Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H. pylori infection in mice☆
Introduction
Helicobacter pylori is now recognised as the most widespread human pathogen. Approximately half of the world's population is infected [1], [2]. Infection with H. pylori is highly associated with chronic active gastritis, peptic ulcers, gastric adenocarcinoma and more rarely, lymphoma of the mucosa-associated lymphoid tissue (MALT) [3], [4], [5]. Current antibiotic-based triple therapies are not practical for global control due to the high cost, problems with patients’ compliance and the emergence of antibiotic-resistant strains [1], [6]. Vaccination against H. pylori has therefore been considered to control H. pylori infection and it has been shown that administration of oral bacterial antigens, mainly urease, together with a mucosal adjuvant can protect mice against Helicobacter infection [6], [7], [8], [9]. Urease is acid stable, appears to be essential for gastric colonisation by H. pylori and is expressed by all clinical strains of H. pylori. Cross-reactivity is present between the ureases of different H. pylori strains or between the ureases of H. pylori and animal strain — H. felis [10]. The urease gene structural subunits A and B (ureAB) have been cloned and expressed in Escherichia coli [11]. Both subunit proteins (UreA and UreB) are immunogenic in combination with a mucosal adjuvant, but UreB seems to be more protective than UreA [12], [13]. Cholera toxin (CT) or E. coli labile toxin (LT) has been used in all successful oral immunisation models. No protection was achieved when antigens were administered without a mucosal adjuvant [14]. CT cannot be given to humans and the use of LT is limited due to its known toxicity in a human volunteers study [15]. A genetically detoxified LT mutant, LTK63, has been used in vaccine studies with promising results, although its safety for human use is still being investigated [9].
The attenuated Salmonella typhimurium aro and phoPC mutants strains expressing UreA and UreB have been used in mouse protection experiments [16], [17]. However, human volunteers orally vaccinated with phoP/phoQ-deleted S. typhi expressing H. pylori UreA and UreB developed gastroenteritis-like syndrome of a variable degree [18]. Live attenuated Salmonella strains are still able to establish a limited infection in man and cannot be used for vulnerable groups, such as infants, elderly people and the immunocompromised patients.
By contrast, the use of Lactococcus lactis to deliver protective antigens to the mucosal surfaces may be an alternative way to avoid these problems. L. lactis does not require mucosal antigens, such as CT or LT and is not capable of establishing an infection in man. L. lactis is a Gram-positive food-grade bacterium and has a long history of widespread use in the food industry for the production of fermented milk products [19]. It does not colonise the digestive tract of man or animals, but can survive passage through the gut [20], [21]. It has been suggested that antigens expressed by L. lactis are presented to the immune system in particulate form and may therefore be less likely to induce oral tolerance than soluble antigens. Furthermore, L. lactis is approximately the same size as biodegradable microparticles that are known to be taken up by M cells and have been shown to be capable of acting as effective oral vaccine vehicles [22]. The use of the live vaccine carrier does not require antigen purification or the combined use of a mucosal adjuvant and it can bypass the degradation and denaturation of antigens in the stomach. L. lactis expressing tetanus toxin fragment C (TTFC) has been used successfully for oral immunisation against tetanus toxin challenge in the mouse model [23]. In addition, oral inoculation of this TTFC expression strain elicited local immunoglobulin A (IgA) responses in the gut secretions [23].
In this study, we used recombinant DNA techniques to clone H. pylori protein UreB and this protein was expressed in L. lactis MG1363 via an expression vector, pTREX1. This recombinant L. lactis expressing UreB was used to vaccinate C57BL/6 mice by the oral route to evaluate the mucosal and systemic antibody responses to UreB in a mouse model. Vaccinated mice were then challenged with H. pylori to determine the extent of any protection provided by vaccination.
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Bacterial strains and growth conditions
L. lactis MG1363 was grown in M17 medium (Oxoid, Basingstoke, UK) containing 0.5% (w/v) glucose (Sigma) (GM17) at 30°C aerobically. H. pylori SS1 was stored at −70°C in Tryptone Soya broth (TSB) (Oxoid) containing 25% glycerol, until required. For mouse challenge experiments, H. pylori was grown on Dent plates (Blood Agar Base No. 2; Oxoid) supplemented with 7% (v/v) defibrinated horse blood (Oxoid), Dent selective supplement (Oxoid) and incubated at 37°C under microaerophilic conditions (4% O2
Cloning and expression of H. pylori UreB into L. lactis MG1363
The gene encoding the urease subunit B of H. pylori (1769 nucleotides) was PCR-amplified and cloned as a SphI-BamHI fragment in the expression plasmid pTREX1 generating pTREX1::ureB. The inserted SphI site at the 3′ end of ureB resulted in an alteration of the amino acid residue at position +2 from lysine to arginine. This modification was expected to have a negligible effect on the conformation of the N-terminus extremity of UreB as they are both positive amino-acid residues.
In vitro and in vivo stability of the ureB construct
The pTREX1::ureB
Discussion
Our results demonstrate for the first time that L. lactis is able to produce substantial quantities of a H. pylori antigen in soluble form. L. lactis has been shown to be able to produce the subunit B of the urease (UreB) organism, H. pylori, in the Gram-positive L. lactis. Although over-expression level of certain proteins and antigens may result in the death of cells [25], the use of the constitutive expression vector, pTREX1, enabled us to reach a UreB production level of 6.25% without
Acknowledgements
We thank Jeremy Wells for the gift of L. lactis MG1363 and pTREX1, Peter Jenks for H. pylori SS1 and Harry Kleanthous (OraVax) for recombinant urease.
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This work was presented in part at the Twelfth International Meeting of the European Helicobacter pylori Study Group Workshop on Gastroduodenal Physiology and Helicobacter pylori, 1–4 September, 1999, Helsinki, Finland.