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Immunization with FliD Confers Protection Against Helicobacter Pylori Infection in Mice

Immunization with FliD confers protection against Helicobacter pylori infection in mice


Nearly half of the world’s population is infected with Helicobacter pylori. Clinical manifestations of this infection range from gastritis and peptic ulcers to gastric adenocarcinoma and lymphomaDue to the limited efficacy of anti-H. pylori antibiotic therapy in the clinical practice, there is increasing interest in the development of a protective vaccine against H. pylori infection. The bacterial protein FliD forms a capping structure on the end of each flagellum which is critical to prevent depolymerization and structural degradation. In this study, the potential of FliD as a prospective H. pylori subunit vaccine was assessed. For this purpose, immunogenicity and protective efficacy of recombinant FliD (rFliD) from H. pylori was evaluated in C57BL/6 mice. Purified rFliD was formulated with different adjuvants and administered via subcutaneous or oral route. Subcutaneous immunization with rFliD elicited mixed Th1/Th2 and Th17 immune responses, with higher titers of specific IgG1 than IgG2a. Splenocytes of immunized mice exhibited strong recall responses, resulting in the secretion of high amounts of IFN-γ and IL-17, and low levels of IL-4. Immunization with rFliD caused a significant reduction in H. pylori bacterial load relative to naïve control mice (p < 0.001), demonstrating a robust protective effect. Taken together, these results suggest that rFliD formulated with CpG or Addavax may be a useful candidate for the development of subunit vaccine against H. pylori infection.

Key words: Helicobacter pylori, immunization, recombinant, adjuvant, protection.

1. Introduction

H. pylori is a spiral-shaped, extra-cellular Gram-negative, and microaerophilic bacterium that has colonized the stomach of approximately 50% of the worldwide human population [1]. In 1994, H. pylori was classified as a class I carcinogen by the World Health Organization [2]. Infection is strongly associated with the development of several gastrointestinal diseases, including duodenal and gastric ulcers, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma (MALT) [3]. Antibiotic therapy, although effective, is associated with several disadvantages including the low patient compliance due to the drugs’ side effects, treatment failure as a result of the emergence of drug-resistant strains, high costs, and the failure to prevent reinfection [4-7]. Therefore, alternative approaches to combat H. pylori infection are currently pursued, including vaccination. Several H. pylori proteinshave been identified as immunogenic in preclinical models, including Urease B (UreB) [8], Vacuolating toxin A (VacA) [9], H. pylori adhesion A (HpaA) [10], neutrophil-activating protein A (NapA) [11], outer membrane protein (Omp) [12], cytotoxin-associated antigen (CagA) [9], heat-shock proteins (Hsp) [1314], OipA [15] catalase [16] and chimeric genes [17-19]. Each of these antigens has the ability to reduce the bacterial load in animal models, but none affords protection against infection. Current strategies to enhance vaccine efficacy include the identification of most suitable immune targets and the combination of immunodominant antigens into multivalent formulations. Gholi et al. [20] demonstrated that FliD,, the flagellar hook-associated protein 2, reacts with approximately 97 percent of sera obtained from patients infected with H. pylori, suggesting that this is a common immune target in the infected human host[20]. FliD plays a crucial role in flagella assembly. Flagellin is important for bacterial motility and is essential for colonization and persistence of H. pylori in the stomach niche [21].

The objective of the current study was to investigate whether recombinant FliD (rFliD) protein is able to protect against H. pylori infection. Owing to inherent safety and the low risk of adverse reactions [22], protein subunit immunization is an attractive vaccination approach. However, immune responses elicited by the administration of pure antigens are usually low. Therefore, we investigated whether formulations of FliD with different adjuvants, namely CpG, Cholera toxin subunit B (CTB), or Addavax, enhance protection of mice against H. pylori infection.

2. Materials and methods

2.1. Bacterial strains

E. coli strains BL21 and TOP10 were used for expression of FliD. Bacteria were routinely grown at 37°C in LB broth or agar. The H. pylori strain SS1 was grown on Brucella agar supplemented with 5% sheep blood, 5 µg/mL trimethoprim, 161.5 µg/mL polymyxin B (Sigma), 10 µg/mL vancomycin (sigma) and 2.5 µg/mL amphotericin B (Sigma), in an anaerobic jar with microaerophilic gas generating kit (Merck, Germany) for 3 days at 37°C. For infection experiments, bacteria were subcultured in brain heart infusion broth (BHI, Merck) containing the aforementioned antibiotics and supplemented with 10% Fetal Bovine Serum (FBS) (Sigma), and grown in microaerophilic conditions for 72 h at 37°C.

