A bacterial flagellin, Vibrio vulnificus FlaB, has a strong mucosal adjuvant activity to induce protective immunity

Abstract

Flagellin, the structural component of flagellar filament in various locomotive bacteria, is the ligand for Toll-like receptor 5 (TLR5) of host cells. TLR stimulation by various pathogen-associated molecular patterns leads to activation of innate and subsequent adaptive immune responses. Therefore, TLR ligands are considered attractive adjuvant candidates in vaccine development. In this study, we show the highly potent mucosal adjuvant activity of a Vibrio vulnificus major flagellin (FlaB). Using an intranasal immunization mouse model, we observed that coadministration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific immunoglobulin A (IgA) responses in both mucosal and systemic compartments and IgG responses in the systemic compartment. The mice immunized with TT plus FlaB were completely protected from systemic challenge with a 200x minimum lethal dose of tetanus toxin. Radiolabeled FlaB administered into the nasal cavity readily reached the cervical lymph nodes and systemic circulation. FlaB bound directly to human TLR5 expressed on cultured epithelial cells and consequently induced NF-kappaB and interleukin-8 activation. Intranasally administered FlaB colocalized with CD11c as patches in putative dendritic cells and caused an increase in the number of TLR5-expressing cells in cervical lymph nodes. These results indicate that flagellin would serve as an efficacious mucosal adjuvant inducing protective immune responses through TLR5 activation.

FIG. 1.

FlaB had mucosal adjuvant activity to induce protective immunity. (A) Recombinant FlaB was analyzed by SDS-PAGE (left) and Western blot analysis (right) with an anti-FlaB antibody. (B) Female 7-week-old BALB/c mice (n = 10) were intranasally given PBS, 3 μg of TT alone, or TT in combination with 1, 5, and 15 μg of FlaB three times in 7-day intervals. Seven days after the last immunization, all of the mice were subcutaneously challenged with a 200× minimum lethal dose of tetanus toxin and observed for 7 days. (C) Seven days after the last immunization, blood and mucosal samples (saliva and vaginal wash) were collected from the mice (n = 5), and TT-specific antibody titers were measured by ELISA. (D) The BALB/c mice were intranasally given 3 μg of TT alone, TT in combination with 7 μg of GST, or 18 μg of GST-FlaB three times in 7-day intervals. Seven days after the last immunization, TT-specific IgG and IgA levels in sera were measured. Values represent the mean endpoint (log₂) antibody titer ± the standard deviation in each group. The experiment was repeated three times with similar results. *, P < 0.01; **, P < 0.05 versus TT-only-immunized group.

FIG. 2.

FlaB stimulated IL-8 production in Caco-2 cells in a dose-dependent manner. Caco-2 cells were treated with indicated concentrations of FlaB for 5 h. (A) IL-8 concentrations in the supernatant were determined by ELISA. *, P < 0.001 compared to control cultures. (B) The level of IL-8 transcripts was assessed by the real-time RT-PCR with β-actin as an internal control. Values of mRNA expression were expressed as the relative increase of IL-8 mRNA compared to that in nontreated Caco-2 cells. Values represent the mean ± the standard deviation in each group. Vv-FlaB, V. vulnificus FlaB.

FIG. 3.

FlaB directly interacted with TLR5 in epithelial cells and FlaB induced TLR5-mediated NF-κB and IL-8 transcription. (A) Caco-2 cells were transfected with a 3× Flag-TLR5-expressing plasmid and further treated with 500 ng of FlaB/ml for 24 h. Cell lysates were subjected to immunoprecipitation (IP) with an anti-FlaB antibody. The immunoprecipitated proteins were resolved by SDS-PAGE and analyzed by Western blotting (WB) with an anti-Flag antibody (top panel). Aliquots of cell lysates were subjected to anti-Flag Western blotting (bottom panel) to evaluate transfection efficiency. Caco-2 cells were transiently cotransfected with hTLR5 expression plasmid and pIL-8-Luc (B) or pNF-κB-Luc (C), along with increasing amounts of purified FlaB. Relative luciferase activities in cell extracts were analyzed by the dual-luciferase reporter assay system and normalized with pCMV-β-galactosidase as a control. All values represent the mean ± the standard error of at least three independent experiments. *, P < 0.02 compared to control cultures. Vv-FlaB, V. vulnificus FlaB.

FIG. 4.

Trafficking of radiolabeled FlaB in mouse. A total of 8 × 10⁶ cpm of ¹³¹I-labeled FlaB (6 μg) in 12 μl was given intranasally to BALB/c mice. In order to detect the distribution of the radiolabeled FlaB in lymphoid organs and blood, we prepared NALT, CLNs, spleens, and blood from the mice at the indicated times. The radioactivity of the sample was measured by using a scintillation counter. The data represent the average counts per minute of three mice ± the standard error in each group.

FIG. 5.

FlaB was colocalized with CD11c⁺ cells in mouse CLNs. BALB/c mice were intranasally administered with 50 μg of FlaB. After 6 h, CLNs were freshly isolated from the mice and were frozen for section. The samples were stained with CD11c-FITC (green) and anti-FlaB antibody, followed by the addition of Texas Red-labeled anti-rabbit IgG antibody (red). Localization of FlaB in the CLNs was determined by confocal microscopic observation. Vv-FlaB, V. vulnificus FlaB.

FIG. 6.

FlaB treatment stimulated TLR5 expression in CLNs and spleen. BALB/c mice were intranasally given PBS or 50 μg of FlaB. After 6 h, CLNs and spleens were freshly isolated from the mice for the evaluation of TLR5 expression in vivo. (A) Frozen sections of CLNs were prepared and stained with CD11c-FITC (green) and anti-TLR5 antibody, followed by Texas Red-labeled anti-rabbit IgG antibody (red). Fluorescence images of cells were captured under a confocal microscope. (B) Tissue lysates were prepared from the spleen and Western blotting was carried out to detect TLR5 expression. The β-actin blot is shown as a loading control. (C) The mRNA expression of TLR5 mRNA in the spleen was analyzed by the RT-PCR analysis. One of three experiments with similar results is shown. Vv-FlaB, V. vulnificus FlaB.