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- MATERIALS AND METHODS
Development of an effective vaccine for controlling H. pylori-associated infection, which is present in about half the people in the world, is a priority. The H. pylori outer inflammatory protein (oipA) has been demonstrated to be a potential antigen for a vaccine. In the present study, use of oipA gene encoded construct (poipA) for C57BL/6 mice vaccination was investigated. Whether co-delivery of IL-2 gene encoded construct (pIL-2) and B subunit heat-labile toxin of Escherichia coli gene encoded construct (pLTB) can modulate the immune response and enhance DNA vaccine efficacy was also explored. Our results demonstrated that poipA administered intradermally (‘gene gun’ immunization) promoted a strong Th2 immune response, whereas co-delivery of either pIL-2 or pLTB adjuvant elicited a Th1-biased immune response. PoipA administered with both pIL-2 and pLTB adjuvants promoted a strong Th1 immune response. Regardless of the different immune responses promoted by the various vaccination regimes, all immunized mice had smaller bacterial loads after H. pylori challenge than did PBS negative and pVAX1 mock controls. Co-delivery of adjuvant(s) enhances poipA DNA vaccine efficacy by shifting the immune response from being Th2 to being Th1-biased, which results in a greater reduction in bacterial load after H. pylori challenge. Both prophylactic and therapeutic vaccination can achieve sterile immunity in some subjects.
Helicobacter pylori is a spiral bacterium that colonizes the gastric mucosa of more than half the world's population. Infection with H. pylori is associated with the development of chronic active gastritis, peptic ulcer and gastric cancer (1). The current management of H. pylori infection relies on antibiotic therapy. This strategy has many drawbacks, including treatment failure due to emergence of bacterial resistance, poor patient compliance, side effects of the antibiotics and high cost of treatment. Two significant drawbacks of antibiotic therapy are the failure to prevent re-infection and the increase in bacterial resistance (2). Therefore, there is an urgent need to identify alternative approaches to prevention and treatment of H. pylori infection.
Some researchers have explored and tested vaccination as a strategy for combating H. pylori and found it to be effective (3–7). A number of candidate antigens, including H. pylori urease A/B subunits, catalase, neutrophil activating protein, and heat shock proteins, administered either as a single antigen or co-delivered with various adjuvants, have been reported to induce protective effects in prophylactic and therapeutic studies (5,8,9).
Recently, researchers identified a novel virulence factor: outer inflammatory protein, designated as oipA, which is encoded by an inflammation-related gene located approximately 100 kb from the Cag pathogenicity island on the chromosome (10). It correlates significantly with the clinical outcome of H. pylori infection and has been demonstrated to be a potent protective antigen (11,12).
A priority in H. pylori vaccine development has been to clarify the types of immune responses that are effective in reducing the bacterial load of H. pylori. Previous studies have demonstrated that a Th2 immune response is required for prevention of H. pylori infection. However, a Th1-biased immune response is required for eradication of H. pylori colonization. Hence, an ideal vaccine against H. pylori would mainly trigger Th1-biased immune responses. Studies that have attempted to shift the immune response after DNA vaccination from a Th2 to a Th1 type have emphasized the role of adjuvants in modulating the type of immune response (13–15).
DNA vaccination is an important immunization strategy that has many of the characteristics of an ideal vaccine, including the elicitation of broad immune responses and long-lasting immunity, simplicity and cost-benefit efficacy (6). However, because DNA vaccines tend to trigger a Th2-biased response, they are unattractive as anti-H. pylori vaccines, which must efficiently prime a Th1 immune response(7). In the present study, we aimed to construct an oipA-encoding DNA vaccine and to deliver it with mouse IL-2 gene and LTB gene encoding plasmids as adjuvants. We also evaluated the efficacy of the DNA vaccine against H. pylori challenge in C57BL/6 mice.
- Top of page
- MATERIALS AND METHODS
In the present study we demonstrated that an H. pylori oipA encoding construct is capable of inducing humoral and cellular responses in immunized mice. The antibody response profiles elicited by the DNA vaccine alone administered intradermally (the gene gun method) showed that it produced a Th2 immune response, while co-delivery of IL-2 and LTB gene encoding constructs promoted a Th1-biased immune response. Regardless of the different immune responses promoted by the various combined vaccination protocols, all immunized mice had reduced bacteria loads after H. pylori challenge. pIL-2 and pLTB enhanced the efficacy of the poipA DNA vaccine by shifting the immune response from a Th2 to a Th1 type, the latter playing an important role in both control and eradication after H. pylori challenge.
