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Evaluations of Alkyl hydroperoxide reductase B cell antigen epitope as a potential epitope vaccine against Campylobacter jejuni.

ABSTRACT: Objective:The present study aimed to screen and find alkyl hydroperoxide reductase (AhpC) B cell dominant epitope of Campylobacter jejuni (C. jejuni). Materials and methods:Bio-informatic algorithms were used to predict B cell epitopes of AhpC. The AhpC protein and chemically synthesized antigenic epitopes of C. jejuni were considered as antigens, and the AhpC antibody was used as the primary antibody, ELISA and dot blot were used to analyze and screen the dominant epitope. The specific IgG of mice serum and IL-4 in splenocyte culture supernatant were detected by ELISA. The protective efficacy was evaluated by animal disease index and tissue histopathological staining of the jejunum. Results:Seven epitopes of AhpC were predicted, one epitope (AhpC4-16) was found to recognize the antibodies of AhpC and had strong antigenicity by ELISA and dot blot analysis. In epitope AhpC4-16 immunized mice, specific IgG of serum and IL-4 in splenocyte culture supernatant were significantly higher. The illness index decreased significantly, the protective rate was 66.67%. Histopathology displayed that the jejunum morphology was better than the control group. Conclusions:These findings suggested that epitope AhpC4-16 showed effective protective role against C. jejuni and is a candidate epitope of vaccine against this pathogen.

SUBMITTER: Lou H

PROVIDER: S-EPMC6734151 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Evaluations of Alkyl hydroperoxide reductase B cell antigen epitope as a potential epitope vaccine against <i>Campylobacter jejuni</i>.

Lou Hongqiang H Li Xusheng X Guo Fangming F Ding Mingxing M Hu Ye Y Chen Haohao H Yan Jie J

Saudi journal of biological sciences 20190529 6

<h4>Objective</h4>The present study aimed to screen and find alkyl hydroperoxide reductase (AhpC) B cell dominant epitope of <i>Campylobacter jejuni</i> (<i>C. jejuni</i>).<h4>Materials and methods</h4>Bio-informatic algorithms were used to predict B cell epitopes of AhpC. The AhpC protein and chemically synthesized antigenic epitopes of <i>C. jejuni</i> were considered as antigens, and the AhpC antibody was used as the primary antibody, ELISA and dot blot were used to analyze and screen the dom ...[more]

PMID: 31516338

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Project description:AIM:To identify alkyl hydroperoxide reductase subunit C (AhpC) homologs in Bacillus subtilis (B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria. METHODS:Two AhpC homologs (AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues (C37S, C47S, C166S, C37/47S, C37/166S, C47/166S, and C37/47/166S for AhpC_H1; C52S, C169S, and C52/169S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahpC genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined. RESULTS:Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis. CONCLUSION:AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.

Project description:Campylobacter jejuni (C. jejuni) is one of the major pathogens contributing to the enteritis in humans. Infection can lead to numerous complications, including but not limited to Guillain-Barre syndrome, reactive arthritis, and Reiter's syndrome. Over the past two decades, joint efforts have been made toward developing a proper strategy of limiting the transmission of C. jejuni to humans. Nevertheless, except for biosecurity measures, no available vaccine has been developed so far. Judging from the research findings, Omp18, AhpC outer membrane protein, and FlgH flagellin subunits of C. jejuni could be adopted as surface protein antigens of C. jejuni for screening dominant epitope thanks to their strong antigenicity, expression of varying strains, and conservative sequence. In this study, bioinformatics technology was adopted to analyze the T-B antigenic epitopes of Omp18, AhpC, and FlgH in C. jejuni strain NCTC11168. Both ELISA and Western Blot methods were adopted to screen the dominant T-B combined epitope. GGS (GGCGGTAGC) sequence was adopted to connect the dominant T-B combined epitope peptides and to construct the prokaryotic expression system of tandem repeats of antigenic epitope peptides. The mouse infection model was adopted to assess the immunoprotective effect imposed by the trivalent T-B combined with antigen epitope peptide based on Omp18/AhpC/FlgH. In this study, a tandem epitope AhpC-2/Omp18-1/FlgH-1 was developed, which was composed of three epitopes and could effectively enhance the stability and antigenicity of the epitope while preserving its structure. The immunization of BALB/c mice with a tandem epitope could induce protective immunity accompanied by the generation of IgG2a antibody response through the in vitro synthesis of IFN-γ cytokines. Judging from the results of immune protection experiments, the colonization of C. jejuni declined to a significant extent, and it was expected that AhpC-2/Omp18-1/FlgH-1 could be adopted as a candidate antigen for genetic engineering vaccine of C. jejuni MAP.

Project description:Campylobacter jejuni is the leading bacterial cause of human enteritis in many industrialized countries. There is no commercial vaccine against C. jejuni available to date. CmeC is an essential outer membrane component of CmeABC multidrug efflux pump that plays a critical role in antibiotic resistance and in vivo colonization of C. jejuni. CmeC is prevalent in C. jejuni strains and is dramatically induced and immunogenic in vivo. In this study, we analyzed CmeC sequence homology, examined in vitro immune protection of CmeC peptide antibodies, and produced full-length recombinant CmeC (rCmeC) for evaluating immunogenicity and protective efficacy of the CmeC subunit vaccine against C. jejuni using chicken model system. Amino acid sequences of CmeC from 24 diverse C. jejuni strains were determined and subjected to alignment, which revealed that CmeC is highly conserved in C. jejuni with a identity ranging from 97.3% to 100%. CmeC peptide antibodies inhibited the function of CmeABC efflux pump and enhanced susceptibility of C. jejuni to bile salts, the natural antimicrobial present in the intestine. Two full-length rCmeC proteins with N- or C-terminal His tag were produced in E. coli; the N-terminal His-tagged rCmeC with high purity and yield was obtained by single step affinity purification. The purified rCmeC was used in two vaccination trials using a chicken model of C. jejuni infection. Stimulation of CmeC-specific serum IgG responses via oral vaccination required immunization with higher doses of rCmeC (200μg) together with 70μg of mucosal adjuvant mLT (modified E. coli heat-labile enterotoxin). Subcutaneous vaccination of chickens with rCmeC remarkably stimulated both serum IgG and IgA responses. However, CmeC-specific intestinal secretory IgA response was not significantly stimulated regardless of vaccination regimen and the rCmeC vaccination did not confer protection against C. jejuni infection. Together, these findings provide further compelling evidence that CmeC is a promising subunit vaccine candidate against C. jejuni infection. However, the CmeC vaccination regimen should be optimized to enhance CmeC-specific mucosal immune response in for protection against C. jejuni.

Dataset Information

Evaluations of Alkyl hydroperoxide reductase B cell antigen epitope as a potential epitope vaccine against Campylobacter jejuni.

Publications

Evaluations of Alkyl hydroperoxide reductase B cell antigen epitope as a potential epitope vaccine against <i>Campylobacter jejuni</i>.

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