The attenuated vector vaccine SH02 against H. pylori was prepared by our laboratory and stored at –80°C in Luria-Bertani (LB) containing 30% glycerol. In each experiment, SH02 was recovered to room temperature from the freezing solution, 50 µL was inoculated into 5 mL antibiotic-free LB medium, and it was incubated with shaking overnight at 37°C. The following day, 2 mL of the bacterial solution was added to 200 mL of antibiotic-free LB medium, incubated with shaking at 37°C for three hours, and induced with 1 mM IPTG for four hours. The expression of the rUreB-HspA fusion protein was assessed by SDS-PAGE and Western Blot. Vector strain was expressed and identified in the same way. The bacteria of SH02 were subsequently collected by centrifugation, washed once with sterile phosphate-buffered saline (PBS), and resuspended with sterile PBS to a final concentration of about 1.25 × 10¹⁰ cfu/mL.

H. pylori strains SS1 were cultured on campylobacter agar base plates (CDRC, Shanghai, China) with 7% fetal bovine serum at 37°C under microaerophilic conditions with 5% O₂, 10% CO₂, and 80% N₂. Three days later, colonies were scraped and washed twice with cold PBS and pelleted by centrifugation at 8000 × g at 4°C for 10 min. Then, the bacterial pellets were resuspended in sterile PBS, the OD value was measured, and the concentration was estimated and adjusted to an appropriate concentration to inoculate mice.

The gene sequences of ureB, hspA, and ureB-hspA were amplified by PCR from the vaccine SH02, cloned into an expression vector pGEX-6P-1(+) plasmid (GE Healthcare, Pittsburgh, USA), and placed between BamHI and NotI restriction sites (UreB-F: 5′-CGCGGATCCAAAAAGATTAGCAGAAAAG-3′, UreB-R: 5′-AAATATGCGGCCGCCTAGAAAATGCTAAAGAG-3′; HspA-F: 5′-CGCGGATCCAAGTTTCAACCATTAG-3′, HspA-R: 5′-AAATATGCGGCCGCTTAGTGTTTTTTGTGATC-3′; UreB-HspA-F: 5′- CGCGGATCCAAAAAGATTAGCAGAAAAG-3′, UreB-HspA-R: 5′- AAATATGCGGCCGCTTAGTGTTTTTTGTGATC-3′). The recombinant plasmid was transformed into Escherichia coli (E. coli) BL21 (DE3) cells (Cwbio, Suzhou, China), and protein expression was subsequently induced with 1 mM IPTG at 20°C before being harvested after 18 hours by centrifugation. The bacterial pellets were resuspended in PBS and sonicated. Any insoluble cellular components were removed by centrifugation, and the rGST-UreB, rGST-HspA, and rGST-UreB-HspA fusion proteins were purified by Price Glutathione Superflow Agarose (Thermo, Rockford, USA) according to the manufacturer’s protocol. Moreover, the GST tag was excised with PreScission Protease (Beyotime, Nanjing, China) and removed with GST-tag Purification resin (Beyotime, Nanjing, China). Finally, the purity and concentration of the recombinant protein rUreB, rHspA, and rUreB-HspA were determined via SDS-PAGE, Western Blot, and BCA assay.

The UreB-HspA expressed by the vaccine strain SH02 and the rUreB-HspA expressed and purified from E. coli were verified by Western Blot analysis. The vector strain without antigen expression, SH02, purified rUreB, rHspA, and rUreB-HspA were denatured and separated by 15% SDS-PAGE, transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Mannheim, Germany), and then blocked with PBST containing 5% skimmed milk at 37°C for one hour to prevent non-specific protein binding. The membranes were incubated with appropriate dilution of UreB monoclonal antibodies (21) or immunized mice sera at 37°C for one hour, washed with PBST three times, and then incubated with HRP-conjugated rabbit anti-mouse IgG (1:5000 dilution, Abcam, Cambridge, UK) at 37°C for one hour. The HRP Western Blot Analysis Kit (Easybio, Beijing, China) was used to detect the binding reaction.