2.2. Cloning, expression and purification of recombinant FliD

Cloning and expression of FliD from H. pylori in BL21 (DE3) and its purification has been described previously [20]. Briefly, the flid gene was amplified by PCR from genomic DNA of H. pylori (Forward: 5’-ATGGCAATAGGTTCATTAAGCT-3’, Reverse: 5’- ATTCTTTTTAGCCGCCGCTT-3’). The amplified DNA fragment was directly inserted into pTZ57R (InsTAclone PCR Cloning Kit, ThermoFisher, USA) and then subcloned into the pET28a+ vector (Novagen, USA) to add a 6xHis tag. To express FliD, 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was used. Upon induction, rFliD was expressed in soluble form and purified under native conditions. Purity of the recombinant protein and its identity was assessed by SDS-PAGE separation and visualized by Coomassie blue staining and Western blotting [23]. Briefly, purified rFliD was size-separated by SDS-PAGE and the protein transferred to a nitrocellulose membrane (BioRad, USA). Next, the membrane was incubated with anti-6xHis peroxidase (Roche, Germany) (1/40,000) for 1 h. Finally, the bound conjugates were detected using 3, 3′-Diaminobenzidine tetrahydrochloride (Sigma). To preclude any adverse effect caused by the endotoxin associated with the recombinant protein, only purified protein with an endotoxin content of less than 0.05 endotoxin units per mg of protein (evaluated by Limulusamebocyte lysate analysis kit, Lonza, Basel, Switzerland) was used. The concentration of recombinant protein was determined by the Bradford method [24].

2.3. Mice

Six- to eight-week-old female C57BL/6 mice maintained under specific pathogen-free conditions were obtained from the Pasteur Institute of Iran. Mice were handled under optimal conditions of temperature, hygiene, humidity and light (cycles of 12 h dark/light). All experimental animal procedures were approved by the ethical committee of Kashan University of Medical Science.

2.4. Immunization and H. pylori inoculation of mice

One hundred and twenty mice were randomly divided into 8 groups (n = 15 each). Five groups were immunized subcutaneously (SC) three times at 2-week intervals with either 30 µg rFliD formulated with CpG (CpG, ODN1826 5ʹ-TCCATGACGTTCCTGACGTT- 3ʹ 20 μg/mouse, synthesized by TAG Copenhagen, Denmark), 30 µg rFliD formulated with Addavax (Invivogen, USA), or PBS, or CpG, Addavax, and PBS alone. The two remaining groups were orally immunized with rFliD and CTB (Sigma, Germany) or CTB alone (Table 1). To enable immunization using a minimal volume, the recombinant antigens were lyophilized and then reconstituted in a volume of 100 µl PBS containing the respective adjuvant.

Two weeks after the final immunization, five mice from each group were challenged orally thrice in 2-day intervals with 5 × 108 CFU mouse-adapted H. pylori strain SS1 in 100 μl brain heart infusion broth. Sera were obtained 0, 15, 30, 45, and 75 days after the first immunization. Five mice were sacrificed to assess immune responses including cytokine production, sIgA secretion, and humoral immune responses at the day of challenge. For measuring sIgA secretion, gastric fluid was collected as described previously [25]. The remaining five mice were bled on day 75 to monitor memory responses.

2.5. Humoral and mucosal immune responses

To measure rFliD-specific serum IgG1, IgG2a and gastric fluid sIgA titers in immunized mice, an enzyme-linked immunosorbent assay (ELISA) was used. 96-well polystyrene plates (Greiner Bio-One, Frickenhausen, Germany) were coated with the purified rFliD (1 µg/ml). After overnight incubation, the plates were washed three times with wash TBST buffer (Tris-buffered saline, pH 7.4, and containing 0.05 % Tween 20) and blocked with 300 µl 10 % FBS in PBS for 2 h at 37°C. Thereafter, plates were incubated with serial dilutions of mouse sera or gastric and intestinal fluid for 2 h at room temperature, and then washed three times as above. HRP-conjugated goat-anti-mouse IgG1, IgG2a or IgA antibodies (BD Pharmingen, USA) were added to the wells for another 90 min at 37°C. After a final washing step, specific reactivity was determined by the addition of 50 µl/well of the enzyme substrate TMB (Pishtaz Teb, Tehran, Iran). The reaction was stopped by adding 30 µl 20 % H2SOto each well. Optical density at 495 nm was measured using an ELISA plate reader (Bio-Tek Instruments, Winooski, USA).

