Oral immunization with recombinant Mycobacterium smegmatis expressing the outer membrane protein 26-kilodalton antigen confers prophylactic protection against Helicobacter pylori infection

Abstract

Helicobacter pylori infection is prevalent worldwide and results in chronic gastritis, which may lead to gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. We have previously reported that oral immunization with recombinant Mycobacterium smegmatis expressing the H. pylori outer membrane protein 26-kilodalton (Omp26) antigen affords therapeutic protection against H. pylori infection in mice. In the present study, we investigated the prophylactic effects of this vaccine candidate on H. pylori challenge in mice. We found that oral immunization with recombinant Mycobacterium Omp26 significantly reduced H. pylori colonization in the stomach compared to inoculation with wild-type M. smegmatis in control mice. Six of the recombinant Mycobacterium-immunized mice (60%) were completely protected from H. pylori infection. The severity of H. pylori-associated chronic gastritis assessed histologically was significantly milder in mice vaccinated with recombinant Mycobacterium than in control animals. Mice immunized with recombinant Mycobacterium showed enhanced antigen-specific lymphocyte proliferation and antibody responses. Moreover, immunization with recombinant Mycobacterium resulted in an increased expression of interleukin-2 and gamma interferon in the stomach and spleen, as determined by reverse transcription-PCR analysis. Our results collectively suggest that vaccination with recombinant Mycobacterium Omp26 confers prophylactic protection against H. pylori infection. The inhibition of H. pylori colonization is associated with the induction of antigen-specific humoral and cell-mediated immune responses.

Fig. 1.

Reduction of H. pylori burden in mice immunized with recombinant Mycobacterium expressing Omp26. Mice were orally immunized with wild-type M. smegmatis (M. s) or a recombinant Mycobacterium strain expressing Omp26 (rM. s) or unimmunized as a control, prior to challenge by H. pylori. The H. pylori colonization in the stomach was assessed as described in Materials and Methods. (A) Bacterial culture assay. The number of viable bacteria (CFU) per milligram of stomach tissue was determined for individual mice in each group. The dotted line designates the detection limit (100 bacteria/mg) for the quantitative culture. The horizontal lines indicate the geometric mean for each group. (B) Rapid urease test. The test was scored as positive if the color changed from yellow to pink-red within 5 min. The positive rate of the urease test was calculated for each group. *, P < 0.05; ns, not significant.

Fig. 2.

Gastric histology in immunized or control mice after H. pylori challenge (HE staining; magnification, ×100). Unimmunized mice (as control) (A) or those immunized with wild-type M. smegmatis (M. s) (B) showed marked gastric mucosal degeneration (arrowhead) and inflammatory cell infiltration (arrow), whereas mice vaccinated with recombinant Mycobacterium (rM. s) (C) displayed no evident inflammatory infiltrate. Representative images for each group are shown. (D) Quantification of the H. pylori-associated gastritis. Gastric inflammation was graded on a scale of 0 to 4, and the mean score was determined for each group. ***, P < 0.001; ns, not significant.

Fig. 3.

Immune responses induced by immunization with recombinant Mycobacterium Omp26. Mice were orally immunized with wild-type M. smegmatis (M. s) or a recombinant Mycobacterium strain expressing Omp26 (rM. s) or unimmunized as a control. Antigen-specific lymphocyte proliferation and antibody responses were determined, as described in Materials and Methods. (A) In vitro lymphocyte proliferation assay. Splenic lymphocytes were isolated from individual mice, and their proliferation in response to ConA or H. pylori extracts was assessed by the MTT assay. The results were expressed as stimulation indices, which were calculated as the ratio of OD values in immunized and unimmunized (control) groups. *, P < 0.05. (B) Measurement of serum antibody responses. Data are expressed as the mean OD₄₅₀ value ± SD. ***, P < 0.001 compared to the M. smegmatis group.

Fig. 4.

Local expression of cytokines in immunized or control mice before (A) and after (B) H. pylori challenge. Total RNA was isolated from the stomach and spleen tissues and subjected to analysis of indicated cytokines by RT-PCR, as described in Materials and Methods. The ratio of cytokine to β-actin was determined by densitometry. Control, unimmunized mice; M. s, mice orally immunized with wild-type M. smegmatis; rM. s, mice orally immunized with recombinant Mycobacterium Omp26. *, P < 0.05 compared to the M. smegmatis control group.