Toxin B subunit Genetically Fused to N-terminal Domains of Flagellin logo Induces Potent Humoral Immune Responses against Pseudomonas aeruginosa in BALB/c Mice Dakterzada F¹, Mohabati Mobarez A¹, Habibi Roudkenar M² ¹Bacteriology department, School of medicine, Tarbiat Modares University, Tehran, Iran. ²Research center, Blood Transfusion Organization, Tehran, Iran.

Introduction Results Pseudomonas aeruginosa is an opportunistic bacterium that has high antibiotic resistance. Immunoprophylaxy may be considered as a desirable way for control of P. aeruginosa infections. Flagellin, structural , consists of conserved domains at its N- and C-termini. N-terminal domains of flagellin play an important role in attachment to TLR5 and probably in the induction of protective immune responses. Cholera toxin B is a safe and effective mucosal adjuvant that has also shown strong adjuvant activities when used subcutaneously. Production of cholera toxin B subunit and N-terminal domains of P. aeruginosa flagellin (1- 161 amino acids) fusion protein (CTB-Flagellin(1-161)) and evaluation of its humoral immune responses were aimed in this study. Materials and Methods

Flagellin(1-161) (Type a flagellin), CTB, and CTB-Flagellin(1-161) recombinant were produced and their antigenicity were confirmed by western blotting. against Flagellin(1-161) were raised and evaluated for the motility inhibition of P. aeruginosa 8821M. Binding activity of CTB and CTB-Flagellin(1-161) was detected by GM1-ELISA. The samples (CTB, Flagellin(1-161), CTB + Flagellin(1-161), and CTB-Flagellin(1-161)) Fig 1. Immunogenicity of the CTB-Flagellin(1-161) fusion protein for induction of a Flagellin-specific serum IgG with and without Freund’s adjuvant were injected to BALA/c mice (five in each group) subcutaneously on days response. titers was defined as the serum dilution that gave OD of 0.1 or the serum dilution for which a one- 0, 14 and 28. The endpoint titers of serum IgG to Flagellin and P. aeruginosa strains 8821M (type a) and 415 (1-161) point-higher dilution (2-fold) gave an OD of <0.1. Nonimmune serum (NI) was used as negative control. (A) PaO1(type b) were determined for each mouse by ELISA. Serums of unimmunized mice were used as negative 415 Immunogenicity analysis of the CTB-Flagellin fusion protein by use of Flagellin as coating antigen. (B) control. Furthermore, the effect of immunized mice antisera on phagocytosis of P. aeruginosa 8821M by murine (1-161) (1-161) Immunogenicity analysis of the CTB-Flagellin fusion protein by use of P. aeruginosa 8821M and PaO1 strains as peritoneal macrophages was assessed by opsonophagocytosis assay. All analyses for statistically significant (1-161) differences were performed using one-way ANOVA and P value <0.05% considered significant. coating antigens.

Results Fig 2. Opsonic killing persentage of mice pulled Rabbit antiserum against Flagellin significantly inhibited P. aeruginosa 8821M motility in vitro (data not (1-161) antiserums of each group against P .aeruginosa shown). CTB and CTB-Flagellin showed high affinity to GM1 receptor (data not shown). All groups except (1-161) 8821M.There was a significant difference between all the group receiving CTB, revealed significant antibody response to Flagellin(1-161) and P. aeruginosa groups and Control (test without adding antiserum homologous 8821M strain (P < 0.05) compared to unimmunized group. The antiserums did not react ), except Falgellin(1-161) and CTB-Flagellin(1-161) groups significantly with heterologous PaO1 strain. There was a significant difference (P < 0.05) between group that do not received FA (P < 0.05). receiving CTB-Flagellin(1-161) fusion protein and Flagellin(1-161) and/or CTB + Flagellin(1-161) groups. Furthermore, In addition, there was a significant difference between antiserum against CTB-Flagellin(1-161) + FA produced significantly higher percentage of opsonic death of P. CTB-Flagellin(1-161) + FA and Flagellin(1-161) + FA and aeruginosa 8821M compared to antiserum against Flagellin(1-161) + FA and/or CTB + Flagellin(1-161) + FA (P < /or CTB + Flagellin + FA groups (P < 0.05). 0.05). (1-161)

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T. Plasmodium vivax ookinete surface protein Pvs25 linked to cholera toxin B subunit induces potent CTB-Flagellin(1-161) antibodies react affectively with homologous strain. transmission-blocking immunity by intranasal as well as subcutaneous immunization. Infect Immun 2010; 78(9): CTB-Flagellin(1-161) may be a promising candidate for combating P. aeruginosa infections. 3773-82.