Introduction

Helicobacter pylori infection is highly prevalent worldwide and is associated with the development of chronic gastritis, peptic ulcer, and gastric cancer (Hirai et al. 1999; Khan and Bemana 2012; Tsimmerman Ia 2009). Current therapies are based on a combination of three or four different antibiotics together with a proton-pump inhibitor. However, there are some drawbacks to this treatment including antibiotic resistance and poor patient compliance (Cameron and Bell 2005; Megraud et al. 2013). Prophylactic and therapeutic vaccine could be an attractive strategy, either as an alternative or a complement to antibiotic treatment against H. pylori infection.

Urease is an important colonization factor and pathogenic factor for H. pylori. Previous studies have found that some monoclonal antibodies (mAbs) against H. pylori urease have the ability to inhibit enzymatic activity (Hirota et al. 2001; Fujii et al. 2004; Qiu et al. 2010), whereas urease-specific polyclonal antibodies generated by immunization with purified H. pylori urease did not inhibit its enzymatic activity at all (Nagata et al. 1992). The UreA183–203 peptide from H. pylori urease A subunit (UreA) is recognized by HpU-2 mAb, which showed the strongest inhibitory effect on the enzymatic activity of the urease suppressing the urease activity by 82 % (Fujii et al. 2004). The UreB327–334 peptide from H. pylori urease B subunit (UreB) is recognized by L2 mAb which could strongly inhibit the enzymatic activity of H. pylori urease (Hirota et al. 2001). Besides, it has been reported three Th cell epitopes (UreA74–90, UreB229–244, and UreB237–251) from UreA or UreB could activate specific CD4+ T cell responses in BALB/c mice (Shi et al. 2007; Lucas et al. 2001). In this study, a multi-epitope vaccine with CTB and tandem copies of B cell epitopes (UreA183–203 and UreB327–334) and Th cell epitopes (UreA74–90, UreB229–244, and UreB237–251) from H. pylori urease A and B subunits named CTB-UE was designed, and its immunogenicity, specificity, ability to induce neutralizing antibodies against H. pylori urease, and prophylactic and therapeutic efficacy were evaluated in BALB/c mice model.

Materials and methods

Animals and bacteria

Specific pathogen-free (SPF) male BALB/c mice, 5–6 weeks of age, were purchased from Comparative Medicine Center of Yangzhou University and bred in an axenic environment. All animal experiments were approved by the Animal Ethical and Experimental Committee of China Pharmaceutical University. The mouse-adapted H. pylori strain SS1 was obtained from the National Center for Disease Control and Prevention (NCDC) and then preserved in our laboratory (collection number: CPU-BS-09). Bacteria were grown and harvested as previously described (Guo et al. 2012b).

Epitope vaccine design and construction

The theoretically optimal combination of the intramolecule adjuvant CTB (GenBank: CAA53976.1), the linkers, and the tandem copies of epitopes from H. pylori urease (GenBank: AF508016.1) was established on the basis of modeling and prediction using RANKPEP, DNASTAR software, and molecular operating environment (MOE). To construct the expression vector pETCUE expressing the fusion protein CTB-UE, a DNA fragment named UE containing tandem copies of the selected B and Th cell epitopes was synthesized. After digestion with KpnI and XhoI, the synthesized UE gene was cloned into the plasmid pETC which contained the CTB gene, generating the expression vector pETCUE. The fusion protein CTB-UE was expressed and purified according to the protocol performed as previously described with some modification (Guo et al. 2012a; Guo et al. 2013). The fusion protein CTB-UE was purified by Ni2+-charged column chromatography (Bio Basic Inc, Markham, Canada) and anion-exchange chromatography using DEAE Sepharose FF (Amersham Pharmacia Biotech AB, Sweden). After purification, the purity of the fusion peptide CTB-UE was analyzed by 12 % SDS-PAGE and computer scan. The samples were dialyzed in 2 l of PBS and finally concentrated and stored at −70 °C.

Western blot

An experimental procedure was performed as previously described with some modifications (Guo et al. 2012a). Purified fusion proteins CTB-UE, previously prepared rCTB, rUreA, and rUreB (Linc-Bio, Shanghai, China) were applied to 12 % SDS-PAGE under denaturing conditions and electrotransferred on to a nitrocellulose membrane at 80 mA for 2 h. Nonspecific binding sites were blocked overnight at 4 °C in blocking buffer (5 % non-fat milk in PBST, pH 7.4, with 0.05 % Tween 20). Membranes were washed three times for 10 min with PBST and further incubated with a 1:1,000 dilution of mouse polyclonal anti-CTB-UE serum or normal mouse serum at 37 °C for 1 h. After washing three times with PBST, the membrane was incubated in horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) at 37 °C for 2 h. The protein bands were visualized with enhanced chemiluminescence detection system (Amersham, UK).

