Microbial Immunity and Vaccines

A Double Mutant Heat-Labile Toxin from Escherichia coli, LT(R192G/L211A), Is an Effective Mucosal Adjuvant for Vaccination against Helicobacter pylori Infection

Louise Sjökvist Ottsjö, Carl-Fredrik Flach, John Clements, Jan Holmgren, Sukanya Raghavan
A. Camilli, Editor
DOI: 10.1128/IAI.01407-12

ABSTRACT

Helicobacter pylori infection in the stomach is a common cause of peptic ulcer disease and is a strong risk factor for the development of gastric adenocarcinoma, yet no effective vaccine against H. pylori infection is available to date. In mice, mucosal vaccination with H. pylori antigens when given together with cholera toxin (CT) adjuvant, but not without adjuvant, can induce protective immune responses against H. pylori infection. However, the toxicity of CT precludes its use as a mucosal adjuvant in humans. We evaluated a recently developed, essentially nontoxic double mutant Escherichia coli heat-labile toxin, LT(R192G/L211A) (dmLT), as a mucosal adjuvant in an experimental H. pylori vaccine and compared it to CT in promoting immune responses and protection against H. pylori infection in mice. Immunization via the sublingual or intragastric route with H. pylori lysate antigens and dmLT resulted in a significant decrease in bacterial load after challenge compared to that in unimmunized infection controls and to the same extent as when using CT as an adjuvant. Cellular immune responses in the sublingually immunized mice known to correlate with protection were also fully comparable when using dmLT and CT as adjuvants, resulting in enhanced in vitro proliferative and cytokine responses from spleen and mesenteric lymph node cells to H. pylori antigens. Our results suggest that dmLT is an attractive adjuvant for inclusion in a mucosal vaccine against H. pylori infection.

INTRODUCTION

Approximately half of the world's population is infected with Helicobacter pylori bacteria in the stomach. While most individuals remain asymptomatic, 10 to 15% develop symptoms such as dyspepsia and peptic ulcers, and chronic infection with H. pylori has been identified as a strong risk factor for the development of gastric adenocarcinoma (1).

In the past decade, several vaccine candidates against H. pylori have been evaluated in animal models (2). We and others have shown that, besides specific H. pylori antigens, an effective adjuvant is needed to induce protection against H. pylori infection after mucosal immunization (2, 3). Thus, immunization with whole-cell or lysate preparations of H. pylori together with adjuvants such as cholera toxin (CT) or heat-labile toxin (LT) and in some cases also mutant forms of the toxins confers protection against H. pylori infection (2, 4). CT, most often used in the preclinical evaluation of mucosal candidate vaccines, promotes strong T cell as well as B cell responses to vaccine components and is a golden standard for testing alternative mucosal adjuvants. However, CT is enterotoxic in humans, causing profuse diarrhea and fluid loss, making it important to find an alternative, nontoxic mucosal adjuvant that could promote a strong protective immune response against H. pylori infection. Clinical trials of candidate H. pylori vaccines have been performed in human volunteers, but so far there has been limited success with regard to protection induced against H. pylori infection (5). Although enhanced immune responses to vaccine components were reported in some studies, the observed adverse effects of the adjuvants used have hindered the further progress to clinical trials (6–9).

A major focus in mucosal adjuvant research for a long time has been the generation of nontoxic derivatives of CT or LT that still retain significant adjuvanticity (10). Mutant LT(R192G) (mLT) has a single amino acid substitution resulting in reduced enterotoxicity compared to native LT, and it was found to be safe and well tolerated (6). However, when it was included in an inactivated H. pylori oral whole-cell vaccine, one-third of the volunteers experienced mild diarrhea (6). In order to further reduce the enterotoxicity of mLT, an additional mutation was introduced (L211A) to create a double mutant, LT(R192G/L211A) (dmLT). This molecule is essentially nontoxic compared to native LT in a patent mouse enterotoxicity assay which measures the increase in intestinal weight resulting from toxin-induced fluid secretion (11). Furthermore, dmLT has been found to strongly potentiate immune responses to various parenterally and mucosally administered vaccines, e.g., tetanus toxoid and experimental whole-cell vaccines against enterotoxigenic Escherichia coli (J. Holmgren et al., unpublished data), Streptococcus pneumoniae, and H. pylori, making it a promising adjuvant to include in future vaccines (11–13).

The objective of the current study was to assess the adjuvant efficiency of dmLT when administered sublingually or intragastrically with H. pylori antigens and to compare it to “gold standard” CT for inducing protective immune responses against H. pylori infection. Our results demonstrate that prophylactic immunization with H. pylori lysate antigens and dmLT confers a reduction of the bacterial loads in the stomachs of H. pylori-infected mice, comparable to that achieved when using CT as adjuvant. Protection was associated with enhanced systemic and local mucosal stomach antibody, interleukin-17 (IL-17), and gamma interferon (IFN-γ) responses and with an influx of immune cells into the stomach. Our data indicate that dmLT is a strong adjuvant for mucosal immunization with H. pylori antigens with properties that should make it attractive for use as an adjuvant also in a vaccine against H. pylori infection in humans.

