ABSTRACT
Background: Some resources have suggested that genetically inactivated pertussis toxoid (PTs) bear a more protective effect than chemically inactivated products. This study aimed to produce new version of PT, by cloning an inactive pertussis toxin S1 subunit (PTS1) in a fusion form with N-terminal half of the listeriolysin O (LLO) pore-forming toxin. Methods: Deposited pdb structure file of the PT was used to model an extra disulfide bond. Codon-optimized ORF of the PTS1 was used to make recombinant constructs of PTS1 and LLO-PTS1 in the pPSG-IBA35 vector. The recombinant PTS1 and LLO-PTS1 proteins were expressed in BL21 DE3 and SHuffle T7 strains of E. coli and purified by affinity chromatography. Cytotoxic effects of the recombinant proteins were examined in the MCF-7 cell line. Results: The purity of the products proved to be more than 85%, and the efficiency of the disulfide bond formation in SHuffle T7 strain was higher than BL21 DE3 strain. No cytotoxicity of the recombinant proteins was observed in MCF-7 cells. Soluble recombinant PTS1 and LLO-PTS1 proteins were produced in SHuffle T7 strain of E. coli with high efficiency of disulfide bonds formation. Conclusion: The LLO-PTS1 with corrected disulfide bonds was successfully expressed in E. coli SHuffle T7 strain. Due to the safety for human cells, this chimeric molecule can be an option to prevent pertussis disease if its immunostimulatory effects would be confirmed in the future.
Subject(s)
Bacterial Toxins/genetics , Heat-Shock Proteins/genetics , Hemolysin Proteins/genetics , Pertussis Toxin/genetics , Recombinant Fusion Proteins/biosynthesis , Cell Proliferation/drug effects , Disulfides/chemistry , Escherichia coli , Humans , MCF-7 Cells , Pertussis Vaccine/biosynthesis , Pertussis Vaccine/chemistry , Protein Engineering , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/pharmacologyABSTRACT
BACKGROUND: Current evidence indicates the resurgence of whooping cough despite high coverage of whole-cell (wP) and acellular (aP) pertussis vaccines. OBJECTIVE: To investigate the cytokine response to a genetically inactivated protein containing the S1 subunit of pertussis toxin (PTS1) with and without the Listeriolysin O (LLO-PTS1), in comparison with current wP and aP vaccines in the mice model. METHODS: Thirty-six female NMRI mice aged 8 to 12 weeks (25 ± 5 g) were divided into six groups, including control (n=6) and five treated groups (n=6/each). Treated groups received intraperitoneal injection of recombinant PTS1, recombinant fusion LLO-PTS1, aP, wP, and sham (phosphate-buffered saline), whereas the control group did not receive anything. After 60 days, the serum levels of IFN-γ, IL-4, and IL-17 cytokines were evaluated by ELISA method. RESULTS: Our findings showed LLO-PTS1 significantly increased IL-17 and IL-4 cytokines compared with wP and aP vaccines. IFN-γ failed to increase substantially in the LLO-PTS1 group compared to others, but it was non-inferior to standard vaccines. CONCLUSION: Our alum free mono-component monovalent recombinant fusion protein (LLO-PTS1) could bear the capacity to stimulate the release of IFN-γ similar to wP and aP vaccines in the mouse model. Besides, it showed better results in stimulating the release of IL-17 and IL-4 response. This study can be regarded as a platform for further probes in booster pertussis vaccine development.