GM1 ganglioside enzyme-linked immunosorbent assay

The adjuvant activity of the CTB component of CTB-UE was assessed by GM1 ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The experimental procedure was adapted from two reference published elsewhere (Menezes et al. 2002; Areas et al. 2004). Briefly, ELISA plates were coated with 1 μg/well of GM1 ganglioside (Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. The wells were blocked with 5 % (m/V) BSA in PBST for 1 h at 37 °C. After washing three times, the fusion proteins were diluted from 100 to 0.78 μg/ml. The fusion proteins were then added to the plates and incubated for 1 h at 37 °C. A proper dilution of anti-CTB polyclonal antibody (Biomade Technology, Qingdao, China) was added to the plates and incubated for 1 h at 37 °C. After washing, HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, USA) was added, and the plate was incubated at 37 °C for 1 h. Substrate tetramethylbenzidine (TMB, Tiangen Biotech, Beijing, China) was then added and incubated at room temperature for 10 min. The absorbance at 450 nm was measured by a microplate reader.

Immunization and infection

An experimental procedure to study the immunogenicity and specificity of the CTB-UE vaccine was performed as previously described with some modifications (Guo et al. 2012a). SPF BALB/c mice (male, 5 to 6 weeks old; n = 6) were immunized with 100 μg of the purified CTB-UE by intraperitoneal injection with Freund's adjuvant. The vaccine antigens in PBS were emulsified with an equal volume of complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA) for the first vaccination and with incomplete Freund adjuvant (Sigma-Aldrich, St. Louis, MO, USA) for the second and third vaccinations at a weekly interval. The vaccine antigens in PBS without adjuvant were for the last booster vaccination. The mice were also immunized with 100 μg of rUreB or PBS using the same method as a control. The rUreB protein was purchased from Shanghai Linc-Bio Science Corporation, and the purity of rUreB was 97.62 %. Antisera were separated on the fifth day after the last booster. The mice were also immunized with rUreB or PBS using the same method as a control.

The experimental procedure of prophylactic vaccination was performed as previously described with some modifications (Zhao et al. 2007; Zhou et al. 2009; Guo et al. 2012b), as shown in Fig. 1. Briefly, SPF BALB/c mice were randomized into three groups (six mice in each group) and vaccinated intragastrically by gavage with 150, 100, or 50 μg of the purified CTB-UE in 300 μl 3 % sodium hydrogen carbonate buffer for four times at 1-week intervals. The mice were also immunized with 150 μg of rUreB or PBS using the same method as a control. At 2 weeks after the final booster vaccination, all mice were inoculated with 0.3 ml H. pylori SS1 dilution (109 CFUs) by oral gavage for four times during a 12-day period with 4 days between each gavage. The mice were bred for 2 weeks before being used to evaluate H. pylori infection.

Fig. 1
figure 1

Prophylactic and therapeutic vaccination. The experimental procedures of prophylactic vaccination were shown as follows. Briefly, SPF BALB/c mice were orally immunized with CTB-UE, rUreB or PBS for four times at 1-week intervals. At 2 weeks after the final booster vaccination, all mice were inoculated with H. pylori SS1 for four times during a 12-day period. The mice were bred for 2 weeks before being used to evaluate H. pylori infection. The experimental procedures of therapeutic vaccination were shown as follows. Briefly, the mice were orally infected with H. pylori SS1. Four weeks after infection, the infected mice were vaccinated intragastrically with CTB-UE, rUreB, or PBS for four times at 1-week intervals. Two weeks after the final immunization, the mice were sacrificed for evaluation of H. pylori infection

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The experimental procedure for therapeutic vaccination was performed as previously described with some modifications (Zhao et al. 2007; Zhou et al. 2009; Guo et al. 2012b), as shown in Fig. 1. Briefly, the mice were given 0.4 mg of omeprazole intragastrically to inhibit the acid secretion before infection and then the mice were orally infected with H. pylori SS1 (109 CFUs) using intubation. Four weeks after infection, the infected mice were vaccinated intragastrically with 150 μg of vaccine antigen (CTB-UE) in 300 μl 3 % sodium hydrogen carbonate buffer for four times at 1-week intervals. The infected mice were also immunized with 150 μg of rUreB or PBS using the same method as control. Two weeks after the final immunization, the immunized and control mice were sacrificed for evaluation of H. pylori infection.

Assessment on immunogenicity and specificity of the CTB-UE vaccine

Antigen-specific antibodies were measured by an ELISA assay (Guo et al. 2012a). First, 96-well microplates were coated overnight with native H. pylori urease, rUreA, or rUreB (Linc-Bio, Shanghai, China) and blocked with 5 % (w/v) BSA. After washing three times, the plates were incubated with 100 μl/well of mouse serum at a dilution of 1:5,000 for 1 h. Specific antibodies were detected with HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgA (General Bioscience Corporation, USA). Substrate TMB was then added and incubated at room temperature for 10 min. The absorbance at 450 nm was measured by a microplate reader.