MATERIALS AND METHODS

Animals.Six- to 8-week old, specific-pathogen-free female C57BL/6 mice were purchased from Taconic (Denmark). The mice were housed in microisolators at the Laboratory for Experimental Biomedicine (EBM) for the duration of the study. All experiments were approved by the ethics committee for animal experiments (Gothenburg, Sweden).

Cultivation of H. pylori SS1 used for infection.The bacteria were cultured in liquid broth as previously described (14). Before infection of mice, the optical density (OD) of the bacteria was adjusted to 1.5, and 300 μl, corresponding to approximately 3 × 108 viable bacteria, was administered intragastrically to each mouse (15).

H. pylori lysate antigens and intragastric and sublingual immunizations.H. pylori lysate antigens from strain Hel 305 (CagA+ VacA+) isolated from a duodenal ulcer patient were prepared as previously described (16). Briefly, the bacteria were grown on Columbia iso agar plates to confluence, and the bacterial harvest from 25 plates was suspended in 5 ml of sterile phosphate-buffered saline (PBS). Each plate corresponded to approximately 1010 bacteria. The bacteria were then pulse sonicated for 2 to 3 h at 50% capacity while being kept on an ice bath. The sonicated bacteria were then centrifuged at 13,000 × g for 20 min at 4°C, and the supernatant was filtered through a 0.2-μm-pore-size filter. The protein content of the lysate antigens was measured using a noninterfering protein assay kit (Calbiochem, San Diego, CA). The antigen preparation was aliquoted, stored at −70°C until further use, and not subjected to multiple freeze-thaw cycles. Thawed aliquots of the H. pylori lysate antigens were immediately freeze-dried and reconstituted to a protein concentration of 40 mg/ml to reduce the volume used for the sublingual immunizations. For intragastric immunizations, the same dose of the antigens was used as for sublingual immunizations. Lyophilized CT from Vibrio cholerae (Sigma-Aldrich, St. Louis, MO) was reconstituted in sterile distilled water to a concentration of 1 mg/ml and stored in aliquots at −70°C until further use. Lyophilized dmLT [LT(R192G/L211A)] from E. coli, prepared as described previously (11), was reconstituted in sterile distilled water to a concentration of 1 mg/ml and stored at 4°C until further use. To ensure the stability of the two adjuvants, repeated freeze-thaw cycles were avoided.

Immunizations and infection with H. pylori.Groups of mice were prophylactically immunized according to the following protocols: (i) sublingual (s.l.) immunization with two biweekly doses of 400 μg H. pylori lysate antigens and 10 μg CT or 10 or 20 μg dmLT, (ii) intragastric (i.g.) immunization with two biweekly doses of 400 μg H. pylori lysate antigens and 10 μg CT or dmLT, and (iii) sublingual immunization with two biweekly 400-μg doses of H. pylori lysate antigens alone or 20 μg dmLT alone. The immunizations were administered under deep anesthesia (Isoflurane; Abbott Scandinavia AB, Solna, Sweden) either by carefully placing 10 μl of H. pylori lysate antigens reconstituted in CT, dmLT, or PBS without bicarbonate buffer through a micropipette under the tongue (s.l. route) or by using a feeding needle, placing 300 μl of H. pylori lysate antigens and CT or dmLT in 3% sodium bicarbonate buffer directly into the stomach (i.g. route). Two weeks after the last immunization, the mice were challenged with live H. pylori SS1 bacteria in brucella broth administered intragastrically using a feeding needle under anesthesia. Animals were sacrificed at 2 to 3 weeks after challenge, and the numbers of H. pylori bacteria in the stomach were determined. In a separate experiment, mice were sublingually immunized with two biweekly doses of 50 μg recombinant HpaA (rHpaA) plus 50 μg rUreB together with 10 μg of CT or 20 μg dmLT. Two weeks after the last immunization, the mice were challenged with live H. pylori SS1 bacteria in brucella broth administered intragastrically using a feeding needle under anesthesia, and they were sacrificed at 4 weeks after challenge. To determine the minimal dose of adjuvant needed to induce immune responses and protection against H. pylori infection, mice were sublingually immunized with two biweekly doses of 200 μg H. pylori lysate antigens and 3.3 μg, 1.1 μg, or 0.3 μg of CT or dmLT. One week after the last immunization, the mice were challenged with live H. pylori SS1 bacteria, and they were sacrificed at 4 weeks after challenge. Finally, to evaluate the effect of increasing the time interval between the last immunization and challenge from 4 to 8 weeks on protection against H. pylori infection using the minimal dose of the adjuvant, mice were sublingually immunized with two biweekly doses of 200 μg H. pylori lysate antigens and 3.3 μg or 0.3 μg dmLT or CT. Animals were sacrificed at 4 weeks after challenge, and the numbers of H. pylori bacteria in the stomach were determined.

