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, 67 (5), 2531-9

Local Secretory Immunoglobulin A and Postimmunization Gastritis Correlate With Protection Against Helicobacter Pylori Infection After Oral Vaccination of Mice

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Local Secretory Immunoglobulin A and Postimmunization Gastritis Correlate With Protection Against Helicobacter Pylori Infection After Oral Vaccination of Mice

T Goto et al. Infect Immun.

Abstract

C57BL/6 mice were orally immunized with five weekly doses of 2 mg, 200 microgram, or 2 microgram of Helicobacter pylori (Sydney strain) whole-cell sonicate combined with cholera toxin. One week after the last vaccination, mice were challenged with 5 x 10(7) CFU of live H. pylori three times at 2-day intervals. At 6 or 18 weeks after the challenge, mice were sacrificed and bacterial cultures and histological studies of the stomach were performed. Vaccination with 2 mg/session or 200 microgram/session inhibited H. pylori colonization by 90 and 100%, respectively. These mice were considered protected. Lower levels of H. pylori-specific immunoglobulin A (IgA) were detected in fecal and saliva samples before challenge. However, a significant increase in IgA secretion in mucosal tissue and a higher labeling index for IgA-positive lumina of pyloric glands were noted in these mice in response to challenge and in a vaccine dose-dependent manner. In protected mice, however, severe gastritis characterized by marked infiltration of inflammation mononuclear cells was noted at 6 weeks after challenge, compared with the gastritis seen in unprotected mice or nonvaccinated, ordinarily infected mice. Marked expression of gamma interferon mRNA was detected in the stomach of all protected mice, and 50% of these mice expressed interleukin 4 (IL-4) or IL-5 mRNA. Our findings suggest that local secretory IgA antibody and severe postimmunization gastritis correlate well with protection of mice against H. pylori infection.

Figures

FIG. 1
FIG. 1
Detection of antibody responses in the feces, saliva, and sera of vaccinated (open bars) and nonvaccinated (filled bars) mice at 1 week after the last vaccination. H. pylori-specific fecal (with undiluted extract) IgA (A), salivary (at a 1:4 dilution) IgA (B), and serum (at a 1:100 dilution) IgA (C) and IgG (D) antibodies were measured by an ELISA. Data represent the mean ± standard deviation optical density at 414 nm for each group (n, 17 to 20 mice). P values were <0.05 (∗), <0.01 (∗∗), and <0.005 (∗∗∗) compared with the values for the corresponding nonvaccinated mice (Student’s t test).
FIG. 2
FIG. 2
Effect of oral vaccination with 5 μg of cholera toxin and 2 mg (group 1), 200 μg (group 2), or 2 μg (group 3) of H. pylori whole-cell sonicate on H. pylori infection. Protection against H. pylori infection was based on the absence of H. pylori, determined by both bacterial culturing and light microscopic examination of stomach sections (Giemsa stain) at 6 weeks after the last challenge (n, 10 mice). An asterisk indicates that the P value was <0.001 compared with the value for the corresponding nonvaccinated/infected mice (Fisher’s exact test).
FIG. 3
FIG. 3
Micrographs of gastric tissues of vaccinated/challenged mice and nonvaccinated/infected mice at 6 weeks after challenge. (A) Antral mucosa of a representative nonvaccinated/infected mouse, showing active chronic inflammation mainly in the submucosal layer (HE stain; original magnification, ×125). (B) Large numbers of spiral bacteria throughout the antral crypts in a representative nonvaccinated/infected mouse (Giemsa stain; original magnification, ×1,000). (C) Fundic mucosa of a representative protected mouse in the vaccinated/challenged group, showing high intensity of infiltration by inflammatory cells composed mainly of mononuclear cells (HE stain; original magnification, ×100).
FIG. 4
FIG. 4
Gastritis scores in vaccinated/challenged and nonvaccinated/infected mice at 6 (open bars) and 18 (filled bars) weeks (W) after the challenge. HE-stained gastric sections were scored based on the overall grade of inflammation (on a scale of 0 to 7), which included the intensity and extent of inflammation (see Materials and Methods). Data represent the mean ± standard deviation gastritis scores for each group (n, 5 to 10 mice). The asterisk indicates that the P value was <0.0005 compared with the value for the corresponding nonvaccinated/infected mice at 6 weeks after the challenge (Student’s t test).
FIG. 5
FIG. 5
Immunohistochemical analysis of gastric tissues from vaccinated/challenged mice (magnification, ×100) (A) and nonvaccinated/infected mice (magnification, ×100) (B) at 6 weeks after the challenge. (C) IgA LI for the gastric mucosa in vaccinated/challenged, nonvaccinated/infected, and normal control mice at 6 weeks after the challenge. The LI represent the percentage of anti-mouse IgA antibody-labeled lumina among 500 pyloric glands of the gastric mucosa. Data represent the mean ± standard deviation LI for each group (n, 10 mice). An asterisk indicates that the P value was <0.05 compared with the value for the corresponding nonvaccinated/infected mice at 6 weeks after the challenge (Student’s t test). A section sign indicates that the P value was <0.001 compared with the value for the corresponding vaccinated/challenged mice (2 μg) at 6 weeks after the challenge (Student’s t test).
FIG. 5
FIG. 5
Immunohistochemical analysis of gastric tissues from vaccinated/challenged mice (magnification, ×100) (A) and nonvaccinated/infected mice (magnification, ×100) (B) at 6 weeks after the challenge. (C) IgA LI for the gastric mucosa in vaccinated/challenged, nonvaccinated/infected, and normal control mice at 6 weeks after the challenge. The LI represent the percentage of anti-mouse IgA antibody-labeled lumina among 500 pyloric glands of the gastric mucosa. Data represent the mean ± standard deviation LI for each group (n, 10 mice). An asterisk indicates that the P value was <0.05 compared with the value for the corresponding nonvaccinated/infected mice at 6 weeks after the challenge (Student’s t test). A section sign indicates that the P value was <0.001 compared with the value for the corresponding vaccinated/challenged mice (2 μg) at 6 weeks after the challenge (Student’s t test).
FIG. 6
FIG. 6
Secondary responses of IgG subclasses in vaccinated mice at 6 weeks after challenge infection. H. pylori-specific serum (at a 1:25 dilution) IgG1 and IgG2a antibodies were quantitated by an ELISA. Data represent the mean ± standard deviation optical density at 414 nm for each group (n, 10 mice). The asterisk indicates that the P value was <0.05 compared with the value for the corresponding nonvaccinated mice at 1 week after the last vaccination (Student’s t test). The section sign indicates that the P value was <0.05 compared with the value for the corresponding unprotected mice in the vaccinated/challenged group and for the nonvaccinated/infected mice at 6 weeks after the challenge (Student’s t test).
FIG. 7
FIG. 7
Expression of IFN-γ, IL-4, and IL-5 mRNAs in the stomachs of nonvaccinated/infected and vaccinated/challenged mice at 6 weeks after the challenge. mRNAs were detected by RT-PCR and expressed as the copy number determined semiquantitatively (see Materials and Methods). Mice were vaccinated with 2 mg (○), 200 μg (◊), and 2 μg (●) of whole-cell sonicate; ⧫, nonvaccinated/infected mice. The ordinate is the logarithmic copy number of each cytokine mRNA.

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