Expression of Helicobacter pylori urease B on the surface of Bacillus subtilis spores (original) (raw)
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PLoS ONE, 2014
The endospores of Bacillus subtilis are now widely used as a platform for presentation of heterologous proteins and due to their safety record and high resistance to harsh environmental conditions can be considered as potential vehicles for oral vaccination. In this research we show that recombinant B. subtilis spores presenting a fragment of the Helicobacter acinonychis UreB protein and expressing the ureB gene under vegetative promoter elicit a strong cellular immune response in orally immunized mice when co-administered with spores presenting IL-2. We show for the first time the successful application of two types of recombinant spores, one carrying an antigen and the other an adjuvant, in a single oral immunization. Citation: Hinc K, Stasiłojć M, Piątek I, Peszyń ska-Sularz G, Isticato R, et al. (2014) Mucosal Adjuvant Activity of IL-2 Presenting Spores of Bacillus subtilis in a Murine Model of Helicobacter pylori Vaccination. PLoS ONE 9(4): e95187.
Asian Journal of Pharmaceutical and Clinical Research, 2013
Helicobacter pylori, a gram-negative, microaerophilic, motile, spiral-shaped bacterium, have been established as the etiologic agent of chronic gastritis and belongs to phylum Proteobacteria. Infection with H. pylori is the primary identified cause of gastric cancer. H.pylori has the capacity to invade the stomach and tolerate the habitat of the stomach. But eventually the organism dies slowly due to the low pH in the stomach. In order to protect itself from the acidic environment it produces loads of urease enzyme. This urease enzyme has drawn attention all round the world as a diagnostic agent in detecting the helicobacter infection. And moreover the antibiotic based drugs are of limited use. Novel methods are being developed for the production of antibodies to specific antigens and thus helping in the process of development of protein based vaccines. UreA (Urease) gene was isolated and ligated into pTZ57R/T cloning vector. The ligated product was then cloned into DH5α strain and allowed to propagate. The plasmids thus cloned were purified and later expressed for the gene of interest in an expression vector. The proteins specific to the gene of interest was then isolated and purified. This proteins purified can in turn be used for protein based vaccines.
Vaccine, 2001
The use of Lactococcus lactis as an antigen delivery vehicle for mucosal immunisation has been proposed. To determine whether L. lactis could effectively deliver Helicobacter pylori antigens to the immune system, a recombinant L. lactis expressing H. pylori urease subunit B (UreB) was constructed. Constitutive expression of UreB by a pTREX1 vector resulted in the intracellular accumulation of UreB to :6.25% of soluble cellular protein. Five different oral regimens were used to vaccinate C57BL/6 mice and the immune response measured. One regimen, which consisted of four weekly doses of 10 10 bacteria, followed after an interval of : 4 weeks by three successive daily doses, was able to elicit a systemic antibody response to UreB in the mice, although subsequently, a similar regimen produced a significant antibody response in only one out of six mice. The other three regimes, in which mice were vaccinated with two or three sets of three consecutive daily doses of recombinant bacteria over 30 days, failed to elicit significant anti-UreB serum antibody responses. In three regimens, the immunised mice were then challenged by H. pylori strain SS1 and no protective effect was observed. These findings suggest that any adjuvant effects of L. lactis are unlikely to be sufficient to produce an effective immune response and to protect against H. pylori challenge, when used to deliver a weak immunogen, such as UreB.
BMC Research Notes, 2014
Background: Helicobacter pylori is a human pathogen and during the process of infection, antigens from the bacterium elicit strong host humoral immune responses. In our previous report, native H. pylori UreG protein showed good reactivity with sera from H. pylori patients. This study was aimed at producing the recombinant form of the protein (rUreG) and determining its seroreactivities.
Isolation of recombinant protective Helicobacter pylori antigens
Infection and immunity, 1999
A total of seven clones producing both new and previously described Helicobacter pylori proteins were isolated from a library of H. pylori genomic DNA. The screening approach by which these proteins were detected relied on the use of antisera raised in mice vaccinated with Helicobacter felis sonicate plus cholera toxin, a regimen which protects mice from H. pylori challenge. This strategy was designed to maximize the possibility of obtaining antigens which might be capable of conferring protection from H. pylori infection. Two of the clones were shown to encode the urease enzyme and the heat shock protein HspB, which have already been identified as protective antigens. The other five clones were sequenced, protein coding regions were deduced, and these sequences were amplified by PCR for incorporation into Escherichia coli expression vectors. The proteins produced from these expression systems were purified to allow testing for protective efficacy in an H. pylori mouse model. All fi...
