Immunological features and efficacy of the reconstructed epitope vaccine CtUBE against Helicobacter pylori infection in BALB/c mice model

Abstract

Urease is an essential virulence factor and colonization factor for Helicobacter pylori, of which the urease B subunit (UreB) is considered as an excellent vaccine candidate antigen. In previous study, an epitope vaccine with cholera toxin B subunit (CTB) and an epitope (UreB321–339) named CtUBE was constructed and the mice were protected significantly after intragastric vaccination with the CtUBE liposome vaccine. However, the fusion protein CtUBE was expressed as inclusion bodies and was difficultly purified. Besides, the immunogenicity and specificity of the CtUBE vaccine was not investigated in a fairly wide and detailed way. In this study, the fusion peptide CtUBE was reconstructed and expressed as a soluble protein with pectinase signal peptide at the N terminus and the 6-his tag at its C-terminal, and then the immunogenicity, specificity, prophylactic, and therapeutic efficacy of the reconstructed CtUBE (rCtUBE) vaccine were evaluated in BALB/c mice model after purification. The experimental results indicated that mice immunized with rCtUBE could produce comparatively high level of specific antibodies which could respond to natural H. pylori urease, UreB, or the minimal epitope UreB327–334 involved with the active site of urease, and showed effectively inhibitory effect on the enzymatic activity of urease. Besides, oral prophylactic or therapeutic immunization with rCtUBE significantly decreased H. pylori colonization compared with oral immunization with rCTB or PBS, and the protection was correlated with antigen-specific IgG, IgA, and mucosal sIgA antibody responses, and a Th2 cells response. This rCtUBE vaccine may be a promising vaccine candidate for the control of H. pylori infection.

Introduction

Helicobacter pylori is a helical Gram-negative bacillus, which was originally discovered by Marshall and Warren (1984) and continues to infect more than half of the world’s population. The prevalence of infection ranges from 20 % in the developed countries to >90 % in the developing world (Graham et al. 1991). H. pylori has been recognized as the major aetiological determinant of various gastroduodenal diseases, such as chronic gastritis, peptic ulcers, and gastric cancer (Hirai et al. 1999; Khan and Bemana 2012; Tsimmerman 2009). H. pylori infections cause very high morbidity and mortality, and they impose a major burden upon health care systems worldwide. Current therapies are based on a combination of three or four different antibiotics together with a proton-pump inhibitor. The high incidence of gastric cancer, high re-infection rates, antibiotic resistance, and the poor patient compliance and relatively high cost of antibiotic treatment make vaccination an attractive strategy (Cheng and Hu 2005; Sack and Gyr 1994), either as an alternative or a complementary to antibiotic treatment in developing countries. The development of an effective prophylactic or therapeutic vaccine will be of enormous scientific value and economic benefits.

H. pylori can produce large amounts of urease that catalyzes the hydrolysis of urea. The active H. pylori urease enzyme consists of a polymeric structure that comprises two subunits, UreA and UreB. Urease can neutralize stomach acid by generating ammonia from urea, which is essential for the survival of H. pylori in the host. Besides, urease is considered an important pathogenic factor. Much evidence shows that ammonia generated by urease can cause local injury to gastroduodenal mucus tissue, especially at intracellular junctions (Eaton et al. 1991; Marshall et al. 1990). Therefore, urease is considered a prime candidate for inclusion in a vaccine formulation against H. pylori. Nonetheless, H. pylori urease-specific polyclonal IgG antibodies generated by immunization with purified urease protein did not inhibit its enzymatic activity at all and further abolished the inhibitory effect of some monoclonal antibodies (MAbs) against H. pylori urease (Hirota et al. 2001; Nagata et al. 1992). This result suggested that there might be two types of urease-specific antibodies; one may help to promote its enzymatic activity and aggravate the gastric disorder, and the other may be beneficial in inhibiting its enzymatic activity and preventing bacterial attachment to the gastric mucosa. Besides, it has recently been reported that poor response to the urease may favor persistence of H. pylori infection (Nagata et al. 1992; Zhao et al. 2007). Therefore it may be favorable to prevent or treat H. pylori-related diseases by epitope vaccine composed of carefully chosen urease epitope which can induce neutralizing antibody.

