Screening and identification of a novel B-cell neutralizing epitope from Helicobacter pylori UreB.

Urease plays a crucial role in the survival and pathogenesis of Helicobacter pylori (H. pylori), and antibody neutralizing the urease activity may be implicated for the protection against H. pylori infection. Previously, a neutralizing monoclonal antibody (MAb) 6E6 against UreB of H. pylori was developed. In this work, we try to identify the B-cell epitope recognized by neutralizing MAb 6E6. Following screening a series of truncated proteins of UreB, an epitope was primarily localized in the aa 200-230 of UreB. Subsequently, we screened the overlapping synthetic peptides covering the aa 200-230 and identified a novel B-cell epitope (U(211-225), IEAGAIGFKIHEDWG) that was recognized by specific MAb 6E6. The newly identified epitope may help understanding of the protective immunity against H. pylori and be implicated for vaccine development.

[Immunization with catalase and UreB two-valence vaccine for preventing Helicobacter pylori infection in mice].

OBJECTIVE: To investigate the effects of the two-valence vaccine consisting of Helicobacter pylori (H. pylori) catalase and urease subunit UreB in preventing H. pyloriinfection in mice. METHODS: C57BL/6 mice were divided into 7 groups and immunized with intragastric administration of catalase and UreB (both 100 microg) plus cholera toxin (CT, 2 microg), catalase (100 microg) plus CT (2 microg), UreB (100 microg) plus CT (2 microg), catalase (100 microg), UreB (100 microg), CT (2 microg), or PBS, respectively, once a week for 4 consecutive weeks. Two weeks after the last immunization, all the mice were challenged by live H. pylori, and sacrificed 4 weeks after the challenge to obtain the gastric mucosa samples for detecting H. pylori using semi-quantitative bacterial culture assay. RESULTS: The total protection rate in mice immunized with the two-valence vaccine, single-valence vaccine of catalase, and single-valence vaccine of UreB was 83.3% (20/24), 41.7% (10/24) and 54.2% (13/24), respectively, and the rate in the other 4 groups were all 0. The H. pyloricolony density in mice with vaccination was significantly lower than that of other 4 groups (P<0.05). The total protection rate and H. pylori colony density differed significantly between the two-valence vaccination group and the single-valence vaccination groups (P<0.05). CONCLUSION: The two-valence vaccine consisting of catalase, UreB and adjuvant has better immunoprotective effects than the single-valence vaccines.

The mucosal adjuvanticity of two nontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes

Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.

Immunization with recombinant Helicobacter pylori urease decreases colonization levels following experimental infection of rhesus monkeys.

Rhesus monkeys, naturally colonized with H. pylori as indicated by culture and histology were immunized with either 40 mg recombinant H. pylori urease administered orally together with 25 microg Escherichia coli heat-labile enterotoxin (LT) or immunized with LT alone. An initial 6 doses were administered over an 8 week period. All five vaccinated monkeys had a greater than two-fold rise in urease-specific serum IgG and IgA level and urease-specific salivary IgA was induced in 3 of 5 vaccinated animals after 6 or 7 doses of vaccine. Vaccination had no measurable therapeutic effect on H. pylori colonization. H. pylori was eradicated from these monkeys with a course of antimicrobials plus omeprazole, a 7th vaccine dose was given (10 months after the 6th dose) and they were rechallenged with H. pylori. Necropsy was performed 23 weeks after rechallenge and H. pylori colonization was determined by histological examination of 12 individual gastric sites. A significant reduction in colonization (p < or = 0.0001; Friedman's analysis of variance) was found in the vaccinated animals. Histopathologic examination of necropsy tissues also revealed a trend towards reduced gastritis and epithelial alterations in the vaccinated group compared to animals receiving LT alone. This study provides the first evidence for effective vaccination of nonhuman primates against H. pylori, and preliminary evidence that a reduction in bacterial density attributable to immunization may lessen gastric inflammation.

Lack of protection against gastric Helicobacter infection following immunisation with jack bean urease: the rejection of a novel hypothesis.

The common mucosal immune system was stimulated by oral immunisation with jack bean urease and the adjuvant cholera toxin. A high level of local antibody and serum antibody was induced in mice following hyperimmunisation with this combination. No cross-reacting antibody was found against either Helicobacter pylori or Helicobacter felis. No protection was observed against oral challenge of immunised mice with living H. felis thus disproving the interesting hypothesis of Pallen and Clayton that plant urease might induce a protective immunity against helicobacter infection.

Identification of the Yersinia enterocolitica urease beta subunit as a target antigen for human synovial T lymphocytes in reactive arthritis.

The local T-cell response to bacterial antigens is involved in the pathogenesis of reactive arthritis (ReA). Here, we have identified a 19-kDa antigen of Yersinia enterocolitica O:9 recognized by Yersinia-specific synovial fluid CD4+ T cells in two patients with Yersinia-induced ReA. N-terminal amino acid sequencing of this protein revealed that it was identical to the 19-kDa urease beta subunit of Y. enterocolitica O:9. This protein has previously been shown to be arthritogenic in preimmunized rats after intra-articular injection. Analysis of the T-cell response to this protein showed that it contains several T-cell epitopes, one of which cross-reacts with other enterobacteria not able to induce ReA. This indicates that the arthritogenicity of the 19-kDa antigen is not a property of the 19-kDa protein alone but is dependent on its expression in bacteria able to induce ReA.

Monoclonal antibodies against urease from Canavalia ensiformis.

Monoclonal antibodies against purified urease (EC 3.5.1.5) from Canavalia ensiformis were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. All culture wells exhibited hybrid growth and 25% of these (ie 45 culture wells) contained anti-urease activity. Two positive hybrid cells were cloned twice by the limiting dilution method and three hybridoma clones (B6F, C4F and B18) secreting monoclonal antibodies were selected at random for purification and characterisation purposes. All three cell lines secreted monoclonal antibodies of IgM class which were purified by gel filtration chromatography on Sephacryl S-200 column with a final recovery of 85% and a purification factor of about 18. The purified preparations were apparently homogeneous on native PAGE running with a M(r) of 920,000 Da. mAbs were highly specific for jack bean urease as determined by Western blotting. The affinity constants (K) for these mAbs ranged from 10(8) to 10(9) l mol-1. mAb B6F inhibited about 65% of urease activity whereas C4F and B18 stimulated the enzyme activity slightly by 20%. The presence of 2-mercaptoethanol in incubation mixtures protected urease from inactivation by B6F. Urease inactivation by B6F could be reversed by addition of 2-mercaptoethanol which reactivated most of the partially inactive enzyme. Gel filtration chromatography of purified urease exhibited two protein peaks with M(r) values of 290,000 and 90,000 Da which revealed antibody activity. This result suggests that the mAb B6F recognizes the trimeric as well as the monomeric forms of urease.

Immunofluorescent localization of urease in the cotyledons of jack bean, Canavalia ensiformis.

Urease has been localized in sections of cotyledons from germinating seeds of jack bean, using FITC-labelled immunoglobulin prepared from urease antiserum raised in rabbits. The complication of lectin binding to the immunoglobulins was resolved by treatment of the sections with specific glycosides. Urease is localized in 2 sites: within the cytoplasm of storage parenchyma cells in spherical granules up to 3 micrometer in diameter, and within the intercellular spaces in spherical granules. Although similar in size, the latter are distinguished from the cytoplasmic granules by the presence of beta-lectin and appear to function as an extracellular lytic compartment or lysosome.