Recombinant Flagellin and Incomplete Freund’s Adjuvant Potentiate the Vaccine Efficacy of the Iron Acquisition Protein (HitA) of Pseudomonas aeruginosa

Objectives: Pseudomonas aeruginosa is a notorious bacterial pathogen that can cause a variety of infections with high morbidity and mortality. The increasing multi-drug resistance of this pathogen makes it urgent to develop an effective vaccine. This study aimed at evaluation of the immunoadjuvant effect of flagellin (FliC) of Salmonella enterica and incomplete Freund’s adjuvant (IFA) on the protective vaccine efficacy of P. aeruginosa recombinant iron acquisition protein (HitA). Methods: In this work, recombinant HitA, FliC and fused FliC-F-HitA proteins were expressed in Escherichia coli after cloning their respective genes into pQE31 plasmid vector. The proteins were purified using metal affinity chromatography. The immunoadjuvant effect of FliC was examined upon mixing as well as fusion with HitA antigen of P. aeruginosa in the presence of incomplete Freund’s adjuvant (IFA). This was tested by active immunization followed by challenge using P. aeruginosa murine infection model. Results: Two weeks after the last immunization dose, serum samples were tested for antibody response which showed significant HitA-specific IgG antibody response in all immunized groups compared to control groups. A significant reduction in bacterial burden of lungs from mice immunized with HitA/FliC/IFA mixture was observed after challenge. Opsonophagocytic assay and liver histopathological examination confirmed the previous results. Conclusion: Overall, HitA recombinant protein is considered a promising vaccine candidate against P. aeruginosa upon mixing with S. enterica flagellin protein FliC and IFA.