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Development of a Trivalent Construct Omp18/AhpC/FlgH Multi Epitope Peptide Vaccine Against Campylobacter jejuni.

ABSTRACT: 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.

SUBMITTER: Lou H

PROVIDER: S-EPMC8793626 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Brucella suis mediates the transmission of brucellosis in humans and animals and a significant facultative zoonotic pathogen found in livestock. It has the capacity to survive and multiply in a phagocytic environment and to acquire resistance under hostile conditions thus becoming a threat globally. Antibiotic resistance is posing a substantial public health threat, hence there is an unmet and urgent clinical need for immune-based non-antibiotic methods to treat brucellosis. Hence, we aimed to explore the whole proteome of Brucella suis to predict antigenic proteins as a vaccine target and designed a novel chimeric vaccine (multi-epitope vaccine) through subtractive genomics-based reverse vaccinology approaches. The applied subsequent hierarchical shortlisting resulted in the identification of Multidrug efflux Resistance-nodulation-division (RND) transporter outer membrane subunit (gene BepC) that may act as a potential vaccine target. T-cell and B-cell epitopes have been predicted from target proteins using a number of immunoinformatic methods. Six MHC I, ten MHC II, and four B-cell epitopes were used to create a 324-amino-acid MEV construct, which was coupled with appropriate linkers and adjuvant. To boost the immunological response to the vaccine, the vaccine was combined with the TLR4 agonist HBHA protein. The MEV structure predicted was found to be highly antigenic, non-toxic, non-allergenic, flexible, stable, and soluble. To confirm the interactions with the receptors, a molecular docking simulation of the MEV was done using the human TLR4 (toll-like receptor 4) and HLAs. The stability and binding of the MEV-docked complexes with TLR4 were assessed using molecular dynamics (MD) simulation. Finally, MEV was reverse translated, its cDNA structure was evaluated, and then, in silico cloning into an E. coli expression host was conducted to promote maximum vaccine protein production with appropriate post-translational modifications. These comprehensive computer calculations backed up the efficacy of the suggested MEV in protecting against B. suis infections. However, more experimental validations are needed to adequately assess the vaccine candidate's potential. HIGHLIGHTS: • Subtractive genomic analysis and reverse vaccinology for the prioritization of novel vaccine target • Examination of chimeric vaccine in terms of allergenicity, antigenicity, MHC I, II binding efficacy, and structural-based studies • Molecular docking simulation method to rank based vaccine candidate and understand their binding modes.

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 (200?g) together with 70?g 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.

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Development of a Trivalent Construct Omp18/AhpC/FlgH Multi Epitope Peptide Vaccine Against Campylobacter jejuni.

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