The peptides of B cell epitopes (UreA183–203 and UreB327–334) were synthesized commercially (TASH, Shanghai, China) by the Fmoc solid-phase method. The synthesized peptides were purified and analyzed by reversed-phase HPLC and then the purified peptides were identified by use of a mass spectrometer. The purity of the epitope peptides UreA183–203 and UreB327–334 were 95.27 and 93.22 %, respectively. The peptides were coated overnight on an ELISA plate at 4 °C (10 μg/ml, 100 μl/well) and the subsequent steps of the assay were performed as described above for the indirect ELISA. All assays were performed in triplicate.

H. pylori urease neutralization assay

Mouse IgG in the anti-serum was purified using a Protein A-Sepharose column (BioVision) according to the manufacturer's instructions. After purification, the concentration of antiserum IgG was measured by a BCA Protein Assay Kit (Tiangen, Beijing). Urease neutralization test was performed as previously described (Guo et al. 2012a).

Urease activity determination

The degree of H. pylori colonization in the mouse stomach was measured by the presence of active urease in the stomach tissue as previously described (Gomez-Duarte et al. 1998). The immunized and control mice were sacrificed for determination of urease activity in the stomachs. Briefly, the antral portion of the stomach was immediately placed inside an Eppendorf tube containing 500 μl of the urease substrate containing 500 mM urea, 0.02 % phenol red, and 0.1 mM DTT. The stomach sample was incubated for 4 h at room temperature and the absorbance was measured at 550 nm (A550).

Quantitative culture of H. pylori from the stomach of mice

To determine the bacterial colonization in the stomach, mice were killed and one half of the stomach cut longitudinally was homogenized in 2 ml of PBS with a tissue homogenizer. Serial dilutions of the homogenates were plated on H. pylori selective plates as previously described with some modifications (Zhao et al. 2007; Zhou et al. 2009; Guo et al. 2012b). After 5 to 6 days under microaerophillic conditions, visible colonies were counted based on typical colony morphology of H. pylori. The bacteria were identified further by microscopy, catalase test, oxidase test, and urease test, according to the methods reported by Ferrero et al. (1998).

Histological analysis

One strip of tissue was cut from each stomach and was fixed with formalin and embedded in paraffin. Then, the tissues were stained with hematoxylin and eosin (HE) and analyzed for H. pylori associated inflammation. The slides were “blinded” and the extent of gastritis was graded as described previously (Guo et al. 2012b; Guy et al. 1998).

Determinations of specific antibody after vaccination

To measure serum antibodies after prophylactic and therapeutic vaccination, blood was collected immediately before sacrifice. Serum antibody titers were determined by ELISA against native H. pylori urease (Linc-Bio, Shanghai, China), which were coated overnight at 4 °C. The serum was isolated and diluted 1:1,000 before assay. To assess specific mucosal sIgA production, one fragment from the stomach, intestine, or feces was homogenized in 1 ml PBS containing 2 mM phenylmethylsulfonyl fluoride, 0.05 mM ethylenediaminetetraacetic acid (EDTA), and 0.1 mg/ml of soybean trypsin inhibitor. The supernatant was collected and diluted 1:5 in PBS for analysis of mucosal secretory IgA (sIgA) antibodies. Specific antibodies were detected with HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgA (General Bioscience Corporation, USA). Substrate TMB was then added and incubated at room temperature for 10 min. The absorbance at 450 nm was measured by a microplate reader.

Proliferation of specific T lymphocytes

Lymphocytes were isolated from spleen with lymphocyte separation medium (Dakewe Biotechnology Company, Shenzhen, China), and cultured (2 × 105 cells/well) with the synthetic peptides (UreA74–90, UreB229–244, or UreB237–251) in RPMI-1640 in 96-well round-bottom plates at 37 °C, 5 % CO2 for 72 h. After that, the cells were incubated with 10 μl of the CCK-8 solution (Dojindo Molecular Technologies Inc) to each well of the plate for 4 h. Measure the absorbance at 450 nm using a microplate reader. All experiments were performed in triplicate. The results were expressed as stimulation indices (SI), defined as the index of lymphocyte proliferation according to formula: SI = the A450 value of stimulated cultures / the A450 value of negative control cultures.

Determinations of cytokine production

Lymphocytes were isolated from spleens with lymphocytes separation medium and cultured (2 × 105 cells/well) with native H. pylori urease (0.5 μg/ml) in RPMI-1640 in 96-well flat-bottom plates at 37 °C in 5 % CO2 for 72 h. Splenic lymphocytes culture supernatants were harvested to assay for interleukin-4 (IL-4) and interferon gamma (IFN-γ) and IL-17 using ELISA kits (R&D System, USA) according to the manufacturer's instructions.