Epitope vaccine based on H. pylori urease is a new strategy for prophylactic and therapeutic vaccination against H. pylori infection, avoiding some crucial drawbacks to the current procedures for vaccine preparation, such as the difficulty of the in vitro culturing of H. pylori, the loss of efficacy due to the genetic variation of the pathogen, as well as the side effects of other unfavorable epitopes in the complete antigen (Sette et al. 2002; Zhou et al. 2009). It has been reported that a mouse monoclonal antibody (mAb), named L2, showed the strong inhibitory effect on the urease activity of H. pylori (Nagata et al. 1992). Further research indicated that the epitope recognized by L2 was involved in the active site of the urease and was a stretch of UreB-derived 19 amino acid residues (UB-33, UreB321–339). The minimal epitope as 8-amino acid residues (F8, UreB327–334) for L2 reactivity was determined by further sequential amino acid deletion of the UreB321–339 from either end (Hirota et al. 2001). In our previous study, we had demonstrated that the mice were protected significantly after intragastric vaccination with the CtUBE epitope vaccine composed of CTB and UreB321–339 (Zhao et al. 2007). In this study, the fusion peptide CtUBE was reconstructed and expressed as an active pentameric form in Escherichia coli and its immunogenicity, specificity, prophylactic, and therapeutic efficacy were evaluated in BALB/c mice model in a fairly wide and detailed way.

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, first developed by Lee et al. (Lee et al. 1997), was obtained from the National Center for Disease Control and Prevention and then preserved in our laboratory (collection number, CPU-BS-09). H. pylori SS1 were cultured on brain–heart infusion (BHI) plates containing 7 % goat blood, trimethoprim (5 μg/ml), polymixin B (5 μg/ml), and vancomycin (10 μg/ml) under microaerophilic conditions at 37 °C for 3–4 days. The bacteria were harvested and re-suspended in BHI and the final concentration was adjusted to 2 × 109 colony forming units (CFU)/ml before inoculation.

Construction of secretory expression vector pETCUB

The fusion gene CtUBE (390 bp) was amplified from the recombinant plasmid pETCtUBE (Zhao et al. 2007) by PCR using a pair of oligonucleotide primers with unique Nco I and Xho I restriction sites, respectively: the forward primer P1-CtUBE (5′-CATGCCATGGGCACACCTCAAAATATTACTGATTTGTGT-3′) and the reverse primer P2-CtUBE (5′-CCGCTCGAGGATCCTTGAATCAGCGAACTGAAC-3). After amplification, the CtUBE fragment was gel-purified using the Universal DNA Purification Kit (TianGen Biotech, Beijing, China), as described by the manufacturer. The PCR fragment was cloned into the Nco I and Xho I restriction sites of E. coli expression plasmid pET-22b, and the secretory expression vector pETCUB encoding the fusion protein rCtUBE with pectinase signal peptide at the N terminus and the 6-his tag at its C-terminal was obtained. The plasmid pETCUB was analyzed by PCR, restriction digest, and sequencing after it was transformed into E. coli DH5α.

Secretory expression and purification of rCtUBE

The fusion protein rCtUBE was expressed and purified according to the protocol performed as previously described (Guo et al. 2012a) with some modification. The secretory expression vectors pETCUB were transformed into E. coli BL21 (DE3) competent cells. The successful transformants were cultured and used to express the fusion protein rCtUBE. Firstly, the rCtUBE protein was purified by Ni2+-charged column chromatography (Bio Basic Inc, Markham, Canada) according to the recommendation of the manufacturer. Secondly, the fusion proteins were then purified by anion-exchange chromatography using DEAE Sepharose FF (Amersham Pharmacia Biotech AB, Sweden) in binding buffer and eluted with elution buffer. After purification, the purity of the fusion peptide rCtUBE 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.

