Immunization with Heat Shock Protein A and γ-Glutamyl Transpeptidase Induces Reduction on the Helicobacter pylori Colonization in Mice

Abstract

The human gastric pathogen Helicobacter pylori (H. pylori) is a successful colonizer of the stomach. H. pylori infection strongly correlates with the development and progression of chronic gastritis, peptic ulcer disease, and gastric malignances. Vaccination is a promising strategy for preventing H. pylori infection. In this study, we evaluated the candidate antigens heat shock protein A (HspA) and H. pylori γ-glutamyl transpeptidase (GGT) for their effectiveness in development of subunit vaccines against H. pylori infection. rHspA, rGGT, and rHspA-GGT, a fusion protein based on HspA and GGT, were constructed and separately expressed in Escherichia coli and purified. Mice were then immunized intranasally with these proteins, with or without adjuvant. Immunized mice exhibited reduced bacterial colonization in stomach. The highest reduction in bacterial colonization was seen in mice immunized with the fusion protein rHspA-GGT when paired with the mucosal adjuvant LTB. Protection against H. pylori colonization was mediated by a strong systemic and localized humoral immune response, as well as a balanced Th1/Th2 cytokine response. In addition, immunofluorescence microscopy confirmed that rHspA-GGT specific rabbit antibodies were able to directly bind H. pylori in vitro. These results suggest antibodies are essential to the protective immunity associated with rHspA-GGT immunization. In summary, our results suggest HspA and GGT are promising vaccine candidates for protection against H. pylori infection.

Fig 1. Candidate antigens and immunization schedule used in this study.

(A) Three His-tagged recombinant proteins were designed to be used as candidate antigens: full length HspA (rHspA), the catalytic domain of GGT (rGGT), and a fusion protein composed of rHspA and rGGT linked by two lysine residues (rHspA-GGT). (B) Recombinant proteins were purified by nickel ion affinity chromatography and gel-filtration chromatography. Recombinant proteins were then analyzed by SDS-PAGE. (C) The schedule for vaccine immunization, determination of cytokines secretion, and antigen-specific antibodies. (D) The schedule for vaccine immunization and analysis of H. pylori colonization.

Fig 2. H. pylori colonization in mouse stomachs after immunization.

BALB/c mice (n = 10) were immunized intranasally on days 0, 14 and 21 with 30 μg antigen, with or without 10μg adjuvant, as indicated in Table 1. The same volume of PBS was used as a negative control. Three weeks after the final vaccination boost, mice were orally challenged four times with H. pylori B6. The level of gastric H. pylori colonization was determined by real-time quantitative PCR four weeks post challenge for each mouse. Data are expressed as mean ± S.D. Significant differences between indicated groups are presented as P values.

Fig 3. Antigen specific antibody responses elicited by immunization.

Two weeks after immunization, mice in each group (n = 5) were bled and the sera were collected for analysis of the specific IgG antibody and its subtype. (A) Specific IgG antibody titers, for antibodies raised against rHspA-GGT and H. pylori 26695 lysate, were measured by ELISA. (B) The levels of specific IgG1 and IgG2a, against rHspA-GGT, were determined from serum samples tested by ELISA. (C) Immunized mice were sacrificed 4 weeks after challenge and the supernatants of homogenized stomachs and intestines were collected for detection of specific sIgA levels, against rHspA-GGT. Data are presented as mean ± S.D (n = 5). (D) Regression analysis of bacterial loads and the level of rHspA-GGT specific serum IgG. **P < 0.01 compared with indicated groups, ***P < 0.001 compared with indicated groups, ns: not significant.

Fig 4. Immunization induced cytokine responses in murine spleen cells.

Two weeks after the last booster, spleen cells of mice (n = 5) in each group were stimulated for 72 hours with antigen rHspA-GGT (5 μg/ml). The supernatants were harvested, and the cytokine levels of (A) IFN-γ, (B) IL-17A, (D) IL-4, and (D) IL-5 were determined by ELISA. Data are expressed as mean ± S.D. *P < 0.05 compared with all other groups or indicated groups, **P < 0.01 compared with indicated groups, ***P < 0.001 compared with indicated groups, ns: not significant.

Fig 5. Indirect immunofluorescence confirmed that anti-serum collected from immunized mice directly binds H. pylori in vitro.

No immunofluorescence was detected in the absence of antigen specific pcAb (A and B). Positive indirect immunofluorescence signals indicate binding of rHspA-GGT pcAb with H. pylori 26695 (C) and the existence of H. pylori (D). This study was performed twice, yielding similar results.