Statistical analyses

All the statistical analyses were performed with the GraphPad Prism 5 software. Data were expressed as mean ± standard deviation (S.D.). Statistical significance was tested using Student's t test. p < 0.05 was considered as statistically significant. (*p < 0.05, **p < 0.01, ***p < 0.001; ns: not significant).

Results

Design of multi-epitope vaccine CTB-UE

In order to retain the immunological function of the B and Th cell epitopes from H. pylori urease, we constructed the multiple-epitope CTB-UE with scientific and reasonable structure on the basis of prediction and modeling using RANKPEP, DNASTAR, and molecular operating environment (MOE) software. The protein structure of the multiple-epitope vaccine CTB-UE is shown in Fig. 2. Multiple-epitope peptide (UE) containing tandem copies of the selected B and Th cell epitopes was fused with the C-terminal of the intramucosal adjuvant CTB and a seven-amino-acid, proline-containing segment (DPRVPSS) was used as a spacer at linkage sites between CTB and UE to decrease the interaction between them. The linker among Th cell epitopes was selected as KK, and the linker among B cell epitopes was GS. The linkers (KK and GS) were designed to retain the immunologic competence of each Th or B epitope and avoid the generation of new epitopes at linkage sites between epitopes.

Fig. 2
figure 2

The designed multi-epitope vaccine CTB-UE. The multi-epitope peptide (UE) contains tandem copies of five different epitopes which are Th cell epitopes (UreA74–90, UreB229–244, and UreB237–251) and B cell epitopes (UreA183–203 and UreA183–203) from H. pylori urease. The Th cell epitopes UreB229–244 and UreB237–251 have a certain overlap, so we took UreB229–251 to represent UreB229–244 and UreB237–251 epitopes. In addition, the linkers (DPRVPSS, KK, and GS) were designed to retain the immunologic competence of each Th or B epitope and avoid the generation of new epitopes at linkage sites among epitopes

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Expression, purification, and antigenicity of CTB-UE

The fusion protein CTB-UE was expressed in E. coli BL21 (DE3) and identified by its molecular mass (33 kDa). The results showed that the fusion protein CTB-UE was mainly expressed in inclusion body and had a high level of expression. After purification by Ni2+-charged column chromatography and anion-exchange chromatography, the purity of the fusion protein CTB-UE, analyzed by 12 % SDS-PAGE (Fig. 3a) and computer scan, was 96.8 %. Western blotting was performed to assess the immunogenicity and immunoreactivity of the CTB-UE fusion protein. As shown in Fig. 3b, the antiserum induced by the fusion protein CTB-UE reacted with rUreA, rUreB, and rCTB, while a negative serum did not, which indicates that CTB-UE has good immunogenicity and immunoreactivity.

Fig. 3
figure 3

Expression, purification, and antigenicity of CTB-UE. a Expression and purification of CTB-UE analyzed by 12 % SDS-PAGE. Lane 1 Protein marker, lanes 2 and 3 the inclusion proteins of E. coli BL21 (DE3) expressing CTB-UE (33 kDa), lane 4 the soluble body proteins of E. coli BL21 (DE3) expressing CTB-UE (33 kDa), lane 5 CTB-UE in inclusion bodies washed with phosphate buffer, lane 6 CTB-UE purified by Ni2+-charged column chromatography, lanes 7 and 8 CTB-UE purified by DEAE Sepharose FF chromatography. b Western blotting of recombinant epitope vaccine. The fusion protein CTB-UE, rCTB, rUreA, and rUreB were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The purified CTB-UE was used to immunize mouse to produce antiserum. 1 Mouse anti-CTB-UE serum was used for detecting immunoreactivity to rUreA and rUreB. 2 Normal mouse serum was used as control

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Immunological features of the CTB-UE vaccine

To assess the adjuvant activity of the CTB component of CTB-UE, A GM1-ELISA was performed. We used rCTB that was able to bind GM1 as positive control. As negative control, the purified rUreB that should not be able to bind GM1 was used. The experimental results indicated that CTB-UE and rCTB were able to bind GM1 in a dose-dependent manner (Fig. 4a). In addition to this, their curves presented the same profile. However, the rUreB protein was incapable of binding GM1.

Fig. 4
figure 4

Assessment on immunological features of the CTB-UE vaccine. The mice were immunized with CTB-UE, rUreB, or PBS by intraperitoneal injection. Data are mean ± S.D. p < 0.05 was considered as statistically significant. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ns not significant. a Assessment on the adjuvanticity of CTB component. A GM1-ELISA was performed to evaluate the adjuvanticity of CTB component. The rCTB was used as a positive control. As negative control, the purified rUreB that should not be able to bind GM1 was used. ELISA plates were coated with 1 μg/well of GM1 ganglioside. b Assessment of antigen-specific IgG antibody responses. ELISA plates were coated with 0.5 μg/well of native H. pylori urease, rUreA, rUreB. The sera were used at a dilution of 1:5,000. c Measurement of IgG1 and IgG2a isotypes and IgA antibodies against H. pylori urease. ELISA plates were coated with 0.5 μg/well of native H. pylori urease. The sera were used at a dilution of 1:5,000. d Assessment of peptide-specific antibody response. ELISA plates were coated with 1 μg/well of UreA183–203 or UreB327–334 peptides. The sera were used at a dilution of 1:2,000. e Inhibition of H. pylori urease activity by specific antibodies. The purified native H. pylori urease was preincubated with a serial dilution of IgG from mice immunized with CTB-UE, rUreB, or PBS. The optical density of the mixture was determined at 550 nm by the indicator of phenol red. The data are expressed as percentage inhibition

