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A plasmid harboring eltB, the gene encoding heat-labile enterotoxin (LTB), was constructed by insertion of eltB into an Asd(+) β-lactamase signal plasmid (pMMP65). This was introduced into the Δlon ΔcpxR Δasd 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.

clinical and vaccine immunology

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