Systemic immune responses to oral administration of recombinant attenuated Salmonella typhimurium expressing Helicobacter pylori urease in mice

Abstract

AIM: To evaluate whether attenuated Salmonella typhimurium producing Helicobacter pylori (H pylori) urease subunit B (UreB) could induce systemic immune responses against H pylori infection.

METHODS: Attenuated S. typhimurium SL3261 was used as a live carrier of plasmid pTC01-UreB, which encodes recombinant H pylori UreB protein. Balb/c mice were given oral immunization with two doses of SL3261/pTC01-UreB at a 3-wk interval. Twelve weeks after oral immunization of mice, serum IgG antibodies were evaluated by ELISA assay. Gamma interferon (IFN-γ) and interleukin 10 (IL-10) in the supernatant of spleen cell culture were also assessed by ELISA.

RESULTS: After oral immunization of mice, serum specific IgG antibodies against UreB in vaccine group were much higher than that in PBS and native Salmonella SL3261 control groups (A₄₅₀, 0.373±0.100 vs 0.053±0.022, 0.142±0.039, respectively, P<0.01). Moreover, IFN-γ in vaccine group was on average 167.53±29.93 pg/mL, which showed a significant increase vs that of PBS control group (35.68±3.55 pg/mL, P<0.01). There was also a tremendous increase of IL-10 in vaccine group compared to PBS and SL3261 control groups (275.13±27.65 pg/mL vs 56.00±7.15 pg/mL, 68.02±15.03 pg/mL, respectively, P<0.01). In addition, no obvious side effects in mice and no change in gastric inflammation were observed.

CONCLUSION: The multiple oral immunizations with the attenuated S. typhimurium expressing H pylori UreB could induce significant systemic immune responses, suggesting it may be used as oral vaccine against H pylori infection.

Key Words: Helicobacter pylori, Salmonella typhimurium, Vaccine, Systemic immune

INTRODUCTION

Helicobacter pylori (H pylori) is one of the most common bacteria worldwide, which infects more than 50% of the human population[1]. It is generally recognized that H pylori infection is a major etiological factor in chronic gastritis, peptic ulcer disease, gastric adenocarcinoma and gastric B-cell lymphoma (MALT). The current therapy, based on the use of a proton-pump inhibitor and antibiotics, is efficacious but faces many problems such as patient compliance, possible recurrence of infection, complex dosing, costs and various side effects, and most importantly, development of antibiotic resistance. These compromise widespread clinical use. As a consequence, new strategies for the prevention and eradication of H pylori infections are being explored. Vaccines are an attractive option, because they are both effective and economic in use. It is widely accepted that, given the worldwide prevalence of H pylori infection, vaccination would be a preferable strategy[2]. A number of trials indicate that attenuated Salmonella typhimurium strains can be used to deliver foreign antigens[3]. So, we had established recombinant attenuated S. typhimurium SL3261 expressing H pylori urease B subunit (UreB), which could elicit strong mucosal immune responses[4]. The purpose of the present study was to determine whether it could induce specific immune responses and whether it could be used as oral vaccine against H pylori infection.

MATERIALS AND METHODS

Animals

Four to six weeks old female Balb/c mice were purchased from the Animal Resources Center, Fourth Military Medical University, Guangzhou. The animals were fed on a commercial diet and given water ad libitum.

Bacterial strains, media, and growth

The attenuated S. typhimurium SL3261 (S. typhimurium WARY hisG 46 aroA del 407 Fusaricres, etc.) kindly provided by Professor DamingRen (Institute of Genetics, Fudan University, Shanghai) were grown routinely at 37 °C in solid or liquid Luria medium.

Immunization of mice

Three groups of mice including controls were used as follows: (1) PBS control group was non-immunized mice that received PBS; (2) native Salmonella control group was mice that received attenuated S. typhimurium SL3261 strain; and (3) the vaccine group (SL3261/pTC01-UreB group) was mice immunized with S. typhimurium SL3261/pTC01-UreB. Prior to immunizations mice were left overnight without solid food and 4 h without water. One hundred microliters of 3% sodium bicarbonate were as given orally using a stainless steel catheter to neutralize the stomach pH. Immediately after stomach neutralization, mice from the PBS control group received 100 μL PBS, and mice from the Salmonella control group and vaccine group, received 5.0×10⁹ cfu of S. typhimurium strain SL3261 or SL3261/pTC01-UreB, respectively, in a total volume of 100 μL. Water and food were returned to the mice after immunization. Three groups of mice were given twice oral dose treatment at a 3-wk interval.