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The capacity of CTB-UE to induce antigen-specific antibodies against native H. pylori urease, rUreA, or rUreB was evaluated by ELISA. Compared with immunization with PBS, immunization with the CTB-UE vaccine significantly increased the levels of specific IgG against native H. pylori urease, rUreA, or rUreB (Fig. 4b). There was no significant difference between CTB-UE- and rUreB-immunized groups on the level of specific IgG antibodies against native H. pylori urease or rUreB, indicating that CTB-UE had good immunogenicity. Besides, H. pylori urease-specific IgG1, IgG2a, and IgA antibodies in sera were detected by an indirect ELISA. Vaccination with the fusion peptide CTB-UE or rUreB induced higher levels of anti-urease IgG1, IgG2a, and IgA antibodies in BALB/c mice, compared to levels in mice vaccinated with PBS (Fig. 4c). Likewise, there was no significant difference between CTB-UE-immunized mice and rUreB-immunized mice on the level of specific IgG1, IgG2a, or IgA antibodies against native H. pylori urease.

To further examine whether the antibodies induced by CTB-UE can recognized the UreA183–203 and UreB327–334 peptides involved with the active site of urease, the UreA183–203 and UreB327–334 peptides were synthesized. Compared with intraperitoneal immunization with rUreB or PBS, immunization with CTB-UE significantly increased the levels of specific IgG against the UreA183–203 or UreB327–334 peptides (Fig. 4d). However, the rUreB vaccine didn't induce specific antibodies against the UreA183–203 or UreB327–334 peptides.

In order to test the neutralizing effect of antibodies induced by CTB-UE, a urease neutralization assay was performed. The inhibition by anti-CTB-UE polyclonal antibodies or anti-rUreB polyclonal antibodies was dose dependent. Besides, the inhibitory effect on H. pylori urease activity by anti-CTB-UE polyclonal antibodies is better than that by anti-rUreB polyclonal antibodies (Fig. 4e). However, antiserum IgG from mice immunized with PBS didn't inhibit the enzymatic activity of H. pylori urease. This result indicated that the antibodies induced by CTB-UE have neutralizing ability.

Assessment on prophylactic effect of CTB-UE

The prophylactic effect of CTB-UE was assessed by urease tests, quantitation of viable bacteria colonies from mice stomachs, and histopathologic assessment of gastritis. The experimental results of the urease test (Fig. 5a), quantitation of culturable H. pylori (Fig. 5b), and gastritis scores (Fig. 5c) showed significant differences between the groups vaccinated with 150 or 100 μg of CTB-UE and PBS, but no differences between the groups vaccinated with 50 μg of CTB-UE and PBS control. Compared with oral immunization with 150 μg of rUreB, oral immunization with 150 μg of CTB-UE showed a better preventive effect. Typical histological findings of gastric mucosa for control and mice immunized with 150 μg of CTB-UE are shown in Fig. 5d. In control mice immunized with PBS, a lot of inflammatory cell infiltrations were observed on the surface and in the gastric mucosa. In mice prophylactically vaccinated with CTB-UE, however, much less or very few inflammatory cell infiltration was detected.

Fig. 5
figure 5

Evaluation of H. pylori infection after prophylactic vaccination. The mice were orally immunized with CTB-UE, rUreB, or PBS and infected with H. pylori. Data are mean ± S.D. p < 0.05 was considered as statistically significant. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ns not significant. a Urease activity in the stomach after oral prophylactic immunization. b Colonization of H. pylori in the stomach after oral prophylactic immunization. The number of bacteria (CFU) per stomach was determined for individual mice in each group by quantitative culture. c Assessment on prophylactic efficacy of the epitope vaccine CTB-UE based on gastritis scores. d Gastric histology in BALB/c mice post-challenge with H. pylori SS1 after prophylactic immunization. A decrease of the gastritis was observed in all of the vaccinated mice after prophylactic vaccination with CTB-UE or rUreB. A typical illustration of HE-stained sections from mice after prophylactic vaccination with CTB-UE shows mild inflammatory infiltrates. But mice vaccinated with PBS showing severe inflammatory infiltrates in the mucosa and submucosa. (HE stain, ×100)

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Assessment on therapeutic effect of rCtUBE