GM1 ganglioside enzyme-linked immunosorbent assay

The adjuvant activity of the CTB component of rCtUBE was assessed by GM1 ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The experimental procedure was adapted from two reference published elsewhere (Areas et al. 2004; Guo et al. 2012a). Briefly, ELISA plates (Corning Costar Corp, MA, USA) were coated with either 1 μg/well of GM1 ganglioside (Sigma, St. Louis, USA) or with 1 μg/well of bovine serum albumin (BSA) in 0.05 M carbonate–bicarbonate buffer (pH 9.6) at 4 °C overnight. The wells were subsequently washed three times, and 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 in PBST, 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, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch, USA) was added, and the plate was incubated at 37 °C for 1 h. The color reaction based on tetramethylbenzidine (TMB, TianGen Biotech, Beijing, China) was terminated after incubation for 10 min at room temperature by the addition of 50 μl of H2SO4 (2 M), and the absorbance at 450 nm was measured by microplate reader.

Immunization and infection

An experimental procedure to study the immunogenicity and specificity of the rCtUBE vaccine was performed as previously described with some modifications (Guo et al. 2012a). BALB/c mice were randomized into three groups (six mice in each group) and were respectively immunized with 100 μg of the purified rCtUBE, rCTB, and PBS by intraperitoneal immunization in presence of Freund’s adjuvant. The fusion proteins in PBS were emulsified with an equal volume of Freund’s Complete Adjuvant (Sigma, St. Louis, USA) for the first vaccination and with incomplete Freund adjuvant (Sigma, St. Louis, USA) for the second and third vaccinations. The fusion proteins in PBS without adjuvant were for the last booster vaccination. Antisera were separated on the fifth day after the last booster.

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). 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 vaccine antigen (rCtUBE) in 300 μl 3 % sodium hydrogen carbonate buffer for four times at 1-week intervals. The mice were also immunized with rCTB 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 3 weeks before being used to evaluate H. pylori infection.

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). 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 (rCtUBE) in 300 μl 3 % sodium hydrogen carbonate buffer for four times at 1-week intervals. The infected mice were also immunized with rCTB 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 rCtUBE vaccine

The epitope peptides of SIKEDVQF (UreB327–334) were synthesized commercially (TASH, Shanghai, China) by the Fmoc solid-phase method. The synthesized peptides were purified and analyzed by reverse-phase HPLC, and then the purified peptides were identified by use of a mass spectrometer. The purity of the epitope peptide was 93.22 %. Antigen-specific antibodies and epitope peptide-specific antibodies were measured by an ELISA assay. The protocol was performed as previously described (Guo et al. 2012a; Kovacs-Nolan and Mine 2006). Briefly, ELISA plates were coated with 0.5 μg/well of natural H. pylori urease (Linc-Bio, Shanghai, China), rUreA, rUreB, or 1 μg/well of UreB327–334 peptides at 4 °C overnight. The plates were washed with PBST, and blocked with 5 % (w/v) BSA in PBS. The plates were then washed with PBST, and incubated with 100 μl of mouse or rat sera, serially diluted in PBS at 37 °C for 1 h. After washing, HRP-conjugated goat anti-mouse IgG (General Bioscience Corporation, USA) was added, and the plates were incubated again for 1 h. The color reaction based on TMB was terminated after incubation for 10 min at room temperature by the addition of 50 μl of H2SO4 (2 M), and the absorbance at 450 nm was measured by microplate reader. Serum samples from mice and rats were assayed in triplicate.

Urease inhibition assay by fusion protein-specific polyclonal antibody

The urease inhibition assay protocol was performed as previously described (Nagata et al. 1992; Qiu et al. 2010). Briefly, the purified natural H. pylori urease (2 μg in 50 μl) was pre-incubated with 50 μl of serial dilutions of antiserum from different groups in 96-well microtiter plates overnight at 4 °C. After pre-incubation, 100 μl of the above reaction solution was mixed with 100 μl of 50 mM phosphate buffer (pH 6.8) containing 500 mM urea, 0.02 % phenol red, and 0.1 mM dithiothreitol (DTT). The plates were incubated at 37 °C. Color development was measured at 550 nm at 30-min intervals over a period of 3 h. Percentage inhibition was determined by the following equation: [(activity without antiserum – activity with antiserum)/(activity without antiserum)] × 100.