Although different vaccination protocols in various animal models of infection and in human trials have been studied (17), the protective mechanism against H. pylori infection is still unclear. Data obtained with transgenic mice suggest that major histocompatibility complex-II restricted CD4-positive T cells may play a fundamental role in protection (18, 19). In addition, it has been proposed that a shift from the Th1 cytokine response induced by H. pylori infection to a Th2 response is a protective mechanism (20). However, recent studies have contradicted the Th2 paradigm and demonstrated the importance of the Th1 response in effective protection following vaccination against H. pylori (21–23). Blanchard et al. have also proposed a role for regulatory T cell in H. pylori vaccination and suggested that the site of T cell activation influences protection (24). Therefore, the definite protective mechanism against H. pylori challenge still needs to be explored. Nevertheless our results show that either poipA alone, or poipA co-delivered with pIL-2 or/and pLTB, promotes protective immunity, via Th2 and Th1-biased immune responses, respectively. However, mice administered poipA co-delivered with pIL-2 or/and pLTB have a significantly reduced bacterial load. In our preliminary study, in which H. pylori-infected mice were vaccinated intradermally with poipA, co-delivered with pIL-2 or/and pLTB, similar results were achieved by therapeutic vaccination (data not shown). Therefore, poipA co-delivered with pIL-2 and pLTB could be a promising candidate for the development of prophylactic and therapeutic vaccines against H. pylori.
Several factors contribute to the immune responses to DNA vaccine, including the properties of the gene encoding the antigen, the route and dose of administration, and co-delivery of adjuvants. Todoroki et al. demonstrated that H. pylori heat shock proteins encoding pcDNA3.1-HspA construct confer less protection against the bacterium than does the pcDNA3.1-HspB construct (3). Though the pcDNA 3.1-HspA construct induced higher IgG and IgA responses, the pcDNA 3.1-HspB construct resulted in a Th0-like response and greatly reduced inflammation in infected mice. In our previous study, intramuscular injection of a high dose (100 microgram per dose) of oipA-encoding pVAX1-oipA construct stimulated Th1-biased responses (unpublished data). However, intradermal delivery of nanogram amounts (about 5 microgram per dose) of the same construct via gene gun favors priming of Th2-biased responses. This is in agreement with the findings of Lin et al. (25). Because DNA vaccination tends to prime Th2 responses preferentially, it has seemed relatively unattractive as a H. pylori vaccine, for which priming of Th1 immune responses is the main requirement.
In order to overcome the inferior, low magnitude, immune response elicited by DNA vaccination, researchers have explored the role of various adjuvants in modulating and enhancing the immune response primed by immunization. The CT from Vibrio cholerae and LT from enterotoxigenic strains of E.coli are potent mucosal adjuvants. It has been found that CT induces a Th2 response (26), whereas LT induces a Th1-biased immune response (27, 28). The B subunits of these toxins are less potent than the active toxins. However, they have practical potential as carrier molecules, having shown less toxicity in both experimental animals and human immunization (29–31). Research has also shown that co-administration of cytokine proteins or cytokine gene-encoded plasmids enhances comprehensive humoral and cellular immune responses (32–35). Recently, synthetic CpGs, which prime a Th1 immune response and have shown some encouraging results in H. pylori vaccines, have been approved for use as adjuvants in humans (21, 23, 36–38). In this study, we used pIL-2 and pLTB as adjuvants in poipA DNA vaccination. Our results indicate that co-delivery of adjuvants modulates the type and magnitude of immune responses. Furthermore, we found that two of the immunized mice developed sterile immunity, demonstrating that effective adjuvants can eradicate the organisms after H. pylori challenge.
The ability of DNA vaccines to induce production of cytokines depends on the use of adjuvants. Our data demonstrate that mice immunized with poipA co-delivered with pIL-2 and/or pLTB have significant increases in IFN-γ secretion with no significant changes in IL-4 secretion, indicated elicitation of a Th1-biased immune type. All mice in all immunization groups had significantly increased IL-2, IL-10, and IL-12 secretion. However, the secretion of IL-2, IL-10, and IL-12 is not specifically associated with either Th1 or Th2 immune responses. Recent studies have also claimed that IL-12 might be an important mediator against H. pylori infection. Akhiani et al. demonstrated that IL-12 is necessary for protection against H. pylori infection in IL-12 knockout mice, which are unable to induce protective immune responses upon challenge (39). Taylor et al. also demonstrated that increases in IL-12 secretion correlate well with long-term protection (40). In addition, IL-10 has proven to be an important regulator of the inflammatory response to H. pylori. Akhiani et al. studied IL-10-deficient mice that had developed severe hyperplastic gastritis (41). They proposed that expression of IL-10 may be an anti-inflammatory marker for predicting the severity of post-immunization gastritis in vaccine studies. The relationship between IL-10 and post-immunization gastritis and the histopathology primed by H. pylori vaccination should be investigated.
pVAX1, the first Food and Drug Administration-approved vector used safely in animal and human DNA vaccines, was constructed by modifying the pcDNA3.1 vector. In the present study, we demonstrated the protective ability of DNA vaccine encoding H. pylori oipA using the pVAX1 plasmid. In addition, we also constructed IL-2 and LTB gene encoding plasmids separately. We did not use a bicistronic vector to construct the DNA vaccine, because we believed that preparing DNA vaccine and adjuvants separately would provide more flexibility. For example, in a prime and boost immunization strategy, an antigen can be administered alone or co-administered with adjuvants. Multiple antigens can be constructed separately and co-delivered with the same adjuvants. To our knowledge, this is the first study showing that a combination of IL-2 and LTB gene encoding constructs shifts immune responses and enhances the efficacy of DNA vaccine. In future studies, various ‘cocktails’ and proportions of DNA vaccines and adjuvants and various routes of administration should be investigated to achieve the most effective protection against H. pylori.