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Pseudomonas aeruginosa is associated with different infections such as urinary tract infections (UTIs). The increased antibiotic resistance reaches the need to develop vaccine against the infections. In the present study, bioinformatics approaches were applied to design a novel multi-epitope of PcrV and OmpE from P. aeruginosa . The raised antibody against the multi-epitope was evaluated and challenge experiment was done to evaluate the efficacy of the multi-epitope. The results of epitope mapping of B-cells indicated 8 regions for PcrV and OmpE. The predicted 3D structure showed C-score = -1 and Z-score = -8.12. Molecular docking indicated high interaction between residues of Toll-like receptor 2 (TLR2) and TLR4 with the multi-epitope. The results of in silico simulation of the immune responses showed elevated levels of B-cell, T-cell, and memory cells. PcrV, OmpE, and the multi-epitope were expressed in pET28a- E. coli BL21 (DE3) and purified by Nickel columns. Our findings indicated that the sera collected from immunized rabbits with the multi-epitope reacted with the multi-epitope, PcrV, and OmpE in western blot. According to the ELISA results, the antibody developed against the multi-epitope showed cross-reactivity with individual proteins PcrV and OmpE. The level of antibody raised against the multi-epitope was significantly higher than the antibody reacted with PcrV or OmpE alone in ELISA. The challenge results confirmed that the load of bacteria was decreased in immunized rabbits as compared to the control. The results present the multi-epitope composed of PcrV and OmpE as a promising candidate against P. aeruginosa . Further evaluations are under investigation in animal model.Communicated by Ramaswamy H. Sarma.