Adjuvant role of Pseudomonas flagellin for Acinetobacter baumannii biofilm associated protein

Figure 1 Western blot analysis of (A) 7 kDa recombinant N Flagellin, and (B) 42 kDa Bap subunit, (C) and (D) sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealing the presence of 7 kDa Flagellin and 42 kDa biofilm associated protein.

Figure 2 Indirect enzyme linked immunosorbent assay of sera of mice injected. A: Indirect ELISA of sera of mice injected with Bap: Rise of antibody titers is significant (P < 0.001); B: Indirect ELISA of sera of mice injected with N-flagellin subunit only: No significant rise of antibody titers was noted; C: Indirect ELISA of sera of mice injected with a single dose of Bap first followed by administration of N-flagellin in subsequent vaccinatio: Significant (P < 0.001) rise of antibody titers was noted; D: Indirect ELISA of sera of mice injected with N-flagellin followed by administration of Bap in the subsequent vaccinations: Significant (P < 0.001) rise of antibody titers was noted; E: Indirect ELISA of mice sera administered with Bap + flagellin subunit: Antibody titers increased significantly (P < 0.001) from the second booster onwards. ELISA: Enzyme linked immunosorbent assay.

Figure 3 Whole cell enzyme linked immunosorbent assay of mice sera immunized with Bap and Bap + flagellin against Acinetobacter baumannii. Significant (P < 0.05) OD values were observed in experimental strain as compared to control group. Bap: Biofilm associated protein.

Figure 4 Survival of mice immunized with biofilm associated protein (group A) and biofilm associated protein-flagellin (group E) challenged with Pseudomonas aeruginosa and Acinetobacter baumannii. A. baumannii: Acinetobacter baumannii; Bap: Biofilm associated protein; P. aeruginosa: Pseudomonas aeruginosa.

Figure 5 Neutralization. A: The group received four doses of 20 μg of the recombinant Bap; B: The group received four doses of 20 μg of the recombinant N-flagellin; C: The group received a single dose of 20 μg recombinant Bap followed by administration of N-flagellin in subsequent vaccinations; D: The group received a single dose of 20 μg of recombinant N-flagellin followed by administration of Bap in subsequent vaccinations; E: The group received combination of Bap and N-flagellin simultaneously in all the vaccinations; F: The control group received combination of PBS and Freund’s adjuvant. A. baumannii: Acinetobacter baumannii; Bap: Biofilm associated protein.

Figure 6 Neutralization test. A: The group received four doses of 20 μg of the recombinant Bap; B: The group received combination of Bap and N-flagellin simultaneously in all the vaccinations; control: The control group received combination of PBS and Freund’s adjuvant. P. aeruginosa: Pseudomonas aeruginosa; Bap: Biofilm associated protein.