ELISA assays

Twelve weeks after final oral immunization of mice, 150 μL of blood were collected retro-orbitally. Anti-UreB IgG antibodies in serum were detected by sandwich ELISA[5].

Cytokine measurement

Spleen cells were isolated by forcing the tissue through nylon cell strainers and resuspended in complete medium containing 15% fetal calf serum as routine method. After 48 h, the supernatant of spleen cell culture was collected. The presence of gamma interferon (IFN-γ) and interleukin 10 (IL-10) in the supernatant was detected by ELISA.

Analysis of anti-urease antibodies in mouse sera by immunoblotting

H pylori strain SS1 whole-cell lysates were analyzed by Western blotting. After SDS-PAGE, a 1:50 dilution of mouse serum in 5% non-fat milk-TBS was added to the strips[5].

Histology

Biopsy specimens from the antrum and the corpus were fixed in 10% buffered formalin, and 4-μm sections were cut. Sections were stained with HE to grade gastritis.

In vitro recombinant attenuated S. typhimurium SL3261/pTC01-UreB stability

S. typhimurium SL3261/pTC01-UreB was grown in Luria broth at 37 °C for 80 generations. For every 10 generations, number of chloromycetin resistant cfu was determined on LB-agar plates supplemented with 170 μg/mL chloromycetin. In the final generation, the expression of UreB protein was evaluated by Western blotting.

Statistical analysis

The differences in anti-UreB IgA and IgG antibodies as well as IFN-γ and IL-10 from immunized and non-immunized mice were evaluated using t-test. Differences were considered significant at P values <0.01.

RESULTS

Serum anti-UreB IgA antibodies in protected mice

As indicated in Table 1, the multiple oral immunizations with SL3261/pTC01-UreB could induce significant H pylori-specific serum IgG responses (P<0.01) (Table 1).

Table 1 Detection of intestinal IgA and serum IgG (A₄₅₀, mean±SD).

	PBS group	SL3261 group	Vaccine group
Serum IgG	0.053±0.022	0.142±0.039	0.373±0.100b

^bP<0.01 vs PBS and SL3261 control groups.

Measurement of cytokine IFN-γ and IL-10

The difference in IFN-γ and IL-10 between immunized mice and non-immunized mice (PBS) was significant (P<0.01). Although the difference in IL-10 between mice immunized with strain SL3261 alone and strain SL3261/pTC01-UreB was also significant (P<0.01), there was no significant increase of IFN-γ between SL3261 group and SL3261/pTC01-UreB group (Table 2).

Table 2 Measurement of IFN-γ and IL-10 (pg/mL, mean±SD).

	PBS group	SL3261 group	Vaccine group
IFN-g	35.68±3.55	118.74±16.84b	167.53±29.93b
IL-10	56.00±7.15	68.02±15.03	275.13±27.65d

^bP<0.01 vs PBS group.

^dP<0.01 vs PBS and SL3261 groups.

Detection of anti-UreB antibodies in mice immunized with the salmonella vaccine strain

The protein of 61 ku, corresponding to UreB, was recognized by serum from the vaccine group mice. This band was not detectable in strips tested from non-immunized mice or mice immunized with Salmonella only. But other non-specific bands were also observed in most lanes.

Histology of mice stomach tissue

Histology showed that the grade of gastritis had no difference between the vaccine group and control groups.

Observation of general character

In the initial stage of immunization, the mice had a jaded appetite and weight loss. No diarrhea and death occurred in immunized mice.

Recombinant attenuated S. typhimurium SL3261/pTC01-UreB stability

After 80 generations of continuous culture, nearly 100% of the SL3261/pTC01-UreB bacterial colonies were chloromycetin resistant and could express UreB protein.

