Mimotopes selected with a neutralizing antibody against urease B from Helicobacter pyloriinduce enzyme inhibitory antibodies in mice upon vaccination
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Abstract:
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.Keywords:
Immunogen
Mimotope
Immunoscreening
Objective To screen and characterize the antigenic mimotopes of advanced oxidation protein products(AOPP) from a random 12-mer phage display peptide library,and to explore the expression or distribution of the mimotope sequences to AOPP.Methods The polyclonal antibodies(PcAbs)against AOPP was used as targets to screen the 12-mer phage display peptide library,and the specificity of phage clones were identified by sandwich ELISA and competitive ELISA.The peptide sequences displayed on positive phage clones were determined by DNA sequencing,and analyzed by bioinformatics.Results Eighteen of phage clones displaying mimotopes of AOPP were isolated in positive rate of 62.07%,which showed a specific reactivity with the PcAbs.The reactivity of PcAbs with positive phage clone No.6 could be well inhibited by AOPP with 0.2μg/ml for IC50,indicating that phage No.6 mimics antigenicity of AOPP epitope.The analysis on amino acid sequence revealed that mimotope sequences contained a common motif LXDMLXD,X is random amino acid.The analysis by bioinformatics showed that the conservative sequences are presented in some fungus and proteins of some parasites.Conclusion The mimotopes of AOPP were obtained by using the phage display technology,which are not presented in protein molecules of normal human and mammalian.
Mimotope
Polyclonal antibodies
Phagemid
Antigenicity
Epitope mapping
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The aim of this study was to evaluate phage display technology for mapping Haemonchus contortus mimotopes. We screened the PhD-7 Phage Display Peptide Library Kit with a sheep polyclonal antibody against H. contortus. After four rounds of selection, 50 phage peptide clones were selected by biopanning and sequenced. Two clones displaying peptide mimotopes of H. contortus proteins were chosen for sheep immunization: clone 6 - mimotope of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and clone 17 - mimotope of a disorganized muscle family member (Dim 1). Twelve sheep were allocated into 3 groups of 4 animals as follow: G1: control group; G2/GAPDH: immunized with clone 6; and G3/Dim1: immunized with clone 17. Four immunizations were performed at intervals of seven days (0, 7, 14, and 21 days). On day 28 post initial vaccination, all groups were orally challenged with 2500 H. contortus infective larvae. The mimotope peptides selected by phage display were recognized by IgG from sheep naturaly infected with H. contortus. The immunization protocol showed an increasein IgG anti-M13 phage titers, but no effect was observed in IgG-specific for the anti-mimotope peptides. This is the first report of successful use of a phage display library for the identification of mimotopes of H. contortus proteins.
Polyclonal antibodies
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To screen and identify the mimotopes for lipopolysaccharide (LPS) epitope, a random phage displayed dodecapeptide library was screened with a monoclonal antibody 2B4 specifically against LPS conservative epitope. The results demonstrate that the peptides screened with 2B4 antibody are mimotopes for LPS conservative epitope.
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Epitope mapping
Linear epitope
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Objective By using specific c7c TNF-α mimotope displayed on phage as a target, we got specific TNF-α binding-peptides clones from 12mer phage display peptide library and developed a novel approach for screening phage peptide library. Methods Using c7c TNF-α mimotope phage clones LCS-7 (c-RRPAQSG-c) as a target to screen 12mer phage peptide library and identify the positive clones by phage ELISA and competive ELISA. Results After 3 rounds of screening, 10 of 20 clones were identified as positive clones and all the 10 phage clones shared the identical amino acid sequence: EHMALTYPFRPP. Conclusion The positive 12mer phage clone peptide screened with cycle 7mer TNF-α simulating phage clone as a target can bind to TNF-α specifically. The results suggest that this method is very specific and easy- for screening of binding epitope from phage display peptide library. [
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clone (Java method)
Phagemid
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Constructing a vaccine against HIV-1, able to induce production of broadly neutralizing antibodies, is crucial. We report here the selection and characterization of RDWSFDRWSLSEFWL peptide mimotope that binds specifically to bNAbs 2F5. The peptide mimotope was selected from 15-mer phage-displayed peptide library by using Mab 2F5 as the selecting agent. The most abundant RDWSFDRWSLSEFWL peptide was inserted into a carrier, an artificial polyepitope immunogen - TBI (T- and B-cell immunogen). TBI-2F5 polyepitope immunogen that includes the mimotope of 2F5 epitope was constructed. It was shown that sera of mice immunized with TBI-2F5 protein recognized TBI protein as well as RDWSFDRWSLSEFWL peptide. The capacity of sera of immunized mice to neutralize HIV-1 was demonstrated using subtype B env-pseudoviruses of HIV-1 QH0692.42 and PVO.4. Based on these results, we conclude that peptide mimotope of 2F5 epitope RDWSFDRWSLSEFWL can be an essential component for a successful HIV-vaccine. Keywords: Humoral response to HIV-1, mAb 2F5, peptide mimotope, Phage display, HIV-1, vaccine against HIV-1.
Mimotope
Immunogen
Peptide vaccine
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Immunogen
Mimotope
Biopanning
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Mimotope
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Linear epitope
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To screen and identify the mimotopes for lipopolysaccharide(LPS) epitope, a random phage displayed dodecapeptide library was screened with a monoclonal antibody 2B4 specifically against LPS conservative epitope. The positive clones were identified by phage ELISA and competitive inhibition assay by either S.typhi T8 61 LPS or E.coli O111:B4 LPS. After three rounds of biopanning,the clones binding with 2B4 antibody were well enriched with positive rate of 80%. The bindings between 12 of positive phage clones and screening antibody were competitively inhibited by the two kinds of LPS,indicating that the positive clones have similar epitope with LPS. The positive peptide sequences were deduced from the corresponding DNA sequences. There were identical sequences among them. The sequences were GPPQWFFSQPQL (5/12,41 7%),LPQYFWNTATTA (3/12,25%),FPQNHWNVPWAT(2/12,16 6%),HSQSFWNAPLAM and AHPWTHGYFPPL (1/12,8 3%) respectively. The results demonstrate that the peptides screened with 2B4 antibody are mimotopes for LPS conservative epitope.
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Biopanning
Linear epitope
Epitope mapping
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