Quantitative culture of H. pylori SS1 from the stomach.To evaluate bacterial colonization in the stomachs of the sacrificed animals, one half of each stomach was homogenized in brucella broth using a tissue homogenizer (Ultra Turrax; IKA Laboratory Technologies, Staufen, Germany). Serial dilutions of the homogenates were plated on blood skirrow agar plates (BD [Becton, Dickinson Biosciences], San Diego, CA). After 7 days of incubation at 37°C under microaerophilic conditions, visible colonies with typical H. pylori morphology were counted, and the urease test was performed for any uncertain colonies. Plates with 10 to 100 colonies were used for calculating the number of bacteria per stomach as previously reported (17).

Serum antibody responses.Blood was collected from the axillary plexus immediately before the mice were sacrificed. Serum antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) against a membrane antigen preparation of H. pylori strain Hel 305 (MP Hel 305), which was coated overnight at room temperature. Levels of IgG antibodies were measured by testing serial dilutions of 1:10 prediluted sera. Bound antibodies were detected using horseradish peroxidase (HRP)-coupled goat anti-mouse IgG (Jackson Immuno Research, West Grove, PA) secondary antibodies. Subsequently, a reaction mixture with the substrate o-phenylenediamine dihydrochloride (OPD), added together with H2O2, was incubated at room temperature for 20 min. The enzymatic reaction was then stopped by adding 1 M sulfuric acid, and the absorbance at 490 nm was measured using a spectrophotometer. The antibody titers are defined as the reciprocal serum dilution giving an absorbance of 0.4 above the background.

Mucosal IgA antibody responses.IgA antibodies in the stomach were determined using the Perfext method (18). Briefly, after sacrifice, mice were extensively perfused with heparinized phosphate-buffered saline (PBS) to remove blood from the organs. Tissue was extracted from the stomach using a 2% saponin–PBS solution as previously described in detail (17). H. pylori-specific IgA antibodies in the supernatants of the saponin extracts were determined by ELISA, using microtiter plates coated with membrane protein preparation (MP) from H. pylori strain Hel 305 and sequential incubations with (i) a 3-fold dilution of saponin extract supernatant at room temperature for 90 min, (ii) an appropriate concentration of HRP-conjugated goat anti-mouse IgA (Southern Technology) at 4°C overnight, and (iii) OPD and H2O2 at room temperature for 30 min. The reaction was stopped by adding 1 M sulfuric acid, and the absorbance at 490 nm was measured in a spectrophotometer.

Cellular immune responses.For the proliferation assays, single-cell lymphocyte suspensions were prepared from the spleen and mesenteric lymph nodes (MLN). Cells were seeded (2 × 105 cells per well) in the presence or absence of H. pylori strain Hel 305 lysate antigens (4 μg/ml) and cultured for 72 h in Iscove's medium (Biochrome, Berlin, Germany) supplemented with 10% heat-inactivated fetal calf serum (Sigma), 50 μM 2-mercaptoethanol (Sigma), 1 mM l-glutamine (Biochrome), and 50 μg/ml gentamicin (Sigma) at 37°C in a 5% CO2 atmosphere. Supernatants were collected and stored at −70°C for subsequent cytokine analysis. The cells were pulsed with 1 μCi of [3H]thymidine (Amersham Bioscience, Buckinghamshire, United Kingdom) for the last 6 to 8 h of culture. The cellular DNA was collected with a cell harvester (Skatron) on glass fiber filters (Wallac) and assayed for 3H incorporation using a liquid scintillation counter (Beckman, LKB, Bromma, Sweden). Cytokines in culture supernatants were measured using the mouse cytometric bead array kit (BD Biosciences) and analyzed according to the manufacturer's instructions.

RNA isolation.The stomach was excised and dissected along the greater curvature. Any loose stomach contents were removed by washing in PBS. One longitudinal strip including the corpus and antrum was cut and placed directly into RNAlater (Qiagen, Hilden, Germany). The samples were kept at 4°C for 24 h and then stored at −70°C. For RNA isolation, the tissue was thawed, transferred to RLT lysis buffer (Qiagen), and homogenized for 2 × 2 min at 30 Hz using a Tissue Lyser II (Qiagen). Total RNA was extracted using the RNeasy minikit (Qiagen) and stored at −70°C.

RT-PCR.RNA (2 μg) was reverse transcribed into cDNA using the Omniscript kit (Qiagen). All real-time PCRs (RT-PCRs) were run in 96-well plates using the standard amplification conditions described for the 7500 RT-PCR system and 4 μl cDNA, 10 μl 2× Power SYBR green master mix (Applied Biosystems, Foster City, CA), and 1 μl of gene-specific oligonucleotide primers (Eurofins MWG Operon, Ebersberg, Germany) (Table 1). The reactions were run in duplicate, and β-actin was used as the reference gene in all experiments. The difference between β-actin and the target gene (ΔCT) was determined, and the relative expression was calculated using the formula 2ΔCT. The values were adjusted so that the mean in the infection control group was set to 1. The negative control (lacking reverse transcriptase) giving the lowest threshold cycle (CT) value was used to determine the detection limit.

