Tetanus toxin fragment C fused to flagellin makes a potent mucosal vaccine

Abstract

Purpose: Recombinant subunit vaccines provide safe and targeted protection against microbial infections. However, the protective efficacy of recombinant subunit vaccines tends to be less potent than the whole cell vaccines, especially when they are administered through mucosal routes. We have reported that a bacterial flagellin has strong mucosal adjuvant activity to induce protective immune responses. In this study, we tested whether FlaB could be used as a fusion partner of subunit vaccine for tetanus.

Materials and methods: We constructed fusion proteins consisted with tetanus toxin fragment C (TTFC), the nontoxic C-terminal portion of tetanus toxin, and a Toll-like receptor 5 agonist from Vibrio vulnificus (FlaB). Mice were intranasally administered with fusion protein and protective immune responses of the vaccinated mice were analyzed.

Results: FlaB-TTFC recombinant protein induced strong tetanus-specific antibody responses in both systemic and mucosal compartments and prolonged the survival of mice after challenge with a supra-lethal dose of tetanus toxin.

Conclusion: This study establishes FlaB as a successful fusion partner for recombinant subunit tetanus vaccine applicable through mucosal route, and it further endorses our previous observations that FlaB could be a stable adjuvant partner for mucosal vaccines.

Keywords: Adjuvant; Flagellin; Subunit vaccine; Tetanus toxin fragment C.

Fig. 1

Development of recombinant fusion proteins. Recombinant fusion vectors were constructed as described in Materials and Methods. The map of the vectors and the DNA fragments of flaB, tetanus toxin fragment C (TTFC), and restriction enzyme sites are shown (A). Purified fusion proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (B) and Western blot analysis (C). Putative structure for the fusion protein was predicted by bioinformatics tools (D). The model was constructed by the Phyre server (http://www.sbg.bio.ic.ac.uk/phyre/) using Protein Data Bank (PDB) data sets. TF, TTFC-FlaB fusion protein; FT, FlaB-TTFC fusion protein.

Fig. 2

Toll-like receptor 5 (TLR5)-mediated nuclear factor-κB (NF-κB) stimulating activity of the recombinant proteins. Fusion protein-mediated TLR5 signaling was determined by NF-κB-luciferase reporter assay. 293 cells were co-transfected with hTLR5 and pNF-κB-Luc and further treated with FlaB (100 ng/mL) or FlaB-tetanus toxin fragment C (FlaB-TTFC) (219 ng/mL) for 24 hours. Relative luciferase activities in cell extracts were analyzed by the dual-luciferase reporter assay system and normalized with pCMV-β-galactosidase as a control. ^***p < 0.001.

Fig. 3

Tetanus toxin fragment C (TTFC)-specific antibody responses after intranasal immunization. Mice were intranasally immunized with phosphate buffered saline (PBS), 1.5 µg TTFC alone (TTFC), 1.5 µg TTFC combined with 1.25 µg FlaB (T+F), or 2.75 µg FlaB-TTFC fusion protein (FT) three times at 1-week interval. One week after the last immunization, serum or mucosal samples were collected and TTFC-specific IgG (A) and IgA (B) titers were determined. Data are presented as the mean±SEM in each group. ^*p<0.05, ^**p<0.01, ^***p<0.001 when compared with the T+F group and FT group.

Fig. 4

Survival (A) and paralytic score (B) of mice challenged with tetanus toxin. Mice were intranasally immunized with phosphate buffered saline (PBS), 1.5 µg tetanus toxin fragment C alone (TTFC), 1.5 µg TTFC combined with 1.25 µg FlaB (T+F), or 2.75 µg FlaB-TTFC fusion protein (FT) three times at 1-week interval. One week after the last immunization, mice were subcutaneously challenged with 600× LD₅₀ tetanus toxin. After the challenge, survival rate and tetanic phenotype were monitored for 7 days. Data represent the results from 7 mice in each experiment group. ^**p<0.01 compared with T+F group with FT group.