Protective efficacy of anti-Helicobacter pylori immunity following systemic immunization of neonatal mice

Abstract

Helicobacter pylori infection of the gastric mucosa is a significant cause of morbidity and mortality because of its etiologic role in symptomatic gastritis, peptic ulcer disease, and gastric adenocarcinoma. Infection occurs in young children; therefore, a prophylactic vaccine would have to be administered within the first year of life, a period thought to be immunologically privileged. We investigated vaccine formulations administered by different routes to confer protective anti-H. pylori immunity in neonatal mice. Neonatal mice immunized with a single dose of vaccine in complete Freund's adjuvant (CFA) generated antigen-specific gamma interferon-, interleukin-2 (IL-2)-, IL-4-, and IL-5-secreting T cells in numbers similar to those in immunized adult mice, while vaccine administered to neonates in incomplete Freund's adjuvant (IFA) induced such cells in reduced numbers compared to those in adult mice. Both IFA and CFA, however, provided partial protection from a challenge with infectious H. pylori when the vaccine was administered subcutaneously. Neonatal immunized mice also had reduced bacterial loads when immunized intraperitoneally with CFA. In all cases, protection was equivalent to that achieved when adult counterparts were immunized. These studies suggest that an efficacious vaccine might be successfully administered to very young children to prevent perinatal infection of H. pylori.

FIG. 1.

Induction of anti-H. pylori (H.p.) immunity by systemic immunization of neonatal mice. (a) Spleen cells collected 28 days after a single i.p. or s.c. immunization with H. pylori lysate or OVA emulsified in IFA were stimulated in vitro with H. pylori lysate and tested by ELISPOT assay for IFN-γ or IL-5. Each value shown is the mean of three individual mice (tested in triplicate) ± the standard deviation. (b) Groups of mice given each vaccine formulation were challenged with infectious H. pylori on day 28 and sacrificed on day 48 for bacterial load assessment. Each symbol represents an individual mouse, and each bar represents the geometric mean of each respective group. A statistical significance of greater than 99.9% relative to OVA-immunized mice is indicated by the triple asterisks. (c) Gastric biopsy samples of the greater curvature were hematoxylin and eosin stained and graded for inflammation. Each value shown is the mean of each immunized-and-challenged group of mice ± the standard deviation.

FIG. 2.

Prechallenge cytokine profiles of spleen cells from mice immunized as neonates (a and b) or adults (c and d) with a single dose of H. pylori (H.p.) antigen and CFA (a and c) or IFA (b and d). Spleen cells were tested by ELISPOT assay for IFN-γ, IL-2, IL-4, and IL-5. Each value shown is the mean of each group (four to six mice each, tested in triplicate) ± the standard deviation.

FIG. 3.

Cytokine profiles of spleen cells from mice immunized as neonates with a single dose of H. pylori (H.p.) antigen and CFA (a) or IFA (b) and challenged with infectious H. pylori 28 days after immunization. Spleen cells were tested on day 56 by ELISPOT assay for IFN-γ, IL-2, IL-4, and IL-5. Each value shown is the mean of each group (five to seven mice each, tested in triplicate) ± the standard deviation.

FIG. 4.

Bacterial loads in the gastric mucosae of mice immunized as neonates (a) or adults (b) with a single dose of H. pylori (H.p.) antigen and CFA or IFA and challenged on day 28. Gastric biopsy samples were examined on day 56 by quantitative culture. Each symbol represents an individual mouse, and each bar represents the geometric mean of each respective group. Statistical significance of differences relative to the respective OVA-immunized control groups is indicated by single (>95% significant), double (>99% significance), or triple (>99.9% significance) asterisks.

FIG. 5.

Mucosal inflammation in the stomachs of mice immunized as neonates (a) or adults (b) with a single dose of H. pylori (H.p.) antigen and challenged with infectious H. pylori on day 28. Strips of the greater curvature of the stomach were surgically removed on day 56, hematoxylin and eosin stained, and graded for inflammation. Each value shown is the mean of each immunized-and-challenged group of mice ± the standard deviation. Statistical significance of differences relative to the respective OVA-immunized control groups is indicated by double (>99% significance) or triple (>99.9% significance) asterisks.