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Table 1

Oligonucleotide primers used for quantitative RT-PCR

Immunohistochemistry.A longitudinal strip from the entire longer curvature of the stomach was taken, fixed in 4% phosphate-buffered formalin, and then embedded in paraffin. Sections 8 μm thick were cut and stained with hematoxylin and eosin. The slides were then examined by light microscopy (magnification, ×100), and the extent of gastritis was graded based on the Sydney gastritis scoring system as described previously (19).

Statistical analysis.Analysis of variance (ANOVA) with Bonferroni's or Dunette's posttest was used to compare multiple groups of mice using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). For all tests, a P value of <0.05 was considered to be statistically significant.

RESULTS

Mucosal immunization with H. pylori antigens and dmLT adjuvant induces protection against H. pylori infection.The objective of the current study was to investigate the adjuvant effect of dmLT and compare it to that of CT in inducing immune responses and protection against challenge with H. pylori bacteria. Two sublingual immunizations with 400 μg H. pylori lysate antigens and either 10 or 20 μg of dmLT and subsequent challenge with H. pylori bacteria at 2 weeks after the last immunization resulted in a significant (P < 0.001; 84-fold and 67-fold, respectively) reduction in the bacterial load 2 to 3 weeks after challenge, compared to that in unimmunized infected mice (Fig. 1A). Sublingual immunization with 20 μg dmLT adjuvant alone or 400 μg H. pylori lysate antigens alone gave no protective effect (Fig. 1A, inset). Furthermore, intragastric immunization with H. pylori lysate antigens and dmLT resulted in a significant (P < 0.05; 17-fold) decrease in the bacterial load compared to that in unimmunized infected mice (Fig. 1B). However, sublingual immunization was found to be superior to intragastric immunization in reducing bacterial loads in the stomachs of mice when using dmLT as an adjuvant (P < 0.001 [84-fold] versus P < 0.05 [17-fold], respectively) as well as CT (P < 0.001 [81-fold] versus P < 0.05 [16-fold]) (Fig. 1A and B). We also wanted to compare the efficacies of dmLT and CT in enhancing immune responses and protection against H. pylori infection at doses lower than 10 μg. In a separate experiment, mice were sublingually immunized two times at a biweekly interval with three different doses (3.3 μg, 1.1 μg, or 0.3 μg) of dmLT or CT together with H. pylori lysate antigens and challenged with H. pylori bacteria 1 week after the last immunization, and bacterial colonization was evaluated 4 weeks after challenge. Significant protection against H. pylori infection was seen when using 3.3-μg, 1.1-μg, and 0.3-μg doses of dmLT or CT compared to unimmunized infection controls (Fig. 1C). To further investigate whether mice challenged 8 weeks after the last sublingual immunization were still protected against H. pylori infection, we immunized groups of mice with 3.3 μg or 0.3 μg of adjuvant dmLT or CT together with H. pylori lysate antigens and challenged them with live bacteria at 8 weeks postimmunization. Unimmunized mice served as infection controls, and bacterial colonization was evaluated 4 weeks after challenge. We observed significant protection against H. pylori infection in immunized mice when challenged 8 weeks postimmunization when using 3.3 μg dmLT (P < 0.05; 22-fold) or 0.3 μg CT (P < 0.05; 25-fold) as an adjuvant (Fig. 1D). As the H. pylori lysate preparation is a cocktail of antigens, we further extended our studies to using two purified recombinant H. pylori antigens, HpaA and UreB (20). Sublingual immunization with a combination of HpaA and UreB together with dmLT (P < 0.01; 44-fold) or CT (P < 0.05; 22-fold) induced significant protection against H. pylori infection (Fig. 1E). In summary, we report a strong adjuvant effect of dmLT for vaccine-induced protection against H. pylori infection, which is fully comparable to that of CT as an adjuvant.

Fig 1
Fig 1

Protection against H. pylori infection by prophylactic sublingual immunization with H. pylori lysate antigens and dmLT or CT as an adjuvant. (A) Groups of mice were sublingually immunized with H. pylori lysate antigens and the indicated concentrations of adjuvant dmLT (black bars) or CT (gray bar) and challenged with live H. pylori bacteria. Unimmunized mice challenged at the same time point served as infection controls (white bar). Data are representative of 3 independent experiments. Inset, mice were sublingually administered H. pylori lysate antigens alone (Lys) or 20 μg dmLT alone (dmLT) or were left unimmunized (Inf) and challenged with live H. pylori bacteria. H. pylori colonization in the stomach was determined by quantitative culture. (B) Mice were intragastrically immunized with H. pylori lysate antigens and the indicated concentrations of adjuvant dmLT (black bar) or CT (gray bar) or were left unimmunized (white bar). Data are representative of 2 independent experiments. (C) Groups of mice were sublingually immunized with H. pylori lysate antigens and the indicated concentrations of adjuvant dmLT (black bars) or CT (gray bars) or were left unimmunized (white bar). Mice were challenged, and bacterial counts were assessed postchallenge. (D) Groups of mice were sublingually immunized with H. pylori lysate antigens and the indicated concentrations of adjuvant dmLT (black bars) or CT (gray bars) or were left unimmunized (white bar). Mice were challenged 8 weeks after the last immunization, and bacterial counts were assessed at 4 weeks postchallenge. (E) Mice were sublingually immunized with 50 μg purified recombinant antigens HpaA and UreB and the indicated concentrations of adjuvant dmLT (black bar) or CT (gray bar) or were left unimmunized (white bar). Evaluation of H. pylori colonization in individual mice was carried out by quantitative culture and expressed as log10 values of bacteria per stomach. Data represents means + standard errors of the means (SEM) (single experiment for the inset and panels C to E). All experiments were with 5 to 7 mice per group. Significant differences in bacterial load compared to that of unimmunized infection controls was assessed by ANOVA with Dunette's posttest (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

