Enhancement of Immune Responses by an Attenuated Salmonella enterica Serovar Typhimurium Strain Secreting an Escherichia coli Heat-Labile Enterotoxin B Subunit Protein as an Adjuvant for a Live Salmonella Vaccine Candidate

Abstract

A plasmid harboring eltB, the gene encoding heat-labile enterotoxin (LTB), was constructed by insertion of eltB into an Asd ؉ ␤-lactamase signal plasmid (pMMP65). This was introduced into the ⌬lon ⌬cpxR ⌬asd Salmonella enterica serovar Typhimurium strain and designated the LTB adjuvant strain. LTB protein production and secretion from the strain were demonstrated with an immunoblot assay and enzyme-linked immunosorbent assay. The LTB strain was evaluated for enhancement of immunity and protection efficacy induced by a previously constructed live Salmonella vaccine candidate. In addition, immunization strategies using the LTB strain were optimized for effective salmonellosis protection. Seventy female BALB/c mice were divided into seven groups (A to G; n ‫؍‬ 10 mice per group). Mice were primed at 6 weeks of age and boosted at 9 weeks of age. All mice were orally challenged with a virulent wild-type strain at week 3 postbooster. Serum IgG and IgA titers from mice immunized with the LTB strain alone or with a mixture of the LTB strain and the vaccine candidate were significantly increased. The secretory IgA titers from mice immunized with the LTB strain alone or with the mixture were at least 2.2 times greater than those of control mice. In addition, all group E mice (primed with the vaccine-LTB mixture and boosted with the vaccine candidate) were free of clinical signs of salmonellosis and survived a virulent challenge. In contrast, death due to the challenge was 100% in control mice, 80% in group A mice (single immunization with the vaccine candidate), 60% in group B mice (primed and boosted with the vaccine candidate), 40% in group C mice (single immunization with the LTB strain), 30% in group D mice (primed and boosted with the LTB strain), and 30% in group F mice (primed and boosted with the vaccine-LTB mixture). These results suggest that vaccination with the LTB strain, especially when added at the prime stage only, effectively enhances immune responses and protection against salmonellosis.

Enhancement of Host Immune Responses by Oral Vaccination to Salmonella enterica serovar Typhimurium Harboring Both FliC and FljB Flagella

“…Bacterial flagellin, the structural component of flagellar filament, is usually assembled in the flagellum, accelerating the bacterial invasion of the host cells, conferring a survival advantage during early infection in mice and activating the host innate immune response [22]. The importance of flagellin-induced gene expression has been acknowledged for Salmonella infection because flagella expression provides a survival advantage in the early stage of infection.…”

“…At 14 days after the challenge, all surviving mice were sacrificed by cervical dislocation and the liver and splenic tissues were aseptically harvested. The harvested spleens and livers were homogenized in PBS and plated on LB agar for CFU determination, and then the challenge strain was isolated after incubation at 37°C for 24 h [22]. …”

“…The serum IgG antibody responses against flagella or lipopolysaccharide (LPS) were investigated by enzyme-linked immunosorbent assay (ELISA) as previously described [22], with some modification. Briefly, 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated with 100 µl of S .…”

Salmonella enterica Serovar Enteritidis Ghosts Carrying the Escherichia coli Heat-Labile Enterotoxin B Subunit Are Capable of Inducing Enhanced Protective Immune Responses

“…5I). The chickens in group B exhibited enhanced induction of the systemic antibody response compared with the chickens in group C, indicating that the presence of LTB may have facilitated increased uptake of antigen by APCs, thus leading to superior induction of IgG antibodies (39). In natural infection, host animals are infected via the oral route by ingesting food contaminated with S. Enteritidis (36).…”

Salmonellaghosts expressing enterotoxigenicEscherichia colik88ab, k88ac, k99, and fasa fimbrial antigens induce robust immune responses in a mouse model

“…A previous study showed that the LTB adjuvant enhanced the efficacy of vaccination (Hur & Lee 2011a). A principal effect of LTB interaction with mammalian cells is the stable cross-linking of GM1 at the cell surface, resulting in the uptake of combined antigens (Freytag & Clements 2005) and the enhancement of both mucosal and systemic immune responses (Moyle et al 2004).…”