Rectal and intranasal immunizations with recombinant urease induce distinct local and serum immune responses in mice and protect against Helicobacter pylori infection

Abstract

To determine the optimal inductive sites for immunization against Helicobacter pylori infection, the protective efficacy of recombinant urease (rUre) was assessed for mice given the vaccine by either the oral (p.o.), intranasal (i.n.), or rectal route. When mice were immunized with rUre (25 microg p.o. or rectally or 10 microg i.n.) plus heat-labile toxin from Escherichia coli as the mucosal adjuvant, all routes afforded protection against challenge with H. pylori, as indicated by a significant reduction in gastric urease activity (P < 0.0005) compared to that of sham-immunized controls. Quantitative H. pylori culture of stomach tissue demonstrated a >97% reduction in bacterial burden in mice immunized by all routes (P < 0.05). Induction of antiurease immunoglobulin A (IgA) levels in gastric luminal secretions after p.o. immunization was greater than after i.n. administration (means, 6.0 and 1.02 ng/ml, respectively) and was dependent upon challenge with H. pylori. However, immunization by the rectal route resulted in the generation of the highest levels of gastric antiurease IgA (mean, 40. 89 ng/ml), which was detectable prior to challenge with H. pylori. Immunohistochemical staining of stomach tissue for cells secreting urease-specific antibody and CD4(+) T cells showed levels of recruitment to be dependent upon challenge with H. pylori and equivalent for all routes. These results identify both the rectum and nasal passages as suitable inductive sites for urease immunization.

FIG. 1

Urease activities of gastric tissues from mice immunized with rUre plus LT by either the p.o., i.n., or rectal (R) route. Mice were immunized with 25 μg of rUre by the p.o. and rectal routes and with 10 μg by the i.n. route. Animals were challenged with H. pylori 2 weeks postimmunization and euthanized 2 weeks postchallenge. Infection was determined by incubating one-fourth of the antrum from each animal in urea broth and measuring the gastric urease activity spectrophotometrically at an A₅₅₀ after a 4-h incubation at room temperature. Mice were considered protected from infection if the urease activity was within 2 standard deviations of the mean value for unchallenged control mice. Mice were significantly protected by all routes compared to LT sham-immunized controls (P < 0.0005; Wilcoxon rank sum test). As no significant differences between results for control groups were apparent, all results for LT sham-immunized animals were pooled for this analysis. Solid lines represent the arithmetic mean for each group studied.

FIG. 2

Quantitative H. pylori culture of gastric tissue, represented on a logarithmic scale, from mice immunized with rUre plus LT by either the p.o., i.n., or rectal (R) route. Gastric tissue from one-fourth of the antrum of each mouse was homogenized in a dounce homogenizer (7 ml), and serial dilutions from each homogenate were plated onto helicobacter-selective agar. Colony counts are presented as CFU per milliliter of the dilutions of the one-quarter antral tissue section of each animal. Only two animals from rUre-immunized groups were determined to have zero bacteria (indicated as 0.1 on a log₁₀ scale). A significant reduction in bacterial burden (2 logs) was observed for all routes when burdens were compared to that of the pooled LT controls (P < 0.05; Wilcoxon rank sum test). Solid lines represent the geometric mean for each group studied.

FIG. 3

Gastric immune responses induced to rUre immunization by route of immunization. (A and B) Recruitment of IgA⁺ B cells (top) and rUre-specific ACC (bottom) and of CD4⁺ (top) and CD8⁺ (bottom) T cells, respectivley, into the gastric mucosae of mice immunized with rUre plus LT is dependent upon challenge with H. pylori. Results for LT controls are shown for rectally immunized animals only. P-I, postimmunization (only results for the rectally immunized group are shown); P-C, postchallenge. Results for sham-immunized control mice are represented with closed circles.