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

The experimental procedure was performed as previously described with some modifications (Zhao et al. 2007; Zhou et al. 2009; Guo et al. 2012b). Briefly, the immunized and control mice were sacrificed for determination of H. pylori load in the stomachs. Half of the stomach was cut longitudinally and was homogenized in 2 ml of PBS with a tissue homogenizer. Serial dilutions of the homogenates were plated on BHI plate containing goat blood and antibiotics as described above. After 5–6 days culture on brain–heart infusion plates containing 7 % goat blood and the foregoing antibiotics under microaerophilic conditions, colonies were counted and the number of CFU per stomach was calculated. 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. For evaluation of gastritis, HE-stained sections were scored based on the degree of infiltrating lymphocytes, plasma cells, and neutrophils (Guy et al. 1998). The scoring grades were defined as follows: 0, none; 1, a few leukocytes scattered in the deep mucosa; 2, moderate numbers of leukocytes in the deep to mid mucosa and occasional neutrophils in the gastric glands (microabscesses); 3, dense infiltrates in the deep to mid mucosa, a few microabscesses; and 4, dense, diffuse infiltrates throughout the lamina propria and into the submucosa, frequent microabscesses.

Determinations of specific antibody after vaccination

Specific antibodies against H. pylori urease were measured by an ELISA assay as previously described with some modifications (Zhao et al. 2007; Zhou et al. 2009; Guo et al. 2012b). The serum was isolated and diluted 1:1,000 in PBS 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 M ethylenediamine tetraacetic acid, 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. Briefly, ELISA plates were coated with 0.5 μg/well of natural H. pylori urease at 4 °C overnight. The plates were washed with PBST, and blocked with 5 % (w/v) BSA in PBS. The plates were then incubated with 100 μl of mouse sera or the supernatant samples, serially diluted in PBS at 37 °C for 1 h. After washing, HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgA (General Bioscience Corporation, USA) was added, and the plates were incubated again for 1 h. The color reaction based on TMB was terminated after incubation for 10 min at room temperature by the addition of 50 μl of H2SO4 (2 M), and the absorbance at 450 nm was measured by microplate reader.

Determinations of cytokine production

Lymphocytes were isolated from spleens with lymphocytes separation medium (Dakewe, Shenzhen, China) and cultured (2 × 105 cells/well) with rCtUBE (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), 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 (SD). Statistical significance was tested using Student’s paired t test. p < 0.05 was considered as statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001; ns: not significant).

Results

Construction, expression, and purification of rCtUBE

The secretory expression vector pETCUB was constructed successfully by inserting the coding sequence of fusion peptide CtUBE into downstream of the pectinase signal peptide of E. coli expression vector pET-22b (Fig. 1a). The fusion protein rCtUBE were expressed in E. coli BL21 (DE3) strains and purified through a Ni2+-charged column chromatography and anion-exchange chromatography. The results showed that the fusion protein rCtUBE was expressed in soluble form (Fig. 1b) and had a moderate level of expression (about 17.4 % of total bacterial protein). After purification, the purity of the fusion protein rCtUBE, analyzed by 12 % SDS-PAGE (Fig. 1b) and computer scan, was 95.3 %.

Fig. 1
figure1

Construction, expression, and purification of rCtUBE. a The secretory expression cassette of fusion peptide rCtUBE. The rCtUBE secretory expression cassette was composed of pectinase signal peptide, CTB, the UreB321–339 epitope and 6-his tag. b The soluble expression and purification of rCtUBE analyzed by SDS-PAGE. Lane 1 the whole bacterial proteins of E. coli BL21 (DE3) expressing rCtUBE by the addition of IPTG (16 kDa). Lane 2 the soluble proteins of E. coli BL21 (DE3) expressing rCtUBE. Lane 3 rCtUBE (5 μg) purified by Ni2+-charged column chromatography; lanes 4 and 5 rCtUBE (2 μg) purified by DEAE Sepharose FF chromatography

Immunological features of the rCtUBE vaccine

A GM1-ELISA was performed to the evaluate adjuvant activity of the CTB component of rCtUBE. We used rCTB that was able to bind GM1 as positive control. As negative control, rCtUBE and rCTB were evaluated using BSA as the coating protein. rCtUBE and rCTB were able to bind GM1 in a dose-dependent manner (Fig. 2a). In addition to this, their curves presented the same profile.