A potent cellular immune response and a systemic antibody response are induced after sublingual immunization with H. pylori antigens and dmLT adjuvant.To further evaluate the cellular immune responses, mesenteric lymph node (MLN) and spleen cells were isolated from groups of sublingually immunized and unimmunized mice both pre- and postchallenge with H. pylori and restimulated in vitro with H. pylori lysate antigens. To demonstrate the adjuvant effect of dmLT, mice were sublingually immunized with 20 μg dmLT alone or H. pylori lysate alone. Sublingual administration of lysate alone gave no significant increase in proliferation of MLN or spleen cells to H. pylori antigens either prechallenge or postchallenge compared to that in naive mice or unimmunized infected mice (Fig. 2A and B). In mice administered dmLT alone, a slight but significant increase of in vitro proliferation to H. pylori antigens was seen both pre- and postchallenge compared to that in naive mice or unimmunized infected mice (Fig. 2A and B). When dmLT was combined with H. pylori lysate antigens, sublingual immunization induced a significant and strong in vitro proliferation in MLN and spleen cells, both pre- and postchallenge, compared to that in naive or unimmunized infection controls (Fig. 2A and B). In a separate experiment, the adjuvant effects of dmLT and CT on the proliferative immune responses in the MLN and spleen were compared to those in unimmunized infection controls. Similar and significant increases of in vitro proliferation of both MLN and spleen cells were seen in mice sublingually immunized with H. pylori lysate antigens and dmLT or CT after challenge with H. pylori bacteria compared to unimmunized infection controls (Fig. 2C and D).

Fig 2
Fig 2

Enhanced in vitro proliferation of spleen and MLN cells isolated from sublingually immunized mice and increased serum IgG antibody response. Mice were sublingually immunized with either H. pylori lysate antigens alone, dmLT alone, or H. pylori lysate antigens and dmLT or CT or were left unimmunized. Mice were sacrificed after immunization (prechallenge) or after infection (postchallenge), and MLN, spleens, and sera were collected. (A and B) MLN (A) and spleen (B) cells from mice prechallenge (hatched bars) or 2 weeks after challenge (black bars) were collected, and single-cell suspensions were prepared and cultured with H. pylori antigens. Results represent a single experiment. (C and D) Groups of mice were sublingually immunized with H. pylori lysate antigens and dmLT (black bars) or CT (gray bars) and challenged with live H. pylori bacteria. Unimmunized mice, challenged at the same time point, served as infection controls (white). MLN (C) and spleen (D) cells were taken, and single cell suspensions were prepared and cultured with H. pylori antigens. Counts per minute of incorporated radioactive thymidine was used as a measure of proliferation of the cells. Results are representative of 2 independent experiments. (E) MLN and spleen cell culture supernatants from the proliferation assays for panels C and D were analyzed for cytokines. Values represent the fold increase of cytokines in 2 independent experiments. No statistical analysis was carried out. Values for nonstimulated wells (no antigen) were subtracted from values from stimulated wells. (F and G) ELISA for detecting H. pylori-specific antibodies in the serum was performed on the same mice as for panels A and B (F) and panels C and D (G). Serum antibodies are expressed as log10 titers. Data represents mean + SEM from 2 independent experiments. All experiments were done with 5 to 7 mice per group. Statistical significance was measured by ANOVA with Bonferroni's posttest (A, B, and F) or Dunette's posttest (C, D, and G) (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

In addition, we evaluated the cytokine secretion pattern of the cells (Fig. 2C and D). Analysis of cell culture supernatants revealed an increase in secretion of cytokines IL-17, IFN-γ, and tumor necrosis factor (TNF) from the MLN and spleen (Fig. 2E) cells isolated from mice sublingually immunized with H. pylori antigens and adjuvant dmLT or CT compared to unimmunized infection controls (Fig. 2E).

