Use of in vivo-regulated promoters to deliver antigens from attenuated Salmonella enterica var. Typhimurium

Abstract

This study describes the construction and analysis of three in vivo-inducible promoter expression plasmids, containing pnirB, ppagC, and pkatG, for the delivery of foreign antigens in the DeltaaroAD mutant of Salmonella enterica var. Typhimurium (hereafter referred to as S. typhimurium). The reporter genes encoding beta-galactosidase and firefly luciferase were used to assess the comparative levels of promoter activity in S. typhimurium in vitro in response to different induction stimuli and in vivo in immunized mice. It was determined that the ppagC construct directed the expression of more beta-galactosidase and luciferase in S. typhimurium than the pnirB and pkatG constructs, both in vitro and in vivo. The gene encoding the C fragment of tetanus toxin was expressed in the aroAD mutant of S. typhimurium (BRD509) under the control of the three promoters. Mice orally immunized with attenuated S. typhimurium expressing C fragment under control of the pagC promoter [BRD509(pKK/ppagC/C frag)] mounted the highest tetanus toxoid-specific serum antibody response. Levels of luciferase expression in vivo and C-fragment expression in vitro from the pagC promoter appeared to be equivalent to if not lower than the levels of expression detected with the constitutive trc promoter. However, mice immunized with BRD509(pKK/ppagC/C frag) induced significantly higher levels of tetanus toxoid-specific antibody than BRD509(pKK/C frag)-immunized mice, suggesting that the specific location of foreign antigen expression may be important for immunogenicity. Mutagenesis of the ribosome binding sites (RBS) in the three promoter/C fragment expression plasmids was also performed. Despite optimization of the RBS in the three different promoter elements, the expression levels in vivo and overall immunogenicity of C fragment when delivered to mice by attenuated S. typhimurium were not affected. These studies suggest that in vivo-inducible promoters may give rise to enhanced immunogenicity and increase the efficacy of S. typhimurium as a vaccine vector.

FIG. 1

Expression plasmids constructed. The promoter regions of nirB, pagC, and katG and the genes encoding β-galactosidase, firefly luciferase, and C fragment were obtained by PCR and ligated to pKK233-2. The four different promoter cassettes combined with the three genes generated 12 different expression plasmids.

FIG. 2

Detection of β-galactosidase expression in vitro. (A) β-Galactosidase activity of BRD509(pKK/pnirB/lacZ) was determined after incubation of cultures at 37°C either shaken, static, or static flushed with nitrogen and sealed. Nitrate (20 mM) (), nitrite (2.5 mM) (░⃞), or no supplement (■) was added to the growth media. (B) BRD509(pKK/ppagC/lacZ) cultures were induced for 2.5 h by the addition of 0.001 to 100 mM MgCl₂. (C) BRD509(pKK/pkatG/lacZ) cultures were induced for 2.5 h by the addition of 0.001 to 100 mM H₂O₂. β-Galactosidase activity is expressed in Miller units. The results presented are representative of a number of separate experiments.

FIG. 3

Detection of luciferase in the PPs of mice. Groups of mice were immunized orally with BRD509(pKK/pnirB/luc) (pnirB), BRD509(pKK/ppagC/luc) (ppagC), BRD509(pKK/pkatG/luc) (pkatG), BRD509(pKK/luc) (ptrc), and BRD509 (509). On days 6, 12, and 18 postimmunization, mice were killed and their PPs were removed. The number of bacteria (calculated by viable count) and the amount of luciferase (by luciferase assay) were determined in each PP sample. The luciferase activity was represented as the number of relative light units (RLU) in PPs per 10⁵ bacteria. Each bar represents the luciferase activity detected in the PPs of one mouse (A) or that detected in a pool of PPs from five mice (B and C). The dashed line on the day 6 graph denotes the mean of the luciferase activities detected in PPs of the control group, mice immunized with BRD509 alone. ∗, the level of luciferase activity is significantly different from that detected in the PPs of mice immunized with BRD509 alone (P < 0.05).

FIG. 4

Expression of C fragment in S. typhimurium. Western blot analysis showing the expression of C fragment by the four promoter cassettes in S. typhimurium BRD509. C-fragment expression was detected by the use of polyclonal anti-tetanus toxoid antiserum and is indicated by the arrows. Lane 1, low-molecular-weight markers; lanes 2 and 3, BRD509(pKK/C frag); lanes 4 and 5, BRD509(pKK/pnirB/C frag); lane 6 and 7, BRD509(pKK/ppagC/C frag); lane 8 and 9, BRD509(pKK/pkatG/C frag); lane 10, BRD509 alone; lane 11, BRD509(pTETtac4) (positive control) (8). Sizes are indicated in kilodaltons.

FIG. 5

In vivo plasmid stability and colonization of S. typhimurium. Groups of mice were orally immunized with BRD509(pKK/pnirB/C frag) (pnirB), BRD509(pKK/ppagC/C frag) (ppagC), BRD509(pKK/pkatG/C frag) (pkatG), BRD509(pKK/C frag) (ptrc), and BRD509. On days 5, 9, and 15 postimmunization, the numbers of bacteria present in the PPs (A) and spleen (B) were determined by isolating bacteria on LB agar (open shapes) or LB agar containing ampicillin (closed shapes). Each point represents the number of bacteria isolated from an individual mouse. ∗, the number of bacteria isolated on LB agar containing ampicillin was significantly lower than the number of bacteria isolated on the corresponding LB agar (P < 0.05).

FIG. 6

LPS- and tetanus toxoid-specific serum antibody responses. Groups of mice were immunized orally with BRD509(pKK/pnirB/C frag) (pnirB), BRD509(pKK/ppagC/C frag) (ppagC), BRD509(pKK/pkatG/C frag) (pkatG), BRD509(pKK/C frag) (ptrc), and BRD509 (509). The amounts of S. typhimurium LPS-specific (A) and tetanus toxoid-specific (B) total immunoglobulin present in serum were determined. Each point represents a serum titer from an individual mouse. The arrows indicate the times of oral immunization. ∗, the anti-tetanus toxoid antibody titer detected in the sera of BRD509(pKK/ppagC/C frag)-immunized mice was significantly different (P < 0.05) from the antibody titer detected in BRD509(pKK/C frag)-immunized mice.