Fig. 2
figure2

Assessment on immunological features of the rCtUBE vaccine. The mice were immunized with rCtUBE, rCTB, or PBS by intraperitoneal injection. Data are mean ± SD; 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. The ELISA was performed by coating a 96-well plate with GM1 or BSA. b Assessment on immunogenicity and immunologic specificity of rCtUBE. ELISA plates were coated with 0.5 μg/well of natural H. pylori urease, rUreA, rUreB, or 1 μg/well of UreB237–334 peptides. The sera were used at a dilution of 1:1,500. c Measurement of IgG1 and IgG2a isotypes and IgA antibodies against H. pylori urease. ELISA plates were coated with 0.5 μg/well of natural H. pylori urease. The sera were used at a dilution of 1:1,500. d Inhibition of H. pylori urease activity by specific antibodies. The purified natural H. pylori urease was pre-incubated with a serial dilution of IgG from mice immunized with rCtUBE or rCTB. The optical density of the mixture was determined at 550 nm by the indicator of phenol red. The data are expressed as percentage inhibition

The capacity of rCtUBE to induce specific antibodies against H. pylori urease, UreA, UreB, or UreB327–334 peptide was evaluated by ELISA. A modest antibody level was observed in sera from mice immunized with the fusion protein rCtUBE. Compared with intraperitoneal immunization with rCTB or PBS, immunization with CtUBE significantly increased the levels of specific IgG (p < 0.001) against natural H. pylori urease, rUreB, or UreB327–334 peptides (Fig. 2b). Besides, H. pylori urease specific IgG1, IgG2a, and IgA antibodies in sera were detected by an indirect ELISA. Vaccination with the fusion peptide rCtUBE or rUreB induced higher levels of anti-urease IgG1, IgG2a, and IgA antibodies in BALB/c mice, compared to levels in mice vaccinated with the rCTB or PBS (Fig. 2c).

In order to test the effect of antibodies induced by rCtUBE, a urease neutralization assay was performed. The purified natural H. pylori urease was pre-incubated with a serial dilution of IgG from mice immunized with rCtUBE or rCTB and the inhibitory effect of the antibodies on the enzymatic activity of H. pylori urease was assayed. The inhibition by anti-rCtUBE polyclonal antibodies was dose-dependent. However, anti-rCTB polyclonal antibodies did not inhibit the enzymatic activity of H. pylori urease obviously (Fig. 2d). This result indicated that the antibodies induced by rCtUBE have neutralization activity.

Assessment on prophylactic effect of rCtUBE

The prophylactic effect of the rCtUBE epitope vaccine was assessed by urease tests, quantitation of viable bacteria colonies from mice stomachs and histopathologic assessment of gastritis. In the prophylactic experiment, the results of the urease test (Fig. 3a), quantitation of culturable H. pylori (Fig. 3b) and gastritis scores (Fig. 3c) showed significant differences between the groups vaccinated with 150 μg of rCtUBE and PBS, but no differences between the groups vaccinated with 50 μg of rCtUBE and PBS control.

Fig. 3
figure3

Evaluation of H. pylori infection after prophylactic vaccination. The mice were orally immunized with rCtUBE, rCTB, or PBS and infected with H. pylori. Data are mean ± SD; 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 rCtUBE based on gastritis scores. d Gastric histology in BALB/c mice post-challenge with H. pylori SS1 after prophylactic immunization (HE stain, ×100). A decrease of the gastritis was observed in all of the vaccinated mice after prophylactic vaccination. A typical illustration of HE-stained sections from mice after prophylactic vaccination shows mild inflammatory infiltrates. But mice vaccinated with rCTB or PBS showing severe inflammatory infiltrates in the mucosa and submucosa

There is significant difference between the groups vaccinated with 100 μg of rCtUBE and PBS in urease activity, but no significant difference in quantitative culture of H. pylori and gastritis score. Typical histological findings of gastric mucosa for control and mice immunized with 150 μg of rCtUBE are shown in Fig. 3d. In control mice immunized with rCTB or PBS, a lot of inflammatory cell infiltrations were observed on the surface and in the gastric mucosa. In mice prophylactically vaccinated with rCtUBE, however, much less or very few inflammatory cell infiltration was detected.