Finally, we evaluated the serum antibody responses to H. pylori antigens in the different groups of sublingually immunized mice compared to unimmunized mice. Sublingual immunization with dmLT alone or H. pylori lysate alone did not induce any significant increase in serum IgG compared to that in unimmunized mice either pre- or postchallenge (Fig. 2F). In contrast, the adjuvant effect of dmLT was evident before and after H. pylori challenge in the mice sublingually immunized with H. pylori lysate antigens and dmLT, with a significant increase in H. pylori-specific serum IgG compared to that in mice administered H. pylori lysate antigen alone or dmLT alone (Fig. 2F). In a separate experiment, mice were sublingually immunized with 10 μg dmLT or CT together with H. pylori lysate antigens and challenged 2 weeks after the last immunization. Two weeks after challenge, serum IgG was analyzed in all groups of mice. Sublingual immunization with H. pylori antigens and either dmLT or CT resulted in highly significant increases in levels of H. pylori-specific IgG in the sera (P < 0.001) compared to that in unimmunized infected controls (Fig. 2G).

Sublingual immunization with dmLT or CT as an adjuvant induces local T cell responses, cytokine secretion, and IgA responses in the stomach.It has previously been shown that Th1 and Th17 responses play essential roles in the vaccine-induced protection against H. pylori infection when using CT as an adjuvant (3, 21). Whether dmLT has a similar ability to induce Th1 and Th17 responses is not known. We thus analyzed the local immune responses in the stomachs of mice sublingually immunized with H. pylori lysate antigens and 10 μg adjuvant dmLT or CT and challenged with live H. pylori. A significant upregulation of CD4 gene transcripts was seen in the stomach at 2 weeks after challenge when mice were immunized with dmLT (P < 0.05) or CT (P < 0.01) as an adjuvant (Table 2). Further analysis of cytokine gene expression revealed an upregulation of IFN-γ, IL-17, and TNF in the stomachs of immunized mice compared to unimmunized infection controls (Table 2).

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Table 2

Cytokine and CD4 gene expression in the stomachs of mice sublingually immunized with H. pylori lysate antigens and dmLT or CT after challenge with H. pylori

We also evaluated the mucosal IgA response to H. pylori antigens and the effect of immunization. Sublingual immunization with dmLT alone or H. pylori lysate antigen alone gave no significant increase of the stomach IgA response, nor was an IgA response detected in infected unimmunized mice (Fig. 3A). Sublingual immunization with H. pylori lysate antigens and 10 μg dmLT resulted in an increased (although not significantly) H. pylori-specific IgA response locally in the stomach, while sublingual immunization with H. pylori lysate antigens and 10 μg CT induced a significant IgA response compared to that in unimmunized infection controls (Fig. 3B). In summary, the results indicate that the cytokine gene expression and IgA responses against H. pylori infection induced in the stomachs of immunized mice were comparable when using dmLT or CT as an adjuvant.

Fig 3
Fig 3

IgA responses in the stomach are increased after sublingual immunization with H. pylori lysate antigens and dmLT or CT. (A) Mice were sublingually immunized with H. pylori lysate antigens alone, dmLT alone, or H. pylori lysate antigens and dmLT or CT or were left unimmunized. Mice were sacrificed after immunization (prechallenge) or after infection (postchallenge), and stomachs were collected. (B) Groups of mice were sublingually immunized with H. pylori lysate antigens together with dmLT (black bar) or CT (gray bar) and challenged with live H. pylori bacteria. Unimmunized mice challenged at the same time point served as infection controls (white bar), and stomachs were collected. ELISA for detecting H. pylori-specific IgA antibodies in the stomach tissue was performed. Data represent mean values + SEM from a single experiment (A) or 2 independent experiments (B). All experiments are done with 3 to 7 mice per group. Significant differences in OD values were assessed by ANOVA with Bonferroni's posttest (A) or Dunette's posttest (B) (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Sublingual immunization with H. pylori lysate antigens and dmLT or CT is associated with local inflammation postchallenge.Previous studies in the H. pylori mouse model have shown that the immunization-induced effector response in the stomach causes enhanced inflammation, also referred to as postimmunization gastritis (22). We were interested in studying whether these effector responses were also induced in the stomachs of mice sublingually immunized with H. pylori lysate antigens and dmLT postchallenge. The histopathology of the stomach sections was evaluated and given a score for parietal cell destruction (1 to 6), chief cell destruction (1 to 6), and cell infiltration (1 to 4), with a total possible score of 16 (19). Indeed, mice sublingually immunized with H. pylori antigens and dmLT had a significantly higher (P < 0.001) inflammation score (Fig. 4A, B, and C) than unimmunized infection controls (Fig. 4A and D). As previously reported (15), mice immunized with H. pylori lysate antigen and CT as an adjuvant also had significantly higher inflammation scores than unimmunized infection controls (Fig. 4A and C).

Fig 4
Fig 4

Sublingual immunization enhances inflammation in the stomach of mice. Mice either sublingually immunized with H. pylori lysate antigens and dmLT (black bar) or CT (gray bar) or unimmunized (white bar) were challenged with live H. pylori bacteria, and stomach tissue was analyzed after challenge. (A) Inflammation score in formalin-fixed stomach tissue stained with hematoxylin and eosin. Data represent mean values + SEM and are representative of 2 independent experiments with 5 to 7 mice per group. (B to D) Wide-field images (magnification, ×100) are shown for mice immunized with H. pylori lysate antigens and dmLT (B) or CT (C) and for unimmunized infection controls (D). Significant differences between group scoring values were assessed by ANOVA with Dunette's posttest (**, P < 0.01; ***, P < 0.001).

