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Antigenic and immunogenic evaluation of Helicobacter pylori FlaA epitopes.

ABSTRACT: OBJECTIVES:Helicobacter pylori are among most common human pathogens affecting at least half of the world's population. Mobility is one of the important primary factors in bacterial colonization and invasion. The purpose of this research is cloning, expression, and purification of FlaA protein specific epitopes in order to evaluate their antigenicity and immunogenicity. MATERIALS AND METHODS:The antigenic region of the flaA gene was bioinformatically predicted using Epitope mapping software's and the predicted epitopes were expressed in a prokaryotic expression vector. The antigen was injected into the animal model (mice BALB/c) and some indicators including IgG1, IgG2a, IgA, IFN-?, and IL 5 were measured. RESULTS:The immunogenicity studies in animal models by measuring serum antibodies (IgG1, IgG2a, and IgA) and cytokines (IFN-? and IL5) revealed that the rFlaA induces a proper immune response in animal models. CONCLUSION:The recombinant FlaA protein is antigenic and immunogenic. Therefore, it might be used in order to design of specific diagnostic kits and recombinant vaccines against H. pylori.

SUBMITTER: Zarei M

PROVIDER: S-EPMC5651478 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Antigenic and immunogenic evaluation of <i>Helicobacter pylori</i> FlaA epitopes.

Zarei Mansoor M Mosayebi Ghasem G Khansarinejad Behzad B Abtahi Hamid H

Iranian journal of basic medical sciences 20170801 8

<h4>Objectives</h4><i>Helicobacter pylori</i> are among most common human pathogens affecting at least half of the world's population. Mobility is one of the important primary factors in bacterial colonization and invasion. The purpose of this research is cloning, expression, and purification of FlaA protein specific epitopes in order to evaluate their antigenicity and immunogenicity.<h4>Materials and methods</h4>The antigenic region of the <i>flaA</i> gene was bioinformatically predicted using ...[more]

PMID: 29085584

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Project description:The analysis of the interaction between Helicobacter pylori (HP) and the host in vivo is an extremely informative way to enlighten the molecular mechanisms behind the persistency/latency of the bacterium as well as in the progression of the infection. An important source of information is represented by circulating antibodies targeting the bacteria that define a specific "disease signature" with prospective diagnostic implications. The diagnosis of some of the HP induced diseases such as gastric cancer (GC), MALT lymphoma (MALT), and autoimmune gastritis (AIG) is not easy because patients do not show symptoms of illness in early-onset stages, at the same time they progress rapidly. The possibility of identifying markers able to provide an early diagnosis would be extremely beneficial since a late diagnosis results in a delay in undergoing active therapy and reduces the survival rate of patients. With the aim to identify the HP antigens recognized during the host immune-response to the infection and possibly disease progression, we applied a discovery-driven approach, that combines "phage display" and deep sequencing. The procedure is based on the selection of ORF phage libraries, specifically generated from the pathogen's genome, with sera antibodies from patients with different HP-related diseases. To this end two phage display libraries have been constructed starting from genomic DNA from the reference HP 26695 and the pathogenic HP B128 strains; libraries were filtered for ORFs by using an ORF selection vector developed by our group (Di Niro et al., 2005; Soluri et al., 2018), selected with antibodies from patients affected by GC, MALT, and AIG and putative HP antigens/epitopes were identified after Sequencing and ranking. The results show that individual selection significantly reduced the library diversity and comparison of individual ranks for each condition allowed us to highlight a pattern of putative antigens specific for the different pathological outcomes or common for all of them. Within the putative antigens enriched after selection, we have validated protein CagY/Cag7 by ELISA assay as a marker of HP infection and progression. Overall, we have defined HP antigenic repertoire and identified a panel of putative specific antigens/epitopes for three different HP infection pathological outcomes that could be validated in the next future.

Project description:Antigen-specific CD4+ T cells play an essential role in effective immunity against Helicobacter pylori (H. pylori) infection. Lpp20, a conserved lipoprotein of H. pylori, has been investigated as one of major protective antigens for vaccination strategies. Our previous study identified two H-2d-restricted CD4+ T cell epitopes within Lpp20 and an epitope vaccine based on these epitopes was constructed, which protected mice in prophylactic and therapeutic vaccination against H. pylori infection. Immunodominant CD4+ T cell response is an important feature of antiviral, antibacterial, and antitumor cellular immunity. However, while many immunodominant HLA-restricted CD4+ T cell epitopes of H. pylori protective antigens have been identified, immunodominant HLA-restricted Lpp20 CD4+ T cell epitope has not been elucidated. In this study, a systematic method was used to comprehensively evaluate the immunodominant Lpp20-specific CD4+ T cell response in H. pylori-infected patients. Using in vitro recombinant Lpp20 (rLpp20)-specific expanded T cell lines from H. pylori-infected subjects and 27 18mer overlapping synthetic peptides spanned the whole Lpp20 protein, we have shown that L55-72 and L79-96 harbored dominant epitopes for CD4+ T cell responses. Then the core sequence within these two 18mer dominant epitopes was screened by various extended or truncated 13mer peptides. The immunodominant epitope was mapped to L57-69 and L83-95. Various Epstein-Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCLs) with different HLA alleles were used as antigen presenting cell (APC) to present peptides to CD4+ T cells. The restriction molecules were determined by HLA class-antibody blocking. L57-69 was restricted by DRB1-1501 and L83-95 by DRB1-1602. The epitopes were recognized on autologous dendritic cells (DCs) loaded with rLpp20 but also those pulsed with whole cell lysates of H. pylori (HP-WCL), suggesting that these epitopes are naturally processed and presented by APC. CD4+ T cells were isolated from H. pylori-infected patients and stimulated with L57-69 and L83-95. These two epitopes were able to stimulate CD4+ T cell proliferation. This study may be of value for the future development of potential H. pylori vaccine.