Assessment on therapeutic effect of rCtUBE

The therapeutic effect of the epitope vaccine rCtUBE 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. 4a), quantitation of culturable H. pylori (Fig. 4b), and gastritis scores (Fig. 4c) showed significant differences between the groups vaccinated with 150 μg of rCtUBE and PBS, but no differences between the groups vaccinated with 100 or 50 μg of rCtUBE and PBS control. Typical histological findings of gastric mucosa for control and immunized mice with 150 μg of rCtUBE are shown in Fig. 4d. In control mice immunized with rCTB or PBS, a lot of inflammatory cell infiltrations were observed in the mucosa and submucosa. But mice vaccinated with the epitope vaccine rCtUBE showed mild inflammatory infiltrates.

Fig. 4
figure4

Evaluation of H. pylori infection after therapeutic vaccination. The mice were infected with H. pylori and orally immunized with rCtUBE, rCTB, or PBS. Data are mean ± SD; 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 rCtUBE based on gastritis scores. d Gastric histology in infected BALB/c mice 4 weeks after therapeutic immunization (HE stain, ×100). A decrease of the gastritis was observed in all of the vaccinated mice after therapeutic vaccination. A typical illustration of HE-stained sections from mice after therapeutic vaccination shows mild inflammatory infiltrates. But mice vaccinated with rCTB or PBS showing severe inflammatory infiltrates in the mucosa and submucosa

Evaluation of antibodies and cytokines after vaccination

The capacity of rCtUBE to induce serum IgG and IgA antibodies was evaluated by ELISA. A modest antibody level was observed in sera from prophylactically and therapeutically vaccinated mice. Compared with oral immunization with PBS, oral immunization with rCtUBE significantly increased the levels of specific IgG and IgA against H. pylori urease in the prophylactic and therapeutic vaccination experiments (Figs. 5a and 6a). Levels of the IgG subtypes IgG1 and IgG2a were further analyzed. Oral immunization with rCtUBE markedly elevated the level of IgG1 against H. pylori urease compared with oral immunization with PBS in prophylactically and therapeutically vaccinated mice, but there was no significantly higher level of specific IgG2a antibodies against H. pylori urease in the group immunized with rCtUBE compared with the control mice vaccinated with PBS (Figs. 5b and 6b). 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. Oral prophylactic (Fig. 5c) or therapeutic (Fig. 6c) immunization with the epitope vaccine rCtUBE markedly elevated the level of specific mucosal sIgA in gastric tissue, intestine mucus, and feces compared with oral immunization with rCTB or PBS.

Fig. 5
figure5

Detection of specific antibodies and cytokines after prophylactic vaccine. Data are expressed as mean ± SD; 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. b Measurement of IgG1 and IgG2a isotypes against H. pylori urease by ELISA. c Evaluation of mucosal IgA 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 prophylactically vaccinated mice. d The concentrations of cytokines in the supernatants of lymphocytes cultures. The splenic lymphocytes from mice immunized with rCtUBE, rCTB, and PBS were stimulated with rCtUBE peptides for 72 h, and the supernatants were collected and cytokine production was determined by ELISA

Fig. 6
figure6

Detection of specific antibodies and cytokines after therapeutic vaccine. Data are expressed as mean ± SD; 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 The concentrations of cytokines in the supernatants of lymphocytes cultures. The splenic lymphocytes from mice immunized with rCtUBE, rCTB, and PBS were stimulated with rCtUBE peptides for 72 h, and the supernatants were collected and cytokine production was determined by ELISA

For Th1, Th2, and Th17 polarization assays, ELISA was utilized to measure the production of IFN-γ, IL-4, and IL-17 in the supernatants of splenic lymphocytes cultures. Stimulation of splenic lymphocytes from mice after prophylactic (Fig. 5d) or therapeutic (Fig. 6d) vaccination with rCtUBE resulted in significantly higher levels of the IFN-γ, IL-4, and IL-17 than stimulation of cells from PBS-immunized mice and naive mice. The level of IL-4 was significantly higher than the levels of IFN-γ or IL-17 in rCtUBE-immunized mice. In addition, there was no significant difference in the levels of the IFN-γ, IL-4, and IL-17 between rCtUBE-immunized mice and rCTB-immunized mice. The results indicated that rCTB could also induce a predominantly Th2-biased response.

