Mice are protected from Helicobacter pylori infection by nasal immunization with attenuated Salmonella typhimurium phoPc expressing urease A and B subunits

Abstract

Live Salmonella typhimurium phoPc bacteria were tested as mucosal vaccine vectors to deliver Helicobacter pylori antigens. The genes encoding the A and B subunits of H. pylori urease were introduced into S. typhimurium phoPc and expressed under the control of a constitutive tac promoter (tac-ureAB) or a two-phase T7 expression system (cT7-ureAB). Both recombinant Salmonella strains expressed the two urease subunits in vitro and were used to nasally immunize BALB/c mice. The plasmid carrying cT7-ureAB was stably inherited by bacteria growing or persisting in the spleen, lungs, mesenteric or cervical lymph nodes, and Peyer's patches of immunized mice, while the plasmid carrying tac-ureAB was rapidly lost. Spleen and Peyer's patch CD4+ lymphocytes from mice immunized with S. typhimurium phopc cT7-ureAB proliferated in vitro in response to urease, whereas cells from mice given S. typhimurium phoPc alone did not. Splenic CD4+ cells from mice immunized with phoPc cT7-ureAB secreted gamma interferon and interleukin 10, while Peyer's patch CD4+ cells did not secrete either cytokine. Specific H. pylori anti-urease immunoglobulin G1 (IgG1) and IgG2A antibodies were detected following immunization, confirming that both Th1- and Th2-type immune responses were generated by the live vaccine. Sixty percent of the mice (9 of 15) immunized with S. typhimurium phoPc cT7-ureAB were found to be resistant to infection by H. pylori, while all mice immunized with phoPc tac-ureAB (15 of 15) or phoPc (15 of 15) were infected. Our data demonstrate that H. pylori urease delivered nasally by using a vaccine strain of S. typhimurium can trigger Th1- and Th2-type responses and induce protective immunity against Helicobacter infection.

FIG. 1

In vitro expression of H. pylori urease A and B subunits by S. typhimurium phoP^c. Bacterial lysates of S. typhimurium phoP^c expressing the two subunits of H. pylori urease were analyzed by Western blotting. The urease A and B subunits were detected with rabbit sera. Lanes: 1 and 2, 3 and 1 μg of S. typhimurium phoP^c tac-ureAB, respectively; 3 and 4, 1 and 3 μg of S. typhimurium phoP^c cT7-ureAB, respectively. The mobilities of the prestained molecular size standards (in kilodaltons) are indicated on the left.

FIG. 2

Recovery of S. typhimurium phoP^c after nasal immunization. Groups of three mice were nasally immunized with 5 × 10⁷ CFU of S. typhimurium phoP^c (top panel), phoP^c tac-ureAB (middle panel), or phoP^c cT7-ureAB (bottom panel) and sacrificed after 2 weeks. The spleen, lungs, cervical and mesenteric lymph nodes (CLN and MLN, respectively) and PP were recovered, homogenized in 15% sucrose–phosphate-buffered saline, and plated on agar plates with (closed bars) or without (hatched bars) ampicillin. Data are expressed as means ± SD of CFU per tissue of three animals.

FIG. 3

Antiurease antibodies in the serum of immunized and immunized and infected mice. Sera were collected from mice after immunization with S. typhimurium phoP^c (○), S. typhimurium phoP^c tac-ureAB (▪), and S. typhimurium phoP^c cT7-ureAB (▵) (A and B) or from mice after immunization and then infection with H. pylori 4 weeks later (C and D) (symbols the same as those for panels A and B). IgG1A (A and C) and IgG2a (B and D) were separately determined by isotype-specific ELISA for each mouse serum (dilution of 1:200). OD, optical density.

FIG. 4

Assessment of H. pylori colonization. Mice were immunized twice and infected 2 weeks later. Bacterial colonization was assessed by the RUT and quantified by spectrophotometric analysis at 550 nm. OD, optical density. P = 0.0007 (phoP^ccT7-ureAB versus phoP^c), Fisher’s exact test.