Generation of an attenuated strain oral vaccine candidate using a novel double selection platform in Escherichia coli

Abstract

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 LTR192G-STaA13Q 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 LT192-STa13 fusion gene to construct the oral vaccine O142 (yaiT::LT192-STa13) (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.

Targeting peptide‐enhanced antibody and CD11c+dendritic cells to inclusion bodies expressing protective antigen against ETEC in mice

“…The production of the IgG2a antibody accompanied the development of IgM and IgA (35,36). The production of specific IgA and IgG against fimbriae (F41) and enterotoxins (LT and STa) was with neutralizing activity through the oral immunization in BALB/c mice (37,38). The specific IgA produced was much more effective in neutralizing the fimbriae and enterotoxins, which in turn, would neutralize the ETEC entering the small intestine.…”

Construction and evaluation of a fluorescence-based live attenuated Escherichia coli delivery system for generating oral vaccine candidate

“…Therefore, deletion, insertion, or mutation of the pseudogene has no influence on growth or biological activity for bacterial strains (Balakirev and Ayala 2003;Echols et al 2002;Harrison et al 2001;Mighell et al 2000). Of the pseudogenes, yaiT gene, inserted by IS element in many E. coli strains, was selected for exogenous gene inserted since it is far away from the origin (84 min on the E. coli K-12 genetic map) and the termination (33 min on the E. coli K-12 genetic map) of chromosomal replication, adding no additional stress for growth and biological activity of the bacterial strain (Liu et al 2014a). After insertion, exogenous gene replicates in company with genome of E. coli, allowing a high and stable expression of the heterologous protein.…”

“…In order to avoid these drawbacks, alternative vaccination approaches are needed, and an alternative approach to prevention of ETEC infection is oral immunization. Orally delivered vaccines have several advantages over other routes of antigen delivery, including convenience, cost-effectiveness, and, most importantly, induction of both local and systemic immune responses (Liu et al 2014a;Lourdault et al 2014). Moreover, oral vaccination typically generates a large amount of secretory IgA (sIgA), which provides the first line in mucosal defense to animals (Wang and Coppel 2008).…”

Expression fusion immunogen by live attenuated Escherichia coli against enterotoxins infection in mice

“…V00612.1) and Strain LT 192 ‐STb (GenBank accession no. ) have been previously described (Liu et al ., ,). The plasmids constructed and used are listed in Table .…”

“…Transform pACBSCE plasmid and pKil‐donor plasmid into E. coli O142:ΔSTa, subsequently, homologous recombination is induced by L‐arabinose, then generated O142 ( yaiT ‐Kil) double selection platform. Use PCR primers (T1 and T4) to verify that pKil‐donor fragment was assembled on the platform, the platform screening capability validation by 30 and 43°C, as previously described (Liu et al ., ,).…”