DISCUSSION

Since the idea of vaccine against H pylori infection was raised in 1990, scientists have placed emphasis on the study of H pylori protein vaccine. Urease is necessary for colonization of H pylori and expressed at high levels by all H pylori strains. Furthermore, because of the high immunogenicity and the high conservation (98%) between different H pylori strains, the urease is regarded as a promising candidate of H pylori vaccine. Studies indicated that oral immunization of natural or recombinant UreB in combination with Cholera toxin (CT) or Escherichia coli labile toxin (LT) could protect mice from H pylori infection[6]. But in all successful vaccination protocols, mucosal adjuvants, i.e., CT or LT, are necessary. One major drawback with these bacterial adjuvants is that they are toxic in humans[7]. This restricts the use of oral protein vaccine. Thus, the development of new vaccine strategies for H pylori is indispensable. Live attenuated S. typhimurium vaccine strains expressing foreign antigens are a promising new generation of vaccines that induce remarkably strong and specific immune responses in the mammalian hosts when given by mucosal immunization routes such as the oral, nasal, rectal and vaginal routes. Moreover, live S. typhimurium used as vector for heterologous antigens do not require antigen purification, and they not only can protect antigen from degradation and denaturation in stomach but also express adjuvant activity that prevents induction of oral tolerance. S. typhimurium SL3261 is an aroA gene mutant that is invasive yet nonvirulent. Human trials indicate that attenuated S. typhimurium strain is well tolerated and highly immunogenic and may be useful for the delivery of foreign antigens and immunoprotection against a variety of pathogens including H pylori. So S. typhimurium SL3261 is an efficient live bacterial vector[8].

The expression of protective immunity against gut pathogens is normally dependent both on local (mucosal) and systemic mechanisms[2]. We cloned successfully the prokaryotic vector pTC01-UreB and established attenuated S. typhimurium SL3261/pTC01-UreB expressing UreB subunit. We have shown that the multiple oral immunizations with SL3261/pTC01-UreB could elicit significantly H pylori-specific mucosal IgA response[4]. The increases in serum IgG and mucosal IgA anti-UreB antibodies indicate that UreB delivered by Salmonella is highly immunogenic and capable of inducing specific humoral immunity and mucosal immunity.

At present, it is generally acknowledged that protective immunization has been associated with a progressive disappearance of Th1 cells and the development of a Th2 response[9]. In a recent study, Guy and coworkers showed strong Th1 and Th2 responses elicited better protection than a predominant Th2 type response only. They thought that an appropriate balance between Th1 and Th2 type responses is required to achieve complete protection[10]. SL3261/pTC01-UreB was vaccinated through the route of mucosal administration. Mucosal tissues favor the development of Th2-type responses[11]. Our results showed that there was significant increase of IFN-γ and IL-10 in SL3261/pTC01-UreB group. These suggest that SL3261/pTC01-UreB can induce strong Th1 and Th2 responses. Nevertheless, the level of IFN-γ had no difference between SL3261/pTC01-UreB group and SL3261control group. And the level of IFN-γ in SL3261 control mice was also higher than in PBS control mice. These facts demonstrate that the strong Th1 response is associated with attenuated S. typhimurium used as vaccine vector, because attenuated S. typhimurium is a kind of invasive pathogens[12].

Genetic stabilization of foreign antigen expression is a crucial step in the development of an immunogenic vaccine strain. Our data showed that there was no pTC01-UreB plasmid loss in SL3261/pTC01-UreB after 80 generations of continuous culture. Moreover, expression of UreB from S. typhimurium SL3261/pTC01-UreB was also obtained at different phases of growth including in the stationary phase. All these clarify that the recombinant plasmid pTC01-UreB is stable in SL3261 and has no obvious toxicity. In addition, the facts that no obvious side effects for mice and no change in gastric inflammation were observed indicate S. typhimurium SL3261/pTC01-UreB is safe. Our results show that the attenuated S. typhimurium expressing H pylori UreB may be used as oral vaccine against H pylori infection. Henceforth, we need to consummate anti- H pylori S. typhimurium vaccine and evaluate its effect.