DISCUSSION

We report in this study that sublingual immunization with H. pylori lysate antigens together with the nontoxic dmLT as an adjuvant conferred protection against H. pylori infection to the same extent as when using CT as an adjuvant. The cellular immune responses to H. pylori in MLN and spleen, and secretion of cytokines IL-17, IFN-γ, and TNF, as well as gene expression for these cytokines in stomach tissue, were elevated to similar levels after immunization when using dmLT and CT as adjuvants. Our data indicate that when given together with an experimental H. pylori vaccine, dmLT is an effective adjuvant, promoting strong B and T cell immune responses to H. pylori antigens and protection against challenge with H. pylori bacteria.

Previous studies evaluating adjuvants in a H. pylori vaccine have used single mutant forms of LT with minimal ADP ribosylating activity, e.g., LTK63 or mLT [LT(R192G)] (6, 23–26). LTK63 administered intragastrically together with formalin-killed H. pylori bacteria was reported to confer protection against H. pylori infection in mice and also to induce protection against reinfection with H. pylori (27). Oral immunization with an H. pylori whole-cell vaccine and the adjuvant mLT (R192G) induced protection against H. pylori infection in mice and also enhanced antibody responses in humans, although in humans there were reported diarrheal side effects from the adjuvant used in the immunization (6, 12, 24). Recently, a lyophilized H. pylori whole-cell vaccine administered orally together with dmLT was shown to be protective in mice, but in that study there was no analysis of T cell responses or any comparison to using CT as a mucosal adjuvant (12). In the present study, we have evaluated in depth the T and B cell responses induced by sublingual immunization with H. pylori antigens and dmLT and have compared the adjuvant effect with that of the gold standard mucosal adjuvant, CT. We also report that when using dmLT as an adjuvant, the sublingual route of immunization was significantly better than the intragastric route in inducing protection against H. pylori infection in mice. Titration of the doses of the adjuvant further supports the comparable adjuvant activities of dmLT and CT, and we report that with both adjuvants the protection induced against H. pylori infection was significant even with increases in the interval between the last immunization and infection. However, we cannot completely rule out that the differences in storage and handling of CT and dmLT could potentially influence the stability of the molecule and subsequently the adjuvant effects against H. pylori infection.

CD4+ T cells have been shown to be essential for vaccine-induced protection against H. pylori (22). Recently it has become clear that after vaccination against H. pylori using CT as an adjuvant, CD4+ T helper cells of both IFN-γ-producing Th1 and IL-17-producing Th17 phenotypes are induced, which after challenge with live H. pylori bacteria migrate into the stomach (3, 21). We and others have also shown that IL-17 is essential for the vaccine-induced protection against H. pylori infection, whereas IFN-γ may be dispensable (3, 21, 28). We now report that sublingual immunization with dmLT and H. pylori lysate antigens strongly increases IFN-γ and IL-17A gene expression in the stomachs of mice and also the production of these cytokines by MLN and spleen cells after in vitro stimulation with H. pylori antigens. Effective induction of IL-17 is desirable when designing a vaccine against H. pylori infection, since one of the functions of IL-17 is to recruit neutrophils, which have been shown to be essential in reducing the bacterial loads in the stomachs of vaccinated mice (28), although DeLyria et al. have reported that in mice immunized with H. pylori lysate antigens and CT, neutrophils can be recruited to the stomach after challenge independent of IL-17A (29). However, IL-17 has multiple roles, and we believe that its main function in H. pylori infection is to promote bacterial clearance by possibly recruiting bactericidal neutrophils and also to stimulate local IgA antibody formation (30). We noted that vaccination with H. pylori antigens together with either dmLT or CT as an adjuvant was associated with enhanced inflammation in the stomach (postimmunization gastritis) after challenge with live bacteria. In the H. pylori model, postimmunization gastritis is considered to be an important component of the local immune response associated with protection in vaccinated mice, and it is known to resolve after eradication of the bacteria (31).

We report that both dmLT and CT induced a significantly stronger protection against H. pylori infection when administered sublingually than when administered intragastrically. The explanation for this difference could be that the adjuvants might be more sensitive to the stomach environment and/or that they are much more diluted when administered intragastrically than when administered sublingually. The sublingual mucosa has few scattered major histocompatibility complex class II-positive (MHC-II+) dendritic cells (DCs) during steady state, but after sublingual administration of CT, a strong influx of such cells can be seen within 2 h (32). The MHC-II+ dendritic cells in the sublingual mucosa sample the antigen and migrate to the cervical lymph nodes, where they present antigen to T cells (32, 33). Whether dmLT induces a similar influx of DCs into the sublingual mucosa is not known; however, it can be envisaged that a mechanism similar to that of CT may contribute to its efficiency as an adjuvant in inducing T cell responses and protection against H. pylori infection. The immune responses induced by the sublingual route of immunization persisted for up to 2 months postimmunization, as we could detect proliferation of spleen and MLN cells (data not shown) and protection against H. pylori infection.

