Absence of suitable mucosal adjuvants for humans prompted us to consider alternative vaccine designs for mucosal immunization. Because adenovirus is adept in binding to the respiratory epithelium, we tested the adenovirus 2 fiber protein (Ad2F) as a potential vaccine-targeting molecule to mediate vaccine uptake. The vaccine component (the host cell-binding domain to botulinum toxin (BoNT) serotype A) was genetically fused to Ad2F to enable epithelial binding. The binding domain for BoNT was selected because it lies within the immunodominant H chain as a β-trefoil (Hcβtre) structure; we hypothesize that induced neutralizing Abs should be protective. Mice were nasally immunized with the Hcβtre or Hcβtre-Ad2F, with or without cholera toxin (CT). Without CT, mice immunized with Hcβtre produced weak secretory IgA (sIgA) and plasma IgG Ab response. Hcβtre-Ad2F-immunized mice produced a sIgA response equivalent to mice coimmunized with CT. With CT, Hcβtre-Ad2F-immunized mice showed a more rapid onset of sIgA and plasma IgG Ab responses that were supported by a mixed Th1/Th2 cells, as opposed to mostly Th2 cells by Hcβtre-dosed mice. Mice immunized with adjuvanted Hcβtre-Ad2F or Hcβtre were protected against lethal BoNT serotype A challenge. Using a mouse neutralization assay, fecal Abs from Hcβtre-Ad2F or Hcβtre plus CT-dosed mice could confer protection. Parenteral immunization showed that the inclusion of Ad2F enhances anti-Hcβtre Ab titers even in the absence of adjuvant. This study shows that the Hcβtre structure can confer protective immunity and that use of Hcβtre-Ad2F gives more rapid and sustained macosal and plasma Ab responses. Copyright © 2006 by The American Association of Immunologists, Inc.