Three groups of six-to-eight-week-old, female, specific-pathogen-free (SPF) BALB/c mice (N = 10/groups) were immunized three times on days 0, 14, and 42. Group A received oral SH02 for all three immunizations, and Group B received oral SH02 for the first two immunizations and a subcutaneous injection of 50 µg 0.05 mL rUreB-HspA + 0.05 mL AddaVax for the third immunization. The control group received oral PBS for all three immunizations. After 20 min of neutralization of gastric acid with 0.5 ml 3% NaHCO₃, all mice were orally inoculated with 2.5 × 10⁹ CFU of SH02 with a volume of 0.2 mL, 0.2 mL PBS, or injected subcutaneously with rUreB-HspA + AddaVax. On day 56, all mice were challenged with 1.3 × 10⁸ CFU of H. pylori strains SS1 with a volume of 0.2 mL. The concentration of SS1 was confirmed by plating 10-fold serial dilutions on campylobacter agar base plates (CDRC, Shanghai, China), and cultured as above for three days.

Before they were challenged, serum was collected for the detection of serum-specific antibody levels, and fresh fecal pellets were collected for the determination of specific secretory IgA (sIgA) levels. The fecal samples were weighed and then suspended in PBS at a ratio of 0.5 mL/100 mg fecal pellets. Moreover, the sample was homogenized by vortexing at room temperature for five min, then being centrifuged at 8000 × g for 10 min, with the supernatant transferred to a new tube containing 1% protease inhibitor cocktail (Bimake, Shanghai, China). Eight weeks following the challenge, all mice were sacrificed and their stomachs were taken out aseptically for determination of H. pylori load and pathological histology analysis. Spleens were simultaneously removed for performing lymphocyte proliferation assays and flow cytometry.

All animals were purchased from the Vital River Laboratory (Beijing, China) and bred under SPF conditions. Animal experiments were confirmed by the Animal Ethics Committees and performed under the regulations of the Animal Care and Use Committee guidelines from the Academy of Military Medical Sciences.

Antigen-specific IgG, IgG1, IgG2a, and IgG2b levels in the sera and sIgA levels in the fecal supernatant were determined by ELISA. The ELISA plates were coated with rUreB-HspA (100 µL, 5 µg/mL) diluted in 0.05 M carbonate coating buffer. The plates were incubated overnight at 4°C, washed three times with PBST, and then blocked with a blocking buffer (PBST containing 5% skimmed milk) for one hour at 37°C. The blocking solution was discarded, and serially diluted serum samples and fecal supernatant were added to the plates. After incubating at 37°C for one hour, the plates were washed again and horseradish peroxidase (HRP) conjugated anti-mouse sIgA, IgG, IgG2b, IgG1 or IgG2a (Abcam, Cambridge, UK) antibody (100 μL/well) was added. The plate was incubated again at 37°C for one hour and washed, and 3,3,5,5-tetramethylbenzene-biphenyl (TMB) substrate (Solarbio, Beijing, China) was subsequently added at 100 μL/well. After the reaction was carried out in the dark for 15–20 min, 1 M H₂SO₄ was added to stop the reaction, and the A450 of each well was measured in SpectraMax i3x (Molecular Devices, San Jose, USA). All samples were analyzed in duplicate, and the means of the replicates were reported in the results.

Stomachs were taken out aseptically, cut along the large bend from the pylorus to the cardia, and placed flat in a sterile petri dish with the mucosa facing upward. The gastric mucosal surface was subsequently flushed with sterile saline through a syringe. Half of the stomach was weighed and transferred to a homogenization tube containing brucella broth at a ratio of 1: 10 and then homogenized with a tissue homogenizer. The colonization level of H. pylori was determined by applying a 10-fold dilution of the gastric homogenate to a campylobacter agar plate (CDRC, Shanghai, China) containing 10 µg/mL amphotericin B, 10 µg/mL vancomycin, 20 U/mL bacitracin, 10 µg/mL polymyxin B, and 5 µg/mL trimethoprim, and culturing for seven days as described above. CFUs were enumerated and bacteriology data were expressed as CFU/g stomach.