Project description:AIM:To clone flagellin genes A (flaA) and B (flaB) from a clinical strain of Helicobacter pylori (H pylori) and to construct prokaryotic expression systems of the genes and identify immunity of the fusion proteins. METHODS:The flaA and flaB genes from a clinical H pylori isolate Y06 were amplified by high fidelity PCR. The nucleotide sequences of target DNA amplification fragments from the two genes were sequenced after T-A cloning. The recombinant expression vector pET32a inserted with flaA and flaB genes was constructed, respectively. The expressions of FlaA and FlaB fusion proteins in E. coli BL21DE3 induced by isopropylthio-beta-D-galactoside (IPTG) at different concentrations were examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of H pylori and immunodiffusion assay using self-prepared rabbit antiserum against FlaA (rFlaA) or FlaB (rFlaB) recombinant proteins were applied to the determination of the fusion proteins immunity. ELISA was used to detect the antibodies against rFlaA and rFlaB in sera of 125 H pylori infected patients and to examine rFlaA and rFlaB expression in 98 clinical isolates of H pylori, respectively. RESULTS:In comparison with the reported corresponding sequences, the nucleotide sequence homologies of the cloned flaA and flaB genes were from 96.28-97.13% and 96.31-97.73%, and their putative amino acid sequence homologies were 99.61-99.80% and 99.41-100% for the two genes, respectively. The output of rFlaA and rFlaB expressed by pET32a-flaA-BL21DE3 and pET32a-flaB-BL21DE3 systems was as high as 40-50% of the total bacterial proteins. Both rFlaA and rFlaB were able to combine with the commercial antibodies against whole cell of H pylori and to induce rabbits to produce specific antibodies with the same 1:2 immunodiffusion titers after the animals were immunized with the two recombinant proteins. Ninety-eight and zero point 4 and 92.80% of the serum samples from 125 patients infected with H pylori were positive for rFlaA and rFlaB antibodies, respectively. One hundred percent and 98.98% of the 98 tested isolates of H pylori were detectable for rFlaA and rFlaB epitopes, respectively. CONCLUSION:Two prokaryotic expression systems with high efficiency of H pylori flaA and flaB genes were successfully established. The expressed rFlaA and rFlaB showed satisfactory immunoreactivity and antigenicity. High frequencies of FlaA and FlaB expression in different H pylori clinical strains and the general existence of specific antibodies against FlaA and FlaB in H pylori infected patients strongly indicate that FlaA and FlaB are excellent antigen candidates for developing H pylori vaccine.

Project description:Helicobacter pylori is one of the most common human pathogens. It causes chronic gastritis and is involved in the pathogenesis of gastroduodenal ulcer disease and possibly gastric carcinoma. Helicobacter mustelae is a bacterium closely related to H. pylori that causes gastritis and ulcer disease in ferrets and is therefore considered an important animal model of gastric Helicobacter infections. Motility, even in a viscous environment, is conferred to the bacteria by several sheathed flagella and is regarded as one of their principal virulence factors. The flagellar filament of H. pylori consists of two different flagellin species expressed in different amounts. The gene (flaA) encoding the major flagellin has recently been cloned and sequenced. Here we report the cloning and sequencing of two highly homologous new flagellin genes from H. pylori 85P and H. mustelae NCTC 12032. The nucleotide sequence of the H. pylori gene proved that it encoded the second flagellin molecule found in H. pylori flagellar filaments. The genes were named flaB. The H. mustelae and H. pylori flaB genes both coded for proteins with 514 amino acids and molecular masses of 54.0 and 53.9 kDa, respectively. The proteins shared 81.7% identical amino acids. The degree of conservation between H. pylori FlaB and the H. pylori FlaA major flagellin was much lower (58%). Both flaB genes were preceded by sigma 54-like promoter sequences. Mapping of the transcription start site for the H. pylori flaB gene by a primer extension experiment confirmed the functional activity of the sigma 54 promoter. To evaluate the importance of both genes for motility, flaA- and flaB-disrupted mutants of H. pylori N6 were constructed by electroporation-mediated allelic exchange and characterized by Western blot (immunoblot) analysis and motility testing. Both mutations selectively abolished the expression of the targeted gene without affecting the synthesis of the other flagellin molecule. Whereas flaA mutants were completely nonmotile, flaB mutants retained motility.

Dataset Information

Antigenic and immunogenic evaluation of Helicobacter pylori FlaA epitopes.

Publications

Antigenic and immunogenic evaluation of <i>Helicobacter pylori</i> FlaA epitopes.

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