Design, purification, and biophysical characterization of a multi-epitope vaccine construct made from outer membrane proteins of Pseudomonas aeruginosa

Pseudomonas aeruginosa is increasingly becoming resistant to multiple drugs and is held responsible for high rate of mortality and morbidity across the globe. This study aims to examine proteins from outer membrane such as OprE, OprF, OprC, and OprG, for the multi-epitope vaccine design. In this article, the prediction of epitopes for helper T-cells, cytotoxic T-cells, and B-cell epitopes was carried out by the application of various immune-informatics tools. All the predicted epitopes were aligned and assembled into a peptide sequence along with linkers and adjuvant sequences. Further, secondary structure and three-dimensional structure were predicted for the multiepitope construct. The vaccine construct designs were evaluated and validated for allergenicity, toxicity, and antigenicity. The validation of the predicted structure was carried out by determining its physiochemical properties by the application of ProtParam. Protein docking and molecular-dynamic simulation confirmed strong and stable interaction between the vaccine construct and Toll-like receptor-4. The vaccine construct was cloned into pET-29b vector and expressed in Escherichia coli. The designed multiepitope vaccine construct was overexpressed and purified by the application of Ni-NTA affinity chromatography and subjected to SDS-PAGE analysis. The circular dichroism spectroscopy analysis revealed it to be stable and structured. The hemolysis assay demonstrated minimal RBC toxicity suggesting that it was safe to use. The designed vaccine construct could activate both humoral and cellular immune responses as demonstrated by the advanced immunoinformatic approach making it a promising vaccine construct for protection against P. aeruginosa.