Since H. pylori colonizes but rarely invades the stomach epithelium, the potential role of antibodies in protection against H. pylori infection has been extensively studied. We show an increase in H. pylori-specific systemic IgG and local IgA antibodies in mice immunized with H. pylori lysate antigens and dmLT to the same levels as in mice immunized with H. pylori lysate antigens and CT. Akhiani et al. have shown that protection in B cell-deficient mice (lacking antibodies) was associated with enhanced gastritis, suggesting that B cells and/or antibodies play a role in regulating inflammation in the stomach (34). In a recent birth cohort study, it was reported that infants who were breast-feeding from mothers with high titers of H. pylori-specific IgA antibodies had a significant delay in acquisition of H. pylori infection in comparison with infants feeding on breast milk with low titers of IgA antibody (35). Thus, although antibodies do not appear to be essential in vaccine-induced protection against H. pylori infection, they may play a role in immune exclusion and probably also in dampening inflammation during H. pylori infection.

Both the CT and dmLT adjuvants used in the current study belong to the AB group of toxins, which are characterized by an A subunit and a B pentamer subunit (AB5 complex with an ADP-ribosyltransferase active A1 component linked to a pentamer of B subunits via an A2 fragment [36]). In CT, the A subunit is proteolytically cleaved by a V. cholerae protease into CTA1 and CTA2 fragments already upon secretion of the toxin from the bacteria, while in LT-producing E. coli, the A subunit is not excised into the analogous LTA1 and LTA2 components until the toxin is being exposed to trypsin in the intestine (37). The cellular uptake of CT or LT through a series of events leads to a transport of A1 (CTA1 or LTA1) into the cytoplasm and ADP-ribosylation of Gs proteins, resulting in activation of adenylate cyclase and increased intracellular levels of cyclic AMP (cAMP). The mutations introduced in dmLT renders it resistant to trypsin cleavage, leading to a dramatically (>100-fold) reduced ADP-ribosyltransferase activity and induction of cAMP production (11). Previous studies have shown a close association between the adjuvant activities of CT, LT, and their derivatives and ADP-ribolysation activity (26, 36, 37).

The question still remains how dmLT with its minimal ADP-ribosylating activity (11, 38) can be as potent as CT in inducing mucosal immune responses. In an attempt to elucidate the adjuvant mechanism of dmLT, Norton et al. (11) reported that the mutations in the dmLT molecule that prevent the proteolytic cleavage of LTA subunit into A1 and A2 subunits also led to rapid degradation of the A subunit in the cytosol of intestinal epithelial cells (11). They also showed that a 1,000-fold-higher dose of dmLT was needed to induce cAMP in Caco-2 cells in vitro, compared to that for native LT (11). Thus, not only did dmLT induce much-reduced cAMP formation in cells, but its A subunit rapidly degraded so as to present no enterotoxicity in a patent mouse fluid secretion assay. It could be that the very short half-life of the detectable A subunit of dmLT within the cells allows stimulation of amounts of cAMP that are low but sufficient for the adjuvanticity without leading to enterotoxicity, as also proposed by Norton et al. (11). Further studies to elucidate the effect of dmLT on different cell types and the adjuvant function of dmLT and CT in the presence or absence of cAMP inhibitors might help to clarify whether some level of cAMP induction is still important for the adjuvant function of dmLT.

In conclusion, we report that sublingual immunization with the nontoxic dmLT adjuvant together with H. pylori antigens can induce protection against H. pylori infection fully comparable to that after immunization using CT as adjuvant. Furthermore, when using dmLT as an adjuvant, the sublingual route of immunization was superior to the intragastric route of immunization in inducing protection against H. pylori infection. Cellular and antibody responses to H. pylori antigens were also similar using dmLT and CT as adjuvants. We conclude that dmLT is an attractive adjuvant for use in a mucosal vaccine against H. pylori infection, since it induces the appropriate immune responses necessary for protection against H. pylori infection and appears to be nontoxic not only in mice (11) but also in human volunteers given dmLT orally together with bicarbonate (L. Bourgeois, personal communication). Further studies will be necessary to determine the feasibility of using dmLT as a mucosal adjuvant in a vaccine against H. pylori infection in humans.

ACKNOWLEDGMENTS

This work was supported by grants from the Swedish Research Council (Medicine), the Marianne and Marcus Wallenberg, Åke Wiberg, Adlerbert, Magnus Bergvall, and Nanna Svartz foundations, and the Swedish Foundation for Strategic Research to the Centre for Mucosal Immunobiology and Vaccines (MIVAC).

We acknowledge the excellent technical assistance provided by Margareta Blomquist and Annelie Ekman.

We declare no conflict of interest.

FOOTNOTES

    • Received 11 December 2012.
    • Returned for modification 26 January 2013.
    • Accepted 16 February 2013.
    • Accepted manuscript posted online 25 February 2013.

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