Mimotopes selected with a neutralizing antibody against urease B from Helicobacter pylori induce enzyme inhibitory antibodies in mice upon vaccination

Abstract

Background: Urease B is an important virulence factor that is required for Helicobacter pylori to colonise the gastric mucosa. Mouse monoclonal antibodies (mAbs) that inhibit urease B enzymatic activity will be useful as vaccines for the prevention and treatment of H. pylori infection. Here, we produced murine mAbs against urease B that neutralize the enzyme's activity. We mapped their epitopes by phage display libraries and investigated the immunogenicity of the selected mimotopes in vivo.

Results: The urease B gene was obtained (GenBank accession No. DQ141576) and the recombinant pGEX-4T-1/UreaseB protein was expressed in Escherichia coli as a 92-kDa recombinant fusion protein with glutathione-S-transferase (GST). Five mAbs U001-U005 were produced by a hybridoma-based technique with urease B-GST as an immunogen. Only U001 could inhibit urease B enzymatic activity. Immunoscreening via phage display libraries revealed two different mimotopes of urease B protein; EXXXHDM from ph.D.12-library and EXXXHSM from ph.D.C7C that matched the urease B proteins at 347-353 aa. The antiserum induced by selected phage clones clearly recognised the urease B protein and inhibited its enzymatic activity, which indicated that the phagotope-induced immune responses were antigen specific.

Conclusions: The present work demonstrated that phage-displayed mimotopes were accessible to the mouse immune system and triggered a humoral response. The urease B mimotope could provide a novel and promising approach for the development of a vaccine for the diagnosis and treatment of H. pylori infection.

Figure 1

SDS-PAGE analysis of expression of pGEX-4T-1/urease B in E. coli. 1: pGEX-4T-1 before induction; 2: pGEX-4T-1 after induction; 3: pGEX-4T-1/urease B before induction; 4: pGEX-4T-1/urease B after induction; 5: protein marker.

Figure 2

SDS-PAGE analysis of purified urease B and Western blot analysis of urease B reactivity with patient serum. (A) Bacteria expressing GST-urease B were lysed by sonication, and the supernatant was applied to a Glutathione Sepharose 4B column. Purified urease B was analysed on a 10% SDS-PAGE. 1: purified urease B; 2: protein marker. (B and C) The gels were transferred to nitrocellulose membranes and probed with diluted human sera (1:50) from a normal donor or an H. pylori-infected patient. Detection was performed with a goat anti-human antibody linked to HRP (1:10,000), followed by staining with 3-amino-9-ethylcarbozole. 1: GST; 2: urease B; 3: protein marker.

Figure 3

Inhibition of urease B enzymatic activity by specific mAb U001. Urease (25 μl) was incubated with 25 μl mAb U001 or U002 (equivalent to 0-25 μg) in 96-well microtitre plates overnight at 4°C, with PBS as a control. On the following day, 50 μl 50 mM phosphate buffer (pH 6.8) that contained 500 mM urea, 0.02% phenol red, and 0.1 mM DTT was added to each well. The colour development was monitored at 550 nm with a microplate reader. The inhibition ratio was determined by the following equation: [(activity without Ab - activity with Ab)/(activity without Ab)] × 100.

Figure 4

Binding specificity of selected phage from the Ph.D.-12 library. Wells of 96-well microtitre plates were coated with mAb U001 and BSA (10 μg/ml, 100 μl) by incubation at 4°C overnight and blocked with 5% BSA in TBS. Affinity-selected phage were added to the wells and allowed to bind at 37°C for 1 h. After the unbounded phage was removed, the bound phage was detected by incubation with peroxidase-labelled murine anti-M13 antibodies. The bound peroxidase was determined by incubation with O-phenylenediamine dihydrochloride and the reaction was determined in an ELISA reader at OD₄₉₂. All the assays were carried out in triplicate and the error bars indicate standard deviation.

Figure 5

Binding specificity of selected phage from the Ph.D.-C7C library. Wells of 96-well microtitre plates were coated with mAb U001 and BSA (10 μg/ml, 100 μl) by incubation at 4°C overnight and blocked with 5% BSA in TBS. Affinity-selected phage were added to the wells and allowed to bind at 37°C for 1 h. After the unbounded phage was removed, the bound phage was detected by incubation with peroxidase-labelled murine anti-M13 antibodies. The bound peroxidase was determined by incubation with O-phenylenediamine dihydrochloride and the reaction was determined in an ELISA reader at OD₄₉₂. All the assays were carried out in triplicate and the error bars indicate standard deviation.

Figure 6

Phage inhibition of mAb U001 binding to urease B protein. Wells of microtitre plates were coated with urease B protein and incubated with the mAb U001 (0.5 μg) plus increasing amounts (10⁹-10¹⁵ pfu) of the selected phage (clones D1 and H1). The binding was monitored by OD₄₉₂ measurements after reaction with the peroxidase-conjugated goat anti-mouse IgG. The inhibition ratio was determined by the following equation: [(activity without Ab - activity with Ab)/(activity without Ab)] × 100.

Figure 7

Western blot analysis of antiserum induced by phagotopes. Two phage (clones D1 and H1) were used to immunise BALB/c mice through intraperitoneal administration. For each immunisation, 10¹² pfu phage (in 50 μl) was used, with TBS and wild-type M13 phage as controls. Three mice were immunised with each sample and pre-immune sera were obtained. The mice were bled once (pre-booster), and they were given a booster in the same manner. The immune mice were bled every 7 days after booster immunisation, and western blotting was performed to test each serum sample. 1: serum from D1 phagotope; 2: serum from H1 phagotope; 3: pre-immune serum; 4: serum from wild M13phage; 5: serum from TBS.

Figure 8

Serum antibodies induced by phagotopes. Two purified phage clones (D1 and H1) were used to immunise BALB/c mice through intraperitoneal administration. For each immunisation, phage (10¹² pfu/50 μl) were used, with TBS and wild-type M13 phage as controls. Three mice were immunised with each sample and pre-immune sera were obtained. The mice were bled once (pre-booster), and they were given a booster in the same manner. The immune mice were bled every 7 days after the booster immunisation, and triplicate ELISAs were performed to test each serum sample with urease B as coated protein. All assays were carried out in triplicate and the error bars indicate standard deviation.

Figure 9

Mimotopes selected out with U001 were input and aligned with ClustalW. (A) Alignment of multiple sequences by ClustalW. (B) The frequency of a given amino acid at each position of the alignment was calculated and displayed in a table. (C) A 3D bar figure was created. The x axis represents the 20 amino acid types and gap, whereas the y axis is the frequency of occurrence and the z axis is the position of the aligned sequences. (D) The alignment could be managed with JalView.