Helicobacter pylori thiolperoxidase as a protective antigen in single- and multi-component vaccines
Highlights
► Helicobacter pylori thiolperoxidase was a protective systemic vaccine antigen. ► H. pylori thiolperoxidase was a protective mucosal vaccine antigen. ► Vaccinating with a combination of antioxidant enzymes did not improve protection. ► The immunogenicity of individual antigens was reduced when given in combination via the nasal route.
Introduction
The development of an effective vaccine against Helicobacter pylori has been an enticing prospect for many years. H. pylori, a Gram-negative pathogenic bacterium of the human stomach, is now understood to be the leading cause of gastritis [1]; the chronic inflammation elicited by its colonisation is directly implicated in the development of gastric adenocarcinoma [2] and gastric MALT (mucosal-associated lymphoid tissue) lymphoma [3]. Estimates suggest that a hypothetical infant vaccination program with a preventative efficacy of 80% would be a highly cost-effective method of control, and even a vaccine with an efficacy as low as 20% may still prove cost-effective in the long-term due to the ability to prevent gastric neoplasia [4].
Vaccine development is driven in large part by the discovery of antigens that induce an effective immune response. Several H. pylori proteins have already been reported as effective vaccine antigens. Urease is the most extensively studied of these antigens [5], [6], [7], [8], [9], [10], but a range of others have been assessed, including VacA cytotoxin [11], heat-shock proteins [12], HpaA [13], NapA [14] and CagA [11]. Each of these antigens, when combined with a suitable adjuvant, is capable of reducing H. pylori colonisation when administered to mice, proving that it is possible to induce a protective effect through vaccination. However, the vaccine formulations described are yet to generate reproducible sterilising immunity.
The H. pylori enzyme thiolperoxidase (Tpx) is a thioredoxin-dependent peroxiredoxin with non-specific peroxidase activity, and is reported to be involved in defence against oxidative damage [15]. The enzyme fulfils many desirable attributes of protective vaccine antigens. It is important for survival in vivo, with Tpx-deficient mutant strains demonstrating increased sensitivity to oxidative stress and failure to colonise mice [16]. Furthermore, the enzyme presents at the surface of the organism [17] and in outer membrane vesicles [18], and is abundant within the proteome [19]. This paper explores the potential of Tpx as a mucosal and systemic vaccine antigen.
In addition, the previous characterisation of two other H. pylori antioxidant enzymes as partially protective vaccine antigens – catalase (KatA) and superoxide dismutase (SOD) [20], [21] – provided the opportunity to assess these antigens in a combination vaccine with Tpx. The functional relationship between these three antigens suggested a potential for synergistic vaccine activity through impairment of different branches of the antioxidant system. As defects in multiple antioxidant enzymes have been shown to cause additional sensitivity to oxidative stress [22], it was proposed that using these antigens in combination may increase the efficacy of the vaccine.
Section snippets
H. pylori culture
H. pylori strains SS1 [23] and J99 [24] were grown on horse blood agar plates (Blood Agar Base No. 2, 2.5 μg/mL amphotericin B (Sigma–Aldrich, Castle Hill, Australia) and Skirrow's Selective Supplements (Oxoid, Basingstoke, UK) and 5% horse blood (Biolab, Mulgrave, Australia) in an anaerobic jar with a microaerophilic gas generating kit (Oxoid) for 2 days at 37 °C. For infection experiments and extraction of genomic DNA, bacteria were subcultured into brain heart infusion broth (BHI; Oxoid)
Tpx is a protective vaccine antigen when delivered via subcutaneous and nasal routes
Evaluation of Tpx as a vaccine antigen was first assessed by subcutaneous immunisation. In addition to Tpx, both SOD and KatA have been demonstrated to be protective antigens against H. pylori [20], [21]. Moreover, as these enzymes are integral components of the H. pylori anti-oxidative enzyme defence system, we hypothesised that a combined vaccination comprised of these three antigens (Tri-Vac) may produce a synergistic protective effect by generating immune responses to different components
Discussion
Antioxidant enzymes are an important defence mechanism for H. pylori, providing vital protection against the damaging effects of reactive oxygen species (ROS). These toxic compounds are generated by a variety of sources in response to infection. Neutrophils and other phagocytes produce ROS during gastritis [34], [35], and gastric epithelial cells have also been shown to generate ROS on exposure to H. pylori [36]. Without a well-developed antioxidant system, H. pylori is susceptible to
Acknowledgements
This work was supported by the Victorian Government's Operational Infrastructure Support Program and by Australian Research Council (ARC) Linkage Project grant LP0560720. PS is supported by a Senior Research Fellowship from the National Health and Medical Research Council of Australia. The funders had no role in the experiments performed, or the preparation of this manuscript.
Contributors: AS, ALE, GZN, YTC and LSO were involved in acquisition of the data. AS, ALE, SE and PS conceived and
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