The other half of the stomach tissue sample was fixed in 10% neutral buffered formalin, then processed and embedded in paraffin. The sections were cut 4 mm thick and stained with hematoxylin-eosin, and the severity of gastritis (infiltration degree of lymphocytes) was determined by an experimental pathologist who did not know the distribution of the experimental group. The extent of gastritis was graded as follows: 0, none; 1, a small amount of lymphocytes infiltrated the lamina propria; 2, a moderate amount of lymphocytes infiltrated the lamina propria; 3, lymphocytes densely infiltrate the lamina propria, and some enter the submucosa; 4, dense, diffuse lymphocytic infiltration of the lamina propria and submucosa.

Spleens were aseptically removed and pressed through a fine nylon mesh using syringe plunges to prepare single cell suspensions. Cells were seeded (4 × 10⁵ cells per well) in RPMI 1,640 medium (Giboc, Grand Island, USA) containing 10% FBS and 100 U/mL Penicillin-Streptomycin in the presence of 10 μg/mL rUreB-HspA (Endotoxin < 100 EU/mg) for 72 hours at 37°C and 5% CO₂. Under the same conditions, the cells without rUreB-HspA were regarded as non-antigen-stimulated cells. The cells were pulsed with 20 μL Cell Counting Kit-8 (CCK8) solution (Dojindo, Kumamoto Ken, Japan) per well for the last four hours of culturing, and the absorbance at 450 nm (A450) was measured in SpectraMax i3x (Molecular Devices, San Jose, USA). The results were expressed as stimulation indexes (SI): SI = OD value of stimulated cultures/OD value of non-stimulated cultures.

Single-cell suspensions of 4 × 10⁶ spleen cells were plated in 12-well plates, stimulated with 2 µg CD28 and 40 µg rUreB-HspA (Endotoxin < 100 EU/mg), supplemented with 100 IU/mL Penicillin-Streptomycin and 10% FBS in 2 mL RPMI 1,640 medium, and incubated for 24 hours at 37°C in 5% CO₂. In the last six hours, protein transport inhibitors Brefeldin A (BD, New Jersey, USA) 2 µL/mL and Monensin (BD, New Jersey, USA) 1.4 µL/mL were added to the cells. After centrifugation to remove the supernatant, 2 μL Fc receptor blocker (Biolegend, California, USA) was added to the cells, which were incubated for 10 min at room temperature, with 5 μL premixed True-Stain Monocyte Blocker (Biolegend, California, USA) and 2 μL CD4 (Biolegend, California, USA) subsequently added before being incubated again at room temperature in the dark for 25 min. After washing the cells with PBS, 1 mL diluted Zombie Aqua solution (Biolegend, California, USA) was added, and the cells were incubated for 10 min and centrifuged. The cells were then fixed, permeabilized, and washed with 2 mL BD Perm/Wash™ Buffer using the Fixation/Permeabilization Kit (BD, New Jersey, USA) according to the manufacturer’s instructions. Following each centrifugation, the cell cluster was ejected. Next, pre-mixed Brilliant Stain Buffer Plus (BD, 10 μL), True-Stain Monocyte Blocker (5 μL), CD3 (Biolegend, California, USA), CD154 (Biolegend, California, USA), IL-17A (Biolegend, California, USA), and IFN-γ (Biolegend, California, USA) (2 μL for each antibody) were added to the cells, thoroughly mixed, and the cells were incubated for 30 min at room temperature in the dark. Finally, the cells were washed with BD Perm/Wash™ Buffer and PBS, resuspended in 0.2 mL PBS, and the number of various types of cells was measured on the Cytek-Aurora flow cytometer.

All data were represented as mean ± standard deviation (SD). D’Agostino-Pearson normality test in Normality and Log normality were used to test whether the data conformed to a normal distribution before statistical analysis. When the experimental data conformed to the normal distribution, one-way ANOVA followed by Bonferroni multiple pairwise comparison tests or Brown-Forsythe and Welch ANOVA followed by Tamhane2 were used to analyze the differences between the groups. The non-parameter Mann-Whitney test was used to analyze non-normally distributed data. All statistical analyses were performed using GraphPad Prism version 8.0.2, and the difference was considered significant at p < 0.05.

