Therapeutic effects of molecularly designed antigen UREB138 for mice infected with Helicobacter pylori.

Helicobacter pylori (H. pylori) is a bacteria that is well known as the principal cause of chronic gastritis and peptic ulcer disease in humans. Because no effective vaccine has yet been established, we designed a new biomolecule as a vaccination antigen capable of preventing the infection of H. pylori. The designed biomolecule involves a 138 stretch (aa 201-aa 338 of beta-subunit of H. pylori urease), which is the functionally important region for urease activity. The region was expressed as a recombinant protein, called UREB138. The therapeutic vaccination was performed using UREB138 in mice persistently infected with H. pylori. The subcutaneous administration of UREB138 remarkably reduced the number of bacteria in the mice stomach compared with the control. Immunization with UREB138 enhanced the urease-specific IgA and IgG1 in the serum. Immunohistochemical staining for IgA in gastric mucosa showed that the number of mice positively stained with IgA was significantly higher in UREB138-immunized mice than in control mice. Furthermore, the expression of interferon-gamma mRNA in the gastric tissues with eradicated bacteria was higher than in the non-eradicated group. The combination of Th1- and Th2-mediated immunity plays a role in reducing the colonization of bacterial numbers of H. pylori.

Catalytic features and eradication ability of antibody light-chain UA15-L against Helicobacter pylori.

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.

Effects of vaccination by a recombinant antigen ureB138 (a segment of the beta-subunit of urease) against Helicobacter pylori infection.

Helicobacter pylori has to counteract acidity during colonization in the stomach. The most important region for the enzymic activity of H. pylori urease, consisting of 138 aa (ureB138), was determined by a comparison of the homology of amino acid sequences, and a structural analysis, between urease of H. pylori and various other species. This region was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which was cleaved by PreScission protease between the GST moiety and ureB138. The ureB138 protein was then purified by gel filtration. The polyclonal antibody (pAb) induced by immunization with the purified ureB138 could suppress urease activity by about 50 %, while the pAb against the H. pylori urease did not show any inhibitory effect at all. Immunohistochemical analysis indicated that the ureB138-specific pAb specifically recognized the H. pylori infecting human gastric tissues. The effects of vaccination of recombinant ureB138 against infection by this organism were also examined. Specific IgG and IgA antibodies against H. pylori urease were induced in the serum of mice immunized with ureB138. A reduction in the number of colonizing H. pylori was observed in mice treated with ureB138 compared to ones treated with BSA and infection control mice. In the protected mice, severe gastritis characterized by marked infiltration of mononuclear cells was noted compared with the gastritis observed in unprotected mice. Immunohistochemical staining for IgA in gastric mucosa showed that the number of mice positively stained with IgA was significantly higher in ureB138-vaccinated mice than in non-vaccinated mice. This indicates that local IgA antibody and severe post-immunization gastritis correlate well with the protection of mice against H. pylori infection.

Recombinant antigen from Helicobacter pylori urease as vaccine against H. pylori-associated disease.

It is well documented that the enzymatic active site of Helicobacter pylori urease is present in the beta-subunit. An important sequence of 135 amino acids of the beta-subunit was determined from the structure of H. pylori urease and by a homology-based study of the urease of other bacteria and plants. The sequence (UreB) was expressed in Escherichia coli as a recombinant fusion protein with glutathione-S-transferase (GST). Seventeen monoclonal antibodies, UA-1-17, were produced using the UreB-GST as the immunogen. The obtained monoclonal antibodies showed a high specificity to UreB, and some of the MAbs cross-reacted with Jack bean urease. About 70% of the established MAbs displayed an inhibitory effect on the enzymatic activity of the urease. Among them, UA-15 MAb could reduce the activity by 53% and it immunologically binds to the bacterium infecting the human stomach mucosa. The antiserum induced by immunization with a recombinant UreB-GST into rabbits displayed a specific binding to mucosal surfaces of the human stomach infected with the pathogen H. pylori. Moreover, the antiserum suppressed the enzymatic activity of H. pylori urease, while the purified H. pylori urease could not induce such an antiserum.

Epitope mapping and features of the epitope for monoclonal antibodies inhibiting enzymatic activity of Helicobacter pylori urease.

Two characteristic monoclonal antibodies (HpU-2 and -18) out of 26 monoclonal antibodies (HpU-1 approximately 26) produced against Helicobacter pylori (H. pylori) urease showed a strong inhibitory effect against the enzymatic activity of the urease. Epitope mapping about some monoclonal antibodies of the HpU-series inhibiting enzymatic activity was performed by using a surface plasmon resonance apparatus and by digesting H. pylori urease with trypsin, followed by mass spectroscopy. The sequences of the epitopes recognized by HpU-2 and -18 were SVELIDIGGNRRIFGFNALVDR (22 mer) and IFGFNALVDR (10 mer), respectively. The former sequence is present as a part of a loop structure at a position close to the C-terminal of the alpha-subunit of H. pylori urease, although it has been suggested that the active site of the urease resides in the beta-subunit. The above peptide (22 mer) was chemically synthesized in a linear and cyclic form, and its conjugate with BSA was immunized in rabbits. The resultant serum induced by the linear form could specifically bind to H. pylori infecting human gastric mucosa. These results suggest that the above sequence (22 mer) must be an important epitope, although it locates in the alpha-subunit but not in the beta-subunit.