Discussion

H. pylori infection of the gastric mucosa remains a cause of significant morbidity and mortality almost 30 years after its discovery (Czinn and Blanchard 2011). Several prophylactic or therapeutic vaccines against H. pylori have been developed, including whole bacteria vaccine (Losonsky et al. 2003; Nystrom et al. 2006; Maeda et al. 2002), recombinant subunit vaccine (Liu et al. 2011), and DNA vaccine (Miyashita et al. 2002; Todoroki et al. 2000), but no major breakthrough has been achieved. Epitope vaccine can induce a specific immune response against H. pylori infection and had a much better specificity and security than other vaccines. H. pylori urease is an important target for prophylactic and therapeutic epitope vaccine development for its outstanding features. In previous study, an epitope vaccine with mucosal adjuvant cholera toxin B subunit and an epitope (UreB321–339) from UreB named CtUBE was constructed and the mice were protected significantly after intragastric vaccination with the CtUBE liposome vaccine(Zhao et al. 2007). In this study, the fusion peptide CtUBE was reconstructed and expressed as a soluble protein with pectinase signal peptide at the N terminus and the 6-his tag at its C-terminal in order for soluble expression and easy purification. Our results showed that oral immunization with rCtUBE could produce comparatively high level of specific antibodies against H. pylori urease, UreB, or the minimal epitope UreB327–334 involved with the active site of urease, and dramatically reduced the bacterial load in the stomachs of mice infected with H. pylori.

The immunological characteristics of rCtUBE vaccine were investigated, including adjuvant activity of the CTB component, immunogenicity, immunologic specificity, and the neutralizing capacity of specific antibodies. The cholera toxin B subunit has been used extensively in vaccine research as a carrier for peptide immunogens due to its immunopotentiating properties, which was considered to be critically dependent on its pentameric structure and its ability to bind to GM1 receptors (Harakuni et al. 2005; Sadeghi et al. 2002). The experimental results of GM1-ELISA confirmed that the CTB component of rCtUBE fusion protein had good adjuvanticity, and the presence of UreB321–339 epitope peptides did not abrogate the binding of the CTB component to its receptor. To examine the immunogenicity and immunologic specificity of the epitope vaccine rCtUBE, many antigens were referred, including natural H. pylori urease, rUreA, rUreB, and the UreB327–334 peptides. Because natural H. pylori urease did not contain protein impurities from E. coli host, it was more truthful and accurate to detect the level and specificity of antibodies using natural H. pylori urease than recombined urease. The results showed that the IgG and IgA antibodies induced by the rCtUBE epitope vaccine could react with natural H. pylori urease or rUreB specifically. In order to examine the epitope-special antibody response, the UreB327–334 epitope peptide was synthesized on a solid support. The results indicated that the rCtUBE epitope vaccine was capable of generating antibodies directed specifically against the UreB327–334 region of UreB. 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). Vaccination with the epitope vaccine CtUBE-induced significant levels of anti-urease IgG1and IgG2a antibodies in BALB/c mice, compared to levels in mice vaccinated with the rCTB or PBS. Therefore, it was speculated that the epitope vaccine CtUBE induced a mixed Th1–Th2 response when Freund’s adjuvant was used. Previous studies have revealed that some monoclonal antibodies (MAb) against H. pylori urease have the ability to inhibit enzymatic activity, whereas urease-specific polyclonal antibodies generated by immunization with purified H. pylori urease did not inhibit its enzymatic activity at all. It has been reported that a B cell epitope from UreB, UreB327–334, is recognized by a mouse monoclonal antibody termed L2, which can strongly inhibited the enzymatic activity of Hp urease. Further research indicates that the UreB327–334 epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease (Hirota et al. 2001). In the present study, the epitope vaccine rCtUBE could generate anti-UreB327–334 antibodies and inhibit the enzymatic activity of H. pylori urease obviously.

