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

School of Biotechnology, Southern Medical University, Guangzhou Dadaobei No,1838, Guangzhou, China.
BMC Biotechnology (Impact Factor: 2.03). 11/2010; 10(1):84. DOI: 10.1186/1472-6750-10-84
Source: PubMed


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.
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.
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.

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Available from: Yundan Wang, Jul 28, 2014
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    • "In the case of mimotope immunization, several studies have shown effective responses in vivo [17]. Importantly, the mimotopes are able to replace the original epitopes for vaccine development [18,19]. Furthermore, active immune responses induced by phage-displayed mimotopes have been verified in many diseases [20-22]. "
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    ABSTRACT: Background Tumor angiogenesis is critical for tumor growth, infiltration and metastasis. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and targeting it is important in reducing angiogenesis. Bevacizumab (Avastin), a monoclonal antibody that reacts directly against VEGF, has been demonstrated to be an effective treatment for various cancers such as rectal cancer, colon carcinoma, and non-small cell lung cancer, etc. Results In the current study, we used the phage display technique to generate mimotopes that complemented the screening Avastin antibody (Ab). The candidate mimotopes of VEGF were isolated from a 12-mer peptide library. The phage displaying peptide DHTLYTPYHTHP (designated as 12P) exhibited high affinity to Avastin. The chemically synthesized 12P was conjugated to keyhole limpet hemocyanin (KLH) by glutaraldehyde (GA) to form vaccine KLH-12 peptide (KLH-12P). This epitope vaccine significantly induced humoral immunity in mice. The blood serum from KLH-12P-immunized mice associated with VEGF and blocked its binding to VEGFR, thus inhibiting vascular endothelial cell proliferation and migration. Conclusions Our data indicate that the isolated mimotope 12P reported here could potentially elicit specific antibodies against VEGF and result in the induction of anti-angiogenesis responses.
    BMC Biotechnology 09/2013; 13(1):77. DOI:10.1186/1472-6750-13-77 · 2.03 Impact Factor
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    • "All mimotope clones displayed in the phage were strongly recognized by the 4H11D10B11 mAb, contrary to what was observed with the M13KE phage (without insert) and the phage carrying a mimotope to TsGST25. This suggests that the interactions between the mAb with the peptide displayed on the phage are specific and support the hypothesis that mimotopes mimic the structure of the linear epitope that recognizes the mAb on TTPI, as has been determined in other studies (Cunha-Júnior et al., 2010; Li et al., 2010). "
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    ABSTRACT: In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193 to 204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of Taenia solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile.
    Experimental Parasitology 05/2013; 134(4). DOI:10.1016/j.exppara.2013.05.010 · 1.64 Impact Factor
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    • "Further studies could reveal if those peptides represents mimotopes of the same antigen or of different antigens. Comparison of selected mimotopes and the native antigen sequence could lead to a better understanding of molecular mechanisms that participate in the immune response and the design of peptides for diagnostic purposes [19]. "
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    ABSTRACT: Background An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients’ sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. Methods Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. Results Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. Conclusions The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.
    BMC Infectious Diseases 01/2013; 13(1):42. DOI:10.1186/1471-2334-13-42 · 2.61 Impact Factor
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