPL-mKate2 by LT192-STb fusion gene, and then it was tested for its feasibility as oral vaccine candidate. Subsequently, BALB/c mice were orogastrically immunized with ER-B. Results showed that mice orally immunized with ER-B produced high levels of specific IgA and IgG antibodies. The induced antibodies demonstrated neutralizing effects to enter toxins LT and STb. In addition, results of cellular immune responses showed that stimulation index values of immunized mice were significantly higher than the control group (P < 0.05) and with a marked shift towards Th 2 immunity. These data indicated that the recombinant E. coli ER-B could be a valuable candidate of future vaccines against ETEC infection.

Previous epidemiological study showed that most of the porcine enterotoxin Escherichia coli (ETEC) strains harbor multiple enterotoxins but lack any of the fimbriae or non-fimbrial adhesion genes. Therefore, effective ETEC vaccines need to aim directly at all the enterotoxin antigens. The objective of this study was to evaluate the simultaneous immune effect of two live attenuated E. coli strains expressing LT-STa and LT-STb fusion immunogen, respectively. The results showed that both local mucosal and systemic immune responses against LT, STa, STb, and F41 were induced in BALB/c mice immunized orally with the recombinant E. coli strains ER- A and ER- B simultaneously. In addition, results of cellular immune responses showed that stimulation index (SI) values of immunized mice were significantly higher than control mice ( P < 0.05) and a marked shift toward type-2 helper T lymphocyte (Th 2) immunity. Moreover, the induced antibodies demonstrated neutralizing effects on LT, STa, and STb producing E. coli infection. These data indicated that the use of recombinant E. coli ER- A and ER- B could be a valuable strategy for future polyvalent vaccine development of ETEC. [ABSTRACT FROM AUTHOR]

Live attenuated bacteria delivered orally are interesting tools for mucosal immunization. The objective of this study was to construct a novel counter-selection platform based on an attenuated wild-type Escherichia coli ( E. coli) strain and to utilize it for the delivery of LT-STa fusion protein as an oral vaccine. First, a counter-selectable marker, namely, PRPL-Kil, was inserted into an attenuated wild-type E. coli strain through the use of the red and G-DOC homologous recombination systems to construct the counter-selection platform, and PRPL-Kil was subsequently replaced by the LT-STa fusion gene to construct the oral vaccine O142 ( yaiT::LT-STa) ( ER- A). Subsequently, BALB/c mice were orogastrically inoculated with ER- A. Our results showed that ER- A could induce the production of specific IgA and IgG against fimbriae (F41) and enterotoxins (LT and STa), with neutralizing activity in BALB/c mice. In addition, assays of cellular immune responses showed that the stimulation index (SI) values of immunized mice were significantly higher than those of control mice ( P < 0.05), and revealed a marked shift toward Th2-mediated immunity. These findings suggest that ER- A is a suitable candidate for an oral vaccine strain to protect animals from enter toxigenic Escherichia coli (ETEC) infection. [ABSTRACT FROM AUTHOR]