Vaccine efficacy against gastric H. pylori infection has been shown in mice, but little is known about the mechanisms of bacterial clearance. Some studies support the view that humoral and local mucosal immune responses are very important for clearing H. pylori (Sijun and Yong 2009). 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 the intragastric route for immunization. Humoral immune responses were examined and revealed that mice vaccinated with 150 μg of rCtUBE acquired a stronger immune response with both IgG and IgA than mice immunized with the rCTB or PBS. In addition, the supernatants of homogenized stomachs, small intestines, and feces were collected for detection of mucosal sIgA against H. pylori urease. Oral immunization with rCtUBE markedly elevated the level of mucosal sIgA against H. pylori urease compared with oral immunization with rCTB or PBS. The role of CD4+ T cells in protective immunity against H. pylori has been widely accepted (Ermak et al. 1998). CD4+ T cells are classified as T helper 1 (Th1), T helper 2 (Th2), and T helper (Th17) cells on the basis of their cytokine secretion. Th1 cells mainly produce IFN-γ and IL-2, Th2 cells predominantly secrete IL-4 and IL-5, and Th17 cells induce IL-17 production (Romagnani 1999). 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 (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. The mice immunized with the epitope vaccine rCtUBE induced a significantly higher level of anti-urease IgG1 antibodies compared to IgG2a antibodies. Therefore, the dominant level of IgG1 antibodies revealed that the epitope vaccine rCtUBE predominantly activated Th2 cells. Further analysis of the Th1, Th2, and Th17 representative cytokines showed that IFN-γ, IL-4, and IL-17 were all significantly induced by rCtUBE and that the level of IL-4 was significantly higher than the level of IFN-γ or IL-17 in mice immunized with rCtUBE. These results indicated that rCtUBE induced a Th2-biased immune response, which might help to produce antibodies specific for H. pylori urease and contribute to the dramatic reduction in the bacterial load in the stomachs of H. pylori infected mice. But the Th2-biased immune response induced by rCTB could not promote the production of specific antibodies and eradicate H. pylori in the stomach of mice.

In conclusion, the rCtUBE vaccine could induce comparatively high level of specific antibodies against natural H. pylori urease, rUreB, or the minimal epitope UreB327–334 involved with the active site of urease, which showed effectively inhibitory effect on the enzymatic activity of H. pylori urease, and protect BALB/c mice from H. pylori infection after oral prophylactic or therapeutic immunization. The protective immune mechanisms of rCtUBE vaccine is possibly mediated by specific serum IgG, IgA, and mucosal sIgA antibodies, and a Th2 cells response. Therefore, the epitope vaccine rCtUBE 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 rCtUBE with other adjuvants, vaccine carriers such as attenuated Salmonella strains and Bacille Calmette Guerin, and different immunization routes. We will also further evaluate the prophylactic and therapeutic effect of rCtUBE or a combination vaccine composed of rCtUBE and CTB-UA (Guo et al. 2012a, b) in Mongolian gerbil. The data from mice 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.

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Acknowledgments

This work was supported by the Science Foundation of China Pharmaceutical University (grant no, JKY2009023), Postgraduate Innovation Project of Jiangsu Province (grant no, CXZZ11_0817), and National Major Special Program of New Drug Research and Development (grant no, 2012ZX09103-301-008). We especially thank Professor Wutong Wu for his contributions in implementation of this experiment. Meanwhile, B.S Catherine J Tsang is thanked for the careful revision of this paper.

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Correspondence to Tao Xi.

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Le Guo and Kunmei Liu contributed equally to this article.

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Guo, L., Liu, K., Zhao, W. et al. Immunological features and efficacy of the reconstructed epitope vaccine CtUBE against Helicobacter pylori infection in BALB/c mice model. Appl Microbiol Biotechnol 97, 2367–2378 (2013). https://doi.org/10.1007/s00253-012-4486-1

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Keywords

  • Helicobacter pylori
  • Epitope vaccine
  • Cholera toxin B subunit
  • Urease B subunit
  • Neutralizing antibody