Prior to each immunization of mice, we took a new cryopreserved candidate vaccine strain SH02 to induce expression and confirmed the expression of the fusion antigen UreB-HspA by SDS-PAGE and Western Blot ( Figure 1A ). The recombinant protein rUreB-HspA was correctly expressed in recombinant clone E. coli BL21 (pGEX-6P-UreB-HspA). After purification and removal of GST tag, rUreB-HspA was verified by SDS-PAGE and Western Blot ( Figure 1B ).

Three groups of mice were immunized and challenged as described in the method, and the detailed immunization schedule was shown in Figure 2A . Two weeks following the final immunization and before the challenge, we first evaluated the antigen-specific antibody produced by immunized mice by Western Blot ( Figure 2B ).

Next, we detected the levels of mucosal and humoral immune responses induced by immunization and the IgG antibody subtypes by ELISA. As expected, compared with the control group, the non-subcutaneous booster injection group (Group A) and the subcutaneous booster injection group (Group B) both produced UreB-HspA-specific IgG antibodies (p < 0.0001). Moreover, the serum antigen-specific IgG titer of Group B was significantly increased, which was markedly different from that of Group A (p < 0.0001). ( Figure 3A ) To further characterize the type of immune responses induced by the vaccine candidate, sera were analyzed for the antigen-specific IgG subtypes (IgG1, IgG2a, and IgG2b). As shown in Figures 3B–D , oral candidate vaccine SH02 (Group A) can cause IgG1 and IgG2a responses, and the subcutaneous injection of rUreB-HspA to boost immunity in Group B can cause IgG1, IgG2a, and IgG2b responses. On the other hand, we tested the antigen-specific mucosal sIgA antibody levels in the feces of mice two weeks following immunization. The results indicated that compared with the control group, groups A and B both produced a significant mucosal immune response (p < 0.01), but there was no difference between group A and group B (p > 0.05) ( Figure 3E ).

Since oral vaccine candidate SH02, both with or without subcutaneous injection of rUreB-HspA, demonstrated strong immunogenicity, we investigated whether immunization can prevent H. pylori infection. Two weeks after the final immunization, the mice were challenged with live H. pylori, and eight weeks following the challenge, the amount of bacterial colonization in the stomach of the mice was evaluated. Compared with the control group, the amount of H. pylori colonization in groups A and B was significantly reduced (p < 0.001). However, there was no significant difference between groups A and B (p > 0.05) ( Figure 4 ).

The histopathological examination of the stomach tissues of the immunized mice sacrificed eight weeks after the H. pylori challenge indicated that the tissue structure of the stomach was not abnormal in the three experimental animal groups. The lymphocytes were located in the lamina propria of the gastric antrum. The control group (9/10) exhibited a wider range of inflammation with the most severe lesions, and Group A (6/10) exhibited milder inflammation than the control group. The inflammation in Group B was the mildest, with only four out of 10 mice exhibiting mild local chronic inflammation. The inflammation scores of all mice were shown in Table 1 , and the histopathological pictures of gastric antrum mucosa in some experimental mice were shown in Figure 5 .

We evaluated the proliferative response of splenic lymphocytes in each group of mice after the H. pylori challenge. Compared with control group mice, the proliferation of lymphocytes in groups A and B increased significantly eight weeks after the challenge ( Figure 6 ). Moreover, the proliferation of lymphocytes in Group B was significantly higher than that of Group A (p < 0.01).

Flow cytometry analysis was performed to compare antigen-specific spleen T lymphocyte responses in the immunized and control mice at eight weeks after the H. pylori challenge. In this study, we used the rUreB-HspA to stimulate splenic lymphocytes, and analyzed the level of CD4+CD154+ T cells and CD4+CD154+ T cells that secrete IFN-γ or IL-17A, which were antigen-specific T lymphocytes. The results demonstrated that the frequency of UreB-HspA-specific CD4+CD154+ T cells, CD4+CD154+IFN-γ+ T cells, and CD4+CD154+IL17A+ T cells in the spleens of mice in groups A and B increased significantly, with Group B significantly higher than Group A ( Figure 7 ).