The therapeutic effect of the epitope vaccine CTB-UE was assessed by urease tests, quantitation of viable bacteria colonies from mice stomachs, and histopathologic assessment of gastritis. In the therapeutic experiment, the results of the urease test (Fig. 6a), quantitation of culturable H. pylori (Fig. 6b), and gastritis scores (Fig. 6c) showed significant differences between the groups vaccinated with 150 μg of CTB-UE and PBS. Besides, oral immunization with 150 μg of CTB-UE showed a better therapeutic effect, compared with oral immunization with 150 μg of rUreB. Typical histological findings of gastric mucosa for control and immunized mice with 150 μg of CTB-UE are shown in Fig. 6d. In control mice immunized with PBS, a lot of inflammatory cell infiltrations were observed in the mucosa and submucosa. But mice vaccinated with the epitope vaccine CTB-UE showed mild inflammatory infiltrates.

Fig. 6
figure 6

Evaluation of H. pylori infection after therapeutic vaccination. The mice were infected with H. pylori and orally immunized with CTB-UE, rUreB, or PBS. Data are mean ± S.D. p < 0.05 was considered as statistically significant. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ns not significant. a Urease activity in the stomach after oral therapeutic immunization. b Colonization of H. pylori in the stomach after oral therapeutic immunization. The number of bacteria (CFU) per stomach was determined for individual mice in each group by quantitative culture. c Assessment on therapeutic efficacy of the epitope vaccine CTB-UE based on gastritis scores. d Gastric histology in infected BALB/c mice 4 weeks after therapeutic immunization. A decrease of the gastritis was observed in all of the vaccinated mice after therapeutic vaccination with CTB-UE or rUreB. A typical illustration of HE-stained sections from mice after therapeutic vaccination with CTB-UE shows mild inflammatory infiltrates. But mice vaccinated with PBS show severe inflammatory infiltrates in the mucosa and submucosa. (HE stain, ×100)

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Evaluation of antibodies after prophylactic and therapeutic vaccination

The capacity of CTB-UE to induce serum IgG and IgA antibodies was evaluated by ELISA. Levels of the IgG subtypes IgG1 and IgG2awere further analyzed. A modest antibody level was observed in sera from prophylactically and therapeutically vaccinated mice. Compared with oral immunization with PBS, oral immunization with 150 μg of CTB-UE or rUreB significantly increased the levels of specific IgG, IgA, IgG1, and IgG2a against native H. pylori urease in the prophylactic and therapeutic vaccination experiments (Fig. 7a, b, Fig. 8a and b). Besides, there was no significant difference between CTB-UE- and rUreB-immunized groups on the level of specific IgG, IgA, IgG1, and IgG2a antibodies against native H. pylori urease, indicating that CTB-UE had good immunogenicity.

Fig. 7
figure 7

Detection of specific antibodies and cytokines after prophylactic vaccine. Data are expressed as mean ± S.D. p < 0.05 was considered as statistically significant. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ns not significant. a Measurement of serum IgG and IgA antibodies against H. pylori urease by ELISA. The sera were used at a dilution of 1:1,000. b Measurement of IgG1 and IgG2a isotypes against H. pylori urease by ELISA. The sera were used at a dilution of 1:1,000. c Evaluation of mucosal IgA antibodies against H. pylori urease. The supernatants of homogenized stomach, intestine, or feces were collected for detection of the levels of mucosal sIgA against H. pylori urease in prophylactically vaccinated mice. d Assessment on proliferation of specific lymphocytes. SI represents the ratio between the proliferation rates of cells stimulated with stimulator and with medium alone. Splenic lymphocytes from mice immunized with CTB-UE, rUreB, or PBS were stimulated with the UreA74–90 (5 μg/ml), UreB229–244 (5 μg/ml), or UreB237–251 (5 μg/ml) peptides, and proliferation was detected by Cell Counting Kit-8. e The concentrations of cytokines in the supernatants of lymphocytes cultures. The splenic lymphocytes from mice immunized with CTB-UE, rUreB, or PBS were stimulated with native H. pylori urease for 72 h, and the supernatants were collected and cytokine production was determined by ELISA

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Fig. 8
figure 8

Detection of specific antibodies and cytokines after therapeutic vaccine. Data are expressed as mean ± S.D. p < 0.05 was considered as statistically significant. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ns not significant. a Measurement of serum IgG and IgA antibodies against H. pylori urease by ELISA. The sera were used at a dilution of 1:1,000. b Measurement of IgG1 and IgG2a isotypes against H. pylori urease by ELISA. The sera were used at a dilution of 1:1,000. c Evaluation of mucosal sIgA antibodies against H. pylori urease. The supernatants of homogenized stomach, intestine, or feces were collected for detecting the levels of mucosal sIgA against H. pylori urease in therapeutically vaccinated mice. d Assessment on proliferation of specific lymphocytes. SI represents the ratio between the proliferation rates of cells stimulated with stimulator and with medium alone. Splenic lymphocytes from mice immunized with CTB-UE, rUreB, or PBS were stimulated with the UreA74–90 (5 μg/ml), UreB229–244 (5 μg/ml), or UreB237–251 (5 μg/ml) peptides, and proliferation was detected by Cell Counting Kit-8. e The concentrations of cytokines in the supernatants of lymphocytes cultures. The splenic lymphocytes from mice immunized with CTB-UE, rUreB, or PBS were stimulated with native H. pylori urease for 72 h, and the supernatants were collected and cytokine production was determined by ELISA