2.6. Cellular immune responses

Fifteen days after the last immunization, the mice were sacrificed and spleens of immunized mice were removed under sterile conditions. To assess cellular immune responses, the spleens were minced and homogenized with a syringe in 10 ml cold PBS containing 5 mM ethylene diamine tetraacetic acid (PBS-EDTA). The cells were washed twice with PBS-EDTA and mononuclear cells (MNCs) were isolated by Ficoll–Paque (GE Healthcare, Uppsala, Sweden) discontinuous gradient centrifugation. The cells were harvested in complete medium (RPMI 1640) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 10 % heat inactivated FBS). The cell concentration was adjusted to 2 × 106 in 2 ml of complete RPMI 1640 medium and the cells were then plated in 24-well flat-bottom plates. Cells were then treated with 10 μg/ml rFliD and incubated at 37°C in 5 % CO2 for 48 h. Control wells received PBS instead of the antigen. Supernatants were collected after 48 h and stored at -70°C for cytokine assay. Levels of interferon-gamma (IFN-γ), interleukin-17 (IL-17), and -4 (IL-4) were measured according to the manufacturer’s instructions (BD Pharmingen).

2.7. Protection experiments

To assess whether immunization of rFliD formulated with different adjuvants could reduce the bacterial load in the stomach of H. pylori challenged mice, H. pylori CFU were quantified six weeks post the last immunization. For this purpose, the copy number of H. pylori DNA within mouse gastric tissues was measured by a quantitative Real-Time PCR as described previously [26]. A recombinant plasmid carrying the H. pylori 16S gene was used as standard to measure copy numbers of the 16S gene in DNA extracted from half of a mouse stomach and the obtained CFU calculated as CFU per stomach.

2.8. Statistical analysis

Statistical analysis was carried out using SPSS computer software, version 16.0. All data were represented as the mean ± standard deviation (S.D). Differences among data of the five groups were analyzed by one factor analysis of variance (ANOVA) and Tukey’s post hoc test in SPSS. P-values <0.05 were considered as statistically significant.

3. Results

3.1. Expression and purification of the recombinant FLiD protein

To obtain sufficient amounts of rFliD for protection experiments and immunological analyses, the H. pylori flid gene was amplified by PCR and fused to a 6x histidine tag in the pET28 bacterial expression vector.  E.coli BL21 cells were transformed with the resulting plasmid and protein expression induced with IPTG. Upon purification with Ni-NTA agarose, rFLiD expression products were verified via SDS-PAGE and Western blot [23]. Consistent with molecular weight prediction, recombinant rFliD had a size of approximately 84 kDa. As assessed by SDS-PAGE, purity was >98% (Fig.1). From one liter of liquid culture, approximately 35 mg of rFliD protein was obtained.

3.2. Humoral and mucosal responses

Specific antibody titers produced in mice immunized with rFliD formulated with different adjuvants were evaluated by ELISA in sera obtained at different days after the first immunization. rFliD induced strong immunoglobulin G (IgG) responses, with IgG1 titers usually exceeding those of the IgG2a subtype (Fig 2). Responses induced in mice orally immunized with rFliD formulated with CTB were markedly lower than in mice immunized subcutaneously with rFliD plus CpG or Addavax. However, the IgG responses of mice immunized with rFliD formulated with CTB were significantly increased 75 days after the initial immunization. Mice vaccinated with rFliD formulated with either CpG or Addavax demonstrated increased IgG1 andIgG2a titers commencing at the second week post-immunization, peaking at the sixth week and remaining elevated until the eleventh week after the first immunization. Compared to all adjuvant formulations, immunization of mice with rFliD alone induced only low titers of both IgG1 and IgG2a antibodies (Fig. 2). To assess the mucosal immune responses elicited by immunization, gastric fluid sIgA production was determined for each immunization formulation. As shown in Fig 2, the three groups immunized with rFliD plus adjuvants showed significantly elevated gastric mucosal sIgA titers compared to the sham control group which only received PBS (P<0.0001). The group immunized with rFliD and PBS exhibited no difference when compared to the PBS control group.

Taken together, these findings suggest that the combination of rFliD with CpG, Addavax, and CTB adjuvants induced systemic and mucosal humoral responses.

