An LTB-entrapped protein in PLGA nanoparticles preserves against enterotoxin of enterotoxigenic Escherichia coli

Objective(s): Enterotoxigenic Escherichia coli (ETEC) is known as the most common bacterial causes of diarrheal diseases related to morbidity and mortality. Heat-labile enterotoxin (LT) is a part of major virulence factors in ETEC pathogenesis. Antigen entrapment into nanoparticles (NPs) can protect them and enhance their immunogenicity.Materials and Methods: In the present study, recombinant LTB protein was expressed in E. coli BL21 (DE3) and purified by an Ni-NTA agarose column. The protein was entrapped in PLGA polymer by the double emulsion method. NPs were characterized physicochemically and the protein release from the NPs was evaluated. ELISA assay was performed for investigation of raised antibody against the recombinant protein in mice. The anti-toxicity and anti-adherence attributes of the immune sera against ETEC were also evaluated.Results: It showed the successful cloning of a 313 bp DNA fragment encoding LTB protein in the pET28a vector. Over-expression in BL21 (DE3) led to the formation of corresponding 15.5 kDa protein bands in the SDS-PAGE gel. Western blotting by using anti-CTX confirmed the purified LTB. Protein-entrapped NPs had a spherical shape with the size of 238 nm mean diameter and 85% entrapment efficiency.  Immunological analyses showed the production of a high titer of specific IgG antibody in immunized animals. The neutralizing antibody in the sera of immunized animals was approved by GM1 binding and Ileal loop assays.Conclusion: The results indicate the efficacy of the entrapped LTB protein as an effective immunogen which induces the humoral responses

Immunoinformatic design of an epitope-based immunogen candidate against Bacillus anthracis

Bacillus anthracis is a highly infectious bacterium and causes anthrax. It infects warm-blooded mammals, likewise its spores have been weaponized and used in biological warfare which has affected many people. Prevention of anthrax remains a great challenge for clinicians. Vaccination is the most hopeful mode and a new generation of epitope-based vaccines has received extended attention. This kind of recombinant vaccine offers an effective and fast response because of the direct targeting of the immune system. Nowadays, collecting epitopes information via in silico tools are highly accurate and simple. In this regard, a variety of the best bioinformatic servers were applied to identify potential B-cell, T-cell, IFN-γ, IL-10, and IL-4 epitopes with no allergenic nor toxic effect. The High-ranked epitopes were selected and fused to each other by GPGPG and EAAAK linkers. They constructed the scaffold along with the L7/L12 adjuvant and 6xHis-tag. The physicochemical properties, secondary and tertiary structures were evaluated, refined, and confirmed using bioinformatics tools. Desirable levels of IgG1, IgG2, T-cytotoxic, T-helper cells, INF-γ, and interleukins were predicted using immune simulation tool. Furthermore, the molecular docking and dynamic simulation between epitopic protein and receptors revealed proper and stable binding affinity. Overall, the engineered recombinant protein can be efficaciously considered as a candidate immunogen

Insilico Design and Construction of a Chimeric Gene Comprising the A Subunit of Vibrio cholera Pilin and the B Subunit of Cholera Toxin

Background and Aims: Cholera is a lethal diarrheal disease that cause by Vibrio cholerae. Cholera toxin and colonization factor pili (tcpA) are the major virulence factor in V.cholerae pathogenesis. The B subunit of the enterotoxin )ctxB) which is responsible for toxin binding to eukaryotic cells and toxin-coregulated pili A (tcpA) that is essential for V.cholerae colonization, have immunogenic properties. Chimeric proteins carrying epitopes, linkers or adjuvant sequences could increase immunogenicity for recombinant antigens and can also elicit broad immune responses. The aim of this study was to design am immunogen against adherence and toxicity of V.cholorae. Materials and Methods: ctxB and tcpA genes were analyzed for rare codons and gene optimization was performed using optimization software In 2017. The half-life and protein instability index was determined. Secondary and tertiary structure was predicted and evaluated. Linear and conformational epitopes were predicted. Recombinant pET28a/chimeric gene plasmid was transformed to E.coli BL21 DE3 and expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG). The protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Chimeric protein instability index was 24.04 and the codon adaptation index of chimeric constructs increased to 0.9. The tertiary predicted structure of the chimeric proteins confirmed and mRNA was stable. Conformational and linear epitopes were seen in both domains of the chimeric protein. Restriction analysis confirmed cloning of the gene into pET28a vector. Expression of recombinant protein in E.coli led to the production of chimeric protein with 37 k Da molecular weight. Conclusions: According to the results of bioinformatics and recombinant protein expression, the design chimeric protein could be used as an immunogen to evaluate the immunity against cholera