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Mucosal secretory IgA (sIgA) production in gastric tissue, intestine mucus, and feces was also tested. A modest level of sIgA was detected in the extracts from H. pylori-infected mice. Compared with oral immunization with PBS, oral prophylactic (Fig. 7c) or therapeutic (Fig. 8c) immunization wit CTB-UE or rUreB markedly elevated the level of specific mucosal sIgA in gastric tissue, intestine mucus, and feces. Besides, there was no significant difference in the levels of specific sIgA antibody between CTB-UE-immunized mice and rUreB-immunized mice.

Evaluation of Th epitope-specific lymphocyte responses

To test whether the specific lymphocyte responses are due to Th epitopes in CTB-UE, splenic lymphocytes from mice orally immunized with CTB-UE, rUreB, or PBS were cultured with synthetic peptides of the Th epitopes (UreA74–90, UreB229–244, or UreB237–251). As shown in (Fig. 7d and Fig. 8d), splenic lymphocytes from mice immunized with CTB-UE proliferated significantly after stimulation with UreA74–90, UreB229–244, or UreB237–251 peptides compared with those from PBS-immunized mice. Besides, splenic lymphocytes from mice immunized with rUreB displayed significantly high proliferation after stimulation with UreB229–244 or UreB237–251 peptides except UreA74–90 peptides. The results show that CTB-UE stimulates Th epitope-specific lymphocyte responses.

Determination of the production of cytokines

The production of IFN-γ, IL-4 and IL17 in the supernatants of splenic lymphocytes cultures was measured by ELISA. Stimulation of splenic lymphocytes from mice after vaccination with CTB-UE or rUreB resulted in significantly higher levels of the IFN-γ, IL-4, and IL17 than stimulation of cells from PBS-immunized mice (Fig. 7e and Fig. 8e). The level of IL-4 or IFN-γ was significantly higher than the level of IL-17 in CTB-UE-immunized mice. In addition, there was no significant difference in the levels of the IFN-γ, IL-4, and IL-17 between CTB-UE-immunized mice and rUreB-immunized mice.

Discussion

H. pylori urease B subunit is an important target for prophylactic and therapeutic vaccine development for its outstanding features. Several prophylactic or therapeutic vaccines based on H. pylori urease B subunit have been developed, being mainly recombinant subunit vaccine (Corthesy et al. 2005; Begue and Moll 2009; Vermoote et al. 2013), but no major breakthrough has been achieved. It has been reported that some MAbs against H. pylori urease A subunit could strongly suppress the enzymatic activity of the urease (Fujii et al. 2004; Hifumi et al. 2006). However, urease-specific polyclonal antibodies generated by immunization with purified H. pylori urease did not inhibit its enzymatic activity at all (Hirota et al. 2001). Besides, a pivotal role of CD4+ T cells in protective immunity against H. pylori has been widely accepted (Ermak et al. 1998). Consequently, we think that an epitope vaccine which can induce specific neutralizing antibodies and cellular immune response against both urease A and B subunits may be a potential candidate for controlling H. pylori infection.

In this study, a multi-epitope vaccine CTB-UE with the intramucosal adjuvant CTB and tandem copies of Th and B cell epitopes from both urease A and B subunits was constructed. Our results showed that CTB-UE could induce comparatively high levels of specific antibodies against native H. pylori urease, UreA, UreB, and the selected B cell epitopes UreA183–203 and UreB327–334 involved with the active site of urease and showed effectively inhibitory effect on the enzymatic activity of H. pylori urease. Besides, oral prophylactic or therapeutic immunization with CTB-UE significantly decreased H. pylori colonization compared with oral immunization with rUreB or PBS, and the protection was correlated with antigen-specific CD4+ T cells and IgG, IgA, and mucosal sIgA antibody responses.

The design of an epitope vaccine is very important, and a number of factors have been shown to influence its overall success at inducing an immune response against the desired peptide sequence (Liu et al. 2004). The linkers (DPRVPSS, KK, and GS) were designed to retain the immunologic competence of each epitope and avoid the generation of new epitopes at linkage sites. The lymphocyte proliferation results showed that splenic lymphocytes from mice immunized with CTB-UE proliferated significantly after stimulation with UreA74–90, UreB229–244, or UreB237–251, and the antibody induced by CTB-UE could recognize the UreA183–203 and UreB327–334 peptides and neutralized urease activity, showing that Th and B cell epitopes all retained their functions. CTB and the multiple copies of epitopes have been shown to be critical factors influencing the magnitude of epitope-specific responses (Liu et al. 2004; Kovacs-Nolan and Mine 2006). The results of indirect ELISA and GM1-ELISA showed that CTB-UE displayed good immunogenicity, and CTB retained the ability to bind to GM1. These results indicate that prediction of the combination order in the epitope vaccine by RANKPEP, DNASTAR, and MOE software is successful.