3.3. Cytokine production assay

In order to further characterize the immune responses, splenocytes from mice immunized with rFliD formulated with different adjuvants, or rFliD alone, or PBS were isolated 45 days after the first immunization. Following incubation with rFliD, cytokine production was assessed. Secretion of IFN-γ, IL-4and IL-17 from splenocytes of immunized mice after re-stimulation was assessed two weeks after the final immunization boost. As shown in Fig 3, the levels of all tested cytokines were significantly increased in mice immunized with rFliD formulated with adjuvants compared to the PBS control group. Splenocytes of mice immunized with rFliD without adjuvants secreted significantly higher amounts of IFN-γ and Il-17 than spenocytes from PBS control mice. These results indicate that rFliD alone is capable of eliciting cellular immune responses of Th1, Th2 and Th17 type. Moreover, these immune responses were markedly enhanced by the use of adjuvants.

It is important to note that there were no discernable differences in the patterns of cytokines secreted by splenocytes from mice immunized with rFliD formulated with the three different adjuvants (Fig. 3).

3.4. Protection assay

To investigate whether immunization with rFliD provided protection against subsequent infection with H. pylori, groups of mice were immunized with rFliD formulated with different adjuvants, rFliD alone or PBS. Two weeks after the last immunization, mice were challenged thrice at one day intervals with 5 × 108 H. pylori SS1. Bacterial DNA load was assessed by real-time PCR four weeks post infection.

The copy numbers of H. pylori DNA in groups immunized with rFliD formulated with adjuvants were significantly lower than in mice immunized with rFliD in PBS or in control mice. The highest degree of protection among animals vaccinated with rFliD plus adjuvants was observed in mice immunized with rFliD plus CpG, albeit without reaching statistical significance (Fig. 4). Compared to the negative control group, even mice immunized with rFliD in PBS showed a significant reduction in CFU, implicating a high immunogenicity of this antigen.

4. Discussion

The development of an effective vaccine against H. pylori has been confounded by several aspects including choice of the antigen, route of vaccination, and selection of adjuvants to boost immunogenicity [2728]. Motility enables H. pylori to reach the gastric epithelium, attach via adhesion factors, and establish infection in the epithelium [29]. Polar flagella is considered an important virulence factor for H. pylori, as studies done with a non-motile fliD mutant demonstrated that FliD, and consequently a functional flagellum, is required for the colonization of mice [30]. Moreover, it has been shown that FliD promotes biofilm formation, thereby contributing toward environmental resilience [31]. The association between the presence of antibodies against FliD and gastric cancer has been documented. Based on these findings, it has been suggested that FliD can serve as biomarker in screening for GC patients [32]. We hypothesized that abundantly expressed proteins that are essential for H. pylori infection may constitute optimal candidates for subunit vaccines. Moreover, rFliD was administered orally or subcutaneously and formulated with Addavax, CpG, or CTB adjuvants. Addavax is a squalene-based oil-in-water emulsion capable of eliciting both cellular and humoral immune responses [3334]. MF59, which has similar composition as Addavax, has been approved for use in the US with a seasonal flu vaccine [35]. By contrast, CpG oligodeoxynucleotides are toll-like receptor 9 (TLR9) agonists known to stimulate innate immune defenses and antigen-specific T cell responses [36]. CTB  is a potent adjuvant which can stimulate innate and antigen-specific responses upon oral or intranasal administration [37].

In the present study, cell-mediated immune responses were assessed in mice vaccinated with rFliD by measuring cytokine production after re-stimulating of immunized mice splenocytes in vitro. Splenocytes from immunized mice restimulated with antigen showed robust IFN-γ secretion, which is indicative of a Th1 type response. In addition, significant higher amounts of IL-4 were produced by splenocytes of rFliD-immunized versus naïve mice, suggesting that the vaccines also elicited a Th2 type of immune response. Furthermore, high quantities of IL-17 were produced by splenocytes of immunized mice upon re-stimulation. Thus, rFLiD-immunized mice appeared to develop immune responses that encompass different immune effector functions. These findings are consistent with earlier studies by other investigators who reported mixed Th1/Th2 responses against a set of distinct H. pylori proteins [19253839]. Although the specific functions of the secreted cytokines in the control of H. pylori infection still need to be elucidated in more detail, Th1 and Th17 cells have been implicated in inducing local inflammation and in promoting protective immune responses [40]. Moreover, Th17 responses are involved in the recruitment of neutrophils, release of anti-microbial peptides and IL-17-driven Th1 immunity (46, 47).

To further characterize the Th1/Th2 profile of the elicited immune responses, production of antigen-specific IgG1 and IgG2a antibodies was examined. Although rFliD-induced humoral immune responses were predominantly of IgG1 isotype, significant levels of IgG2a titers were also detected. Specific antibodies against rFliD were also produced in mice immunized with rFliD and PBS, but titers were significantly lower in comparison to mice immunized with rFliD and adjuvants. In addition, subcutaneous immunization with rFLiD formulated with Addavax or CpG elicited more pronounced systemic humoral responses than oral immunization in the presence of CTB.

