Zonula occludens toxin acts as an adjuvant through different mucosal routes and induces protective immune responses

Abstract

Zonula occludens toxin (Zot) is produced by Vibrio cholerae and has the ability to increase mucosal permeability by reversibly affecting the structure of tight junctions. Because of this property, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. Here we show that Zot acts as a mucosal adjuvant to induce long-lasting and protective immune responses upon mucosal immunization of mice. Indeed, the intranasal delivery of ovalbumin with two different recombinant forms of Zot in BALB/c mice resulted in high Ag-specific serum immunoglobulin G titers that were maintained over the course of a year. Moreover, His-Zot induced humoral and cell-mediated responses to tetanus toxoid in C57BL/6 mice and protected the mice against a systemic challenge with tetanus toxin. In addition, we found that Zot also acts as an adjuvant through the intrarectal route and that it has very low immunogenicity compared to the adjuvant Escherichia coli heat-labile enterotoxin. Finally, by using an octapeptide representing the putative binding site of Zot and of its endogenous analogue zonulin, we provide evidence that Zot may bind a mucosal receptor on nasal mucosa and may mimic an endogenous regulator of tight junctions to deliver Ags in the submucosa. In conclusion, Zot is a novel and effective mucosal adjuvant that may be useful for the development of mucosal vaccines.

FIG. 1.

Persistence of serum Ab response to Ova in BALB/c mice. Mice received five weekly intranasal immunizations with Ova alone or with Ova in the presence of the indicated adjuvants and were then kept in the animal house for 11 months without receiving any further immunizations. Serum samples were collected 1 week after the last immunization (day 35, open bars) and 1 year after the beginning of the immunization protocol (striped bars). Data are expressed as Ab GMTs ± standard errors for five mice in each group.

FIG. 2.

Adjuvant effect and immunogenicity of His-Zot in C57BL/6 mice. Mice were intranasally immunized five times with TT alone or with TT in the presence of His-Zot or LT, and their sera were analyzed for anti-TT Ab titers (A) and for anti-His-Zot and anti-LT Ab titers (B). Data are expressed as described in the legend for Fig. 1.

FIG. 3.

Proliferative responses (A) and cytokine secretion (B) in C57BL/6 mice after intranasal immunization with TT alone (open bars) or with TT and His-Zot (striped bars). Three mice in each group were analyzed, and the data are mean values ± standard deviations. Stimulatory indices of >3 were considered to be positive.

FIG. 4.

Zot acts as an adjuvant through the intrarectal route. C57BL/6 mice were immunized four times intrarectally with TT alone (unfilled circles) or with TT in the presence of His-Zot (filled circles) or LT (squares). The data are expressed as described in the legend for Fig. 1.

FIG. 5.

Inhibition of His-Zot adjuvant activity by the octapeptide FZI/0. C57BL/6 mice were intranasally immunized five times with TT alone or with TT and His-Zot in the presence or absence of the peptide FZI/0. An additional group of mice received TT and FZI/0 to test the potential toxic effect of the inhibitor; however, the octapeptide did not affect the response to the Ag alone (data not shown). Numbers identify individual mice in each group.