Jundishapur Journal of Microbiology, 2019
Background: Helicobacter pylori is the main cause of stomach ulcers and gastric cancer. Hence, the diagnosis, treatment, and prevention of H. pylori infection can considerably reduce the fatality. Objectives: This study aimed to construct a dual-antigen protein by combining the antigenic regions of UreB and FlaA of H. pylori and determine its antigenicity as a promising vaccine and serodiagnosis candidate. Methods: The antigenic regions of FlaA and UreB were detected by immunological bioinformatics, amplified and joined together by polymerase chain reaction (PCR) with special primers containing linker sequences. Then, it was cloned into pET-32a and after expression and purification of the recombinant multi-epitope protein (rFlaA-UreB), its antigenicity was evaluated by immunoblotting using the sera of infected patients. Results: DNA sequencing and enzyme digestion analysis showed the rFlaA-UreB gene was successfully inserted into pET32a. The recombinant protein was produced and purified via affinity chromatography and its molecular weight was similar to what had been predicted. Moreover, data indicated that rFlaA-UreB was recognized by all patients' sera and its sensitivity and specificity were high. Conclusions: Although the developed recombinant multi-epitope protein was very smaller and lighter than the natural forms of these two critical antigens, they all had close antigenic properties. Therefore, this recombinant protein can be an important antigen in the diagnosis and vaccination against H. pylori.
International Journal of Applied Pharmaceutics, 2020
Around half of the world's population faces Helicobactor pylori (H. pylori) infection. Enormous progress has been made to understand the bacterial pathogenesis process and pathogen interaction with eukaryotic cells but infectious diseases are still the cause of premature death of humans around the world. H. pylori is categorized under class I carcinogen by the WHO based on clinical study results. This review paper discusses various attempts made to establish an efficient vaccine to manage H. pylori infection. Some of the problems in developing an efficient vaccine against H. pylori are recurrent or persistent infection, insufficient knowledge about the action specifically in case of probiotics, development of antibiotic resistance, and cost of therapy are noted. This research may come up with transient Nicotiana benthamiana with suitable H. pylori genes expressed as antigenic proteins, which can be used for further studies to develop a vaccine for gastric ulcer/cancer and generate good scientific data that can be helpful for scientists and researchers in this field.
PLoS ONE, 2013
A mucosal vaccine against Helicobacter pylori infection could help prevent gastric cancers and peptic ulcers. While previous attempts to develop such a vaccine have largely failed because of the requirement for safe and effective adjuvants or large amounts of well defined antigens, we have taken a unique approach to combining our strong mucosal CTA1-DD adjuvant with selected peptides from urease B (UreB). The protective efficacy of the selected peptides together with cholera toxin (CT) was first confirmed. However, CT is a strong adjuvant that unfortunately is precluded from clinical use because of its toxicity. To circumvent this problem we have developed a derivative of CT, the CTA1-DD adjuvant, that has been found safe in non-human primates and equally effective compared to CT when used intranasally. We genetically fused the selected peptides into the CTA1-DD plasmid and found after intranasal immunizations of Balb/c mice using purified CTA1-DD with 3 copies of an H. pylori urease T cell epitope (CTA1-UreB3T-DD) that significant protection was stimulated against a live challenge infection. Protection was, however, weaker than with the gold standard, bacterial lysate+CT, but considering that we only used a single epitope in nanomolar amounts the results convey optimism. Protection was associated with enhanced Th1 and Th17 immunity, but immunizations in IL-17A-deficient mice revealed that IL-17 may not be essential for protection. Taken together, we have provided evidence for the rational design of an effective mucosal subcomponent vaccine against H. pylori infection based on well selected protective epitopes from relevant antigens incorporated into the CTA1-DD adjuvant platform.
Journal of Medical Microbiology, 2004
The potential therapeutic effects of Helicobacter pylori-specific immunoglobulin (IgY-Hp) derived from egg yolk and identification of the immunodominant H. pylori proteins have previously been reported. In this study, the urease epitope that is recognized by IgY-Hp was identified and used as an immunogen to produce urease-specific IgY (IgY-HpU). Epitope regions were mapped and peptides of selected epitope regions were synthesized. The IgY-Hp titre against synthetic peptides was evaluated using ELISA analysis. Hens were immunized with synthetic peptides conjugated with BSA. Urease activity was quantified by measuring the optical density of an indicator dye. Of the five synthetic peptides assayed, a peptide representing 15 amino acid residues of UreB (UB-3; aa 396-410, DNDNFRIKRYLSKYT) was specifically recognized by the IgY-Hp. Immunization of hens with BSA-conjugated UB-3 resulted in the generation of IgY-HpU. IgY-HpU markedly reduced H. pylori urease activity by 80 % as compared to control IgY (IgY-BSA). The availability of the synthetic UreBderived peptide enabled the production of highly specific anti-urease IgY, which had a significant inhibitory effect on H. pylori urease activity. Therefore, specific IgY-HpU produced using the synthetic peptide may be an effective tool against infection by H. pylori.