To assess the mucosal humoral immune response induced by immunization, gastric sIgA production was evaluated for each vaccine formulation. Immunizations with rFliD and Addavax, CpG, or CTB induced significant levels of antigen-specific gastric sIgA compared to control mice. These findings suggest that immunization with rFliD and adjuvant, regardless of subcutaneous or oral route of delivery, efficiently induced gastric mucosal immune responses.

We next looked at the protective capability of rFliD immunization by challenging immunized mice with live H. pylori. Our results demonstrate that mice immunized with rFliD in combination with any of the tested adjuvants conferred significant protective immunity to mice as compared to the control group. Immunization with non-adjuvanted rFliD resulted in partial protection against H. pylori which was not statistically significant in comparison to the naïve control. Taken together, our results suggest that FliD is capable of eliciting strong and protective immune responses in mice when combined with adjuvants. Our results also demonstrated that the most pronounced immune correlates of protection are elicited by subcutaneous rather than oral immunization. Since both CpG and Addavax have been approved for human application, they may form the basis of future subunit vaccines against H. pylori [4142].  Additional experiments are needed to assess whether vaccine efficacy can be further increased by incorporating additional H. pylori antigens.


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Table 1. Vaccine formulations and route of application.

Route Vaccine Number of mice
Subcutaneous rFliD+ CPG 15
rFliD+Addavax 15
rFliD+PBS 15
CpG 15
Addavax 15
PBS 15
Oral rFliD+CTB 15
CTB 15

Figure legends       

Fig. 1. Expression and purification of recombinant FliD. (a) Analysis of rFliD protein expression by SDS-PAGE. Protein expression in bacteria transformed with the flid gene was induced by the addition of IPTG to the media. Four hours after induction, the bacteria were harvested, the bacterial lysates run over Nickel-NTA columns and bound proteins eluted with Imidazole. Aliquots of the different fractions were size-separated by SDS-PAGE and the resulting gel stained with Coomassie-Blue. Lane 1: molecular weight marker, lane 2: bacterial lysate after IPTG induction, lane 3: flow through of Nickel-NTA column, lanes 4 and 5: column eluate with buffer containing 20 mM Imidazole, lanes 6 and 7: eluate with buffer containing 40 mM Imidazole, Lane 8: eluate  with buffer containing 1 M Imidazole. (b) Western blot analysis of rFliD using a monoclonal antibody directed against the 6xHis-tag attached to rFliD. Lane 1: Molecular weight marker, lane 2: purified rFliD, lane 3: lysate of untransformed bacteria. Expected size of rFliD: 84 kDa.

Fig. 2. Analysis of the rFliD-specific antibody response in immunized animals. (a) Kinetics of the IgGand IgG2a responses after immunization with rFliD plus different adjuvants. Animals were bled retroorbitally on the indicated days and specific IgG1 and IgG2a antibody titers against rFliD  were evaluated by ELISA. Titer values represent the mean ± SD of sera from three analyses of five animals each. (b) Gastric scrapings were collected 45 days post first immunization and rFliD-specific sIgA titers analyzed by ELISA. Ψ: Comparison of antigen-specific anti-rFliD IgG1 and IgG2a in mice immunized with rFliD formulated with and without adjuvant. ∮: Comparison of anti-rFliD IgA in mice immunized with rFliD versus PBS. n.s.: not significant, *: < 0.05, **: p < 0.01, ***: p < 0.001.

Fig. 3. Cytokine production by splenocytes from immunized mice after restimulation with rFliD. Spleen cells of mice from all five groups were stimulated in vitro with 10 μg/ml rFliD for 48 h. Cytokine concentrations in culture supernatant were measured by sandwich ELISA. The data are the mean ±SD of five individual mice from each group with two repeats. Significance was evaluated between mice immunized with rFliD +/- adjuvants and mice treated with PBS. ***: p < 0.001.

Fig. 4. H. pylori colonization of stomach in immunized mice. C57BL/6 mice were immunized on day 0, 15 and 30 with three doses of 30 μg rFliD with CpG, Addavax, CTB or PBS. The control groups only received adjuvants or PBS. Two weeks after final vaccination (on day 45), mice were challenged orally with H. pylori. Four weeks post challenge, levels of gastric H. pylori colonization were determined by real-time quantitative PCR. Significance (ANOVA) was evaluated with reference to the PBS control. n.s.: not significant, ***: p < 0.001.

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