Despite extensive research on vaccine-induced protection in mice, there are still unanswered questions with regard to the contribution of different effector cells and molecules involved in protective immunity to H pylori. Some studies support the view that a humoral immune response is important for clearing H. pylori (Nystrom and Svennerholm 2007; Lee et al. 1995; Ferrero et al. 1997). We consider that neutralization of urease activity can break down the microenvironment colonized by H. pylori, and the inhibition of bacterial adhesion may contribute to clearance of H. pylori. Therefore, we selected two B cell epitopes UreA183–203 and UreB327–334 involved with the active site of urease for constructing the vaccine CTB-UE. The inhibitory effect on H. pylori urease activity by anti-CTB-UE polyclonal antibodies is better than that by anti-rUreB polyclonal antibodies (Fig. 4e), probably reflecting the contributions of the two B cell epitopes. In addition, there was no significant difference between CTB-UE- and rUreB-immunized groups on the level of specific IgG, IgA, sIgA, IgG1, and IgG2a antibodies against native H. pylori urease after prophylactic and therapeutic vaccination (Fig. 7a, b, c, Fig. 8a, b and c). However, oral immunization with 150 μg of CTB-UE showed a better preventive and therapeutic effect compared with oral immunization with 150 μg of rUreB (Figs. 5 and 6), indicating that the reduction of H. pylori colonization is also associated with the specific humoral immune responses, especially neutralizing antibody response. There might be two types of inhibition mechanisms against H. pylori urease activity; one assumed to suppress the urease activity via an allosteric effect generated by the specific antibodies specific for the UreA183–203 fragment within UreA, which might cause a distortion of the conformation of the urease, and thus affect enzyme activity. The other mechanism involves the antibodies specific for the UreB327–334 fragment located at the active site of urease, resulting in the reduction of urease activity.

Whether Th1, Th2, or Th17 responses contribute to protective immunity remains controversial (Mohammadi et al. 1997; DeLyria et al. 2009; Sayi et al. 2009; DeLyria et al. 2011). We believe that there are multiple mechanisms for activating vaccine-based protective immunity against H. pylori due to the complexity and complementarity of the immune system network. In this study, we selected three Th epitopes from UreA and UreB to construct the epitope vaccine CTB-UE. The lymphocyte cell proliferation assay showed that three of the Th epitopes could stimulate high proliferative responses, and analysis of the cytokines showed that IFN-γ, IL-4, and IL-17 were all significantly induced by CTB-UE and the level of IL-4 or IFN-γ was significantly higher than the level of IL-17 in CTB-UE immunized mice. These results indicate that CTB-UE induced amixed Th1-Th2 immune response, which might contribute to the dramatic reduction in the bacterial load in the stomachs of H. pylori-infected mice. Besides, oral immunization with CTB-UE significantly increased the levels of specific IgG1 and IgG2a against native H. pylori urease in the prophylactic and therapeutic vaccination experiments compared with oral immunization with PBS. For mice, it is generally accepted that the IgG1 response reflects helper activity of Th2 CD4+ T cells, where IgG2a results from Th1 activity (Abbas et al. 1996). Therefore, it was also speculated that CTB-UE induced a mixed Th1–Th2 response. Therefore, we think the immune responses induced by the CTB-UE differ from those induced by natural H. pylori infection, since natural infection with H. pylori polarizes to Th1-type responses, whereas our vaccine induced a mixed Th1–Th2 response.

In conclusion, we have designed and constructed an epitope vaccine CTB-UE against urease A and B subunits, and oral prophylactic or therapeutic immunization with it significantly reduced H. pylori colonization in the stomachs of infected mice. The prophylactic and therapeutic immune protection of CTB-UE is possibly mediated by specific serum IgG, IgA and mucosal sIgA antibodies, and a mixed Th1–Th2 cells response. Therefore, CTB-UE is worth investigating as a novel and promising approach in the development of an oral vaccine against H. pylori. Ongoing studies will evaluate the efficacies of CTB-UE with other adjuvants, vaccine carriers such as lactic acid bacteria and Bacille Calmette Guerin (BCG), and different immunization routes. We will also further evaluate the prophylactic and therapeutic effect of CTB-UE in Mongolian gerbil. The data from mouse models will provide much information for the further development of prophylactic or therapeutic vaccines against H. pylori and should lead to studies of the epitope-based vaccine for human use.