Development of A Novel Anti-Adhesive Vaccine Against Pseudomonas Aeruginosa Targeting the C-Terminal Disulfide Loop of the Pilin Protein

Type iv pili (t4p) are major virulence factors of pseudomonas aeruginosa (p. aeruginosa) that are associated with primary adhesion, biofilm formation and twitching motility. this study focuses on the introduction of a novel biologically active subunit vaccine derived from the disulfide loop (dsl) of p. aeruginosa pilin. we investigated the expression of the novel pila in-frame with pet26b vector, which contains three domains, that each domain contains three tandem repeats. the flexible (ggggs) and (ggggs)3 linkers were linked between the three tandem repeats and each pila domain, respectively. the recombinant construct (pet26b/pila) was transformed and expressed in escherichia coli bl21 (de3). the reactivity of specific antiserum against pila was assessed by elisa method. the biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis and twitching inhibition assays. the pet26b/pila plasmid was confirmed by enzymatic digestion. the purified pila protein was confirmed by immunoblot analysis. the checkerboard titration showed that the optimal dilution of the antibody to react with antigen was 1:8. the results of opsonophagocytosis assay revealed that the antibodies raised against pila promoted phagocytosis of the pao1 and 6266e strains to some extent (17.5% and 16.3%, respectively), so the twitching inhibition test confirmed this result. taken together, these are the preliminary results based on a first chimerical structure failure to induce antibodies that promote the opsonization and eradication of the pathogen. therefore, the biological activity of the pila protein showed that it should be introduced with other proteins or target antigens against p. aeruginosa in the future studies.