Botulism results from absorption of the toxin into the systemic circulation from either a mucosal surface or a wound. BoNT intoxication is typically attributed to oral ingestion of contaminated food either with the toxin or bacteria producing the toxin [6
]. Although natural exposure via the inhalational route is unlikely [18
], illicit aerosol releases have occurred [28
]; thus, there is a precedence to stimulate mucosal immune responses to aid in conferring protection by neutralizing the toxin at the mucosal surface. While most studies have focused on BoNT/A [13
], few studies have evaluated BoNT/B-based vaccines [31
]. Since BoNT/B is often implicated in human illness, the present study focuses on generating a protective mucosal vaccine.
Efforts to develop an efficacious vaccine for botulism are warranted because the current pentavalent toxoid vaccine has a number of disadvantages [23
], including the limited ability to stimulate mucosal Abs when given parenterally [30
] and the required yearly boosts to maintain protective Ab levels [13
]. To address these shortcomings, recombinant vaccine approaches would best preserve neutralization epitopes, and consideration of mucosal immunization approaches may best serve to induce both mucosal and systemic immunity. Immunization of both mucosal and systemic immune compartments could then facilitate the clearance of the toxin from tissues and inhibit its absorption to mucosal surfaces [31
Current efforts to develop BoNT vaccines have focused primarily on immunizing with recombinant Hc polypeptides capable of evoking protective immunity against BoNT challenge [10
]. Hc is an efficient immunogen that combined with the ability to penetrate the epithelial membranes raises the possibility that the Hc can serve as a mucosal vaccine against botulism. Furthermore, as previously shown for protection against BoNT/A [23
] and suggested by others [36
], the entire Hc fragment is not required for protection and can be further reduced to contain the C-terminal β-trefoil subdomain. Our previous study provided the first evidence the Hcβtre fragment retains its immunogenicity and protective capacity when delivered nasally [23
]. In addition, since β-trefoil structure is conserved among the seven BoNTs, it is plausible this conserved structure can evoke cross-reactive Abs against the other serotypes. In addition, we sought to develop a second animal model for BoNT intoxication using antisera developed in rabbits. When testing commercially prepared Hc against Hcβtre/A-Ad2F in rabbits formulated with CT or a non-toxin adjuvant, we found the Hcβtre/A-Ad2F vaccine was superior to Hc in conferring protection against BoNT/A challenge in a mouse neutralization assay when using antisera developed in rabbits (manuscript in preparation). These results suggest that the Hcβtre-Ad2F immunogen is effective in eliciting BoNT-specific Abs.
While next generation vaccines may significantly improve vaccine efficacy, these are often poorly immunogenic when applied mucosally [33
]. To enhance their immunogenicity, vaccines would require an effective adjuvant to bolster protective immunity. Currently, a major obstacle to adapting mucosal vaccines for humans is the lack of a mucosal adjuvant suitable for use in humans. Nasal application of adenovirus-based vaccines offers one approach to stimulate robust systemic and mucosal Ab responses, as well as cell-mediated immunity [25
]. In fact, recombinant adenovirus bearing the Hc transgene has been shown to effectively stimulate neutralizing Abs to BoNT/C [37
]. However, one concern limiting the use of adenoviral vaccine vectors is that preexisting immunity to adenoviruses induced by natural exposure can potentially neutralize adenoviral vaccines via reactivity against its hexon protein [24
], thus, preventing the development of a protective immune response [38
]. Admittedly, studies have shown that i.n. immunization seems to avoid the stimulation of systemic neutralizing Abs [39
]. However, another concern is the possible retrograde uptake of replication-deficient adenovirus vectors into the olfactory bulb [24
], suggesting perhaps a natural consequence of wild-type Ad infection. Although adenoviruses have been found in the CNS, they are not generally considered as "neurotropic" and isolation of them from brain tissues is rare [40
To circumvent such concerns, efforts were focused on developing a targeting moiety to enable mucosal vaccination based upon the adenovirus adhesin or F protein instead of the whole virus. In support of our previous study against BoNT/A [23
], our current results clearly demonstrated that the inclusion of the targeting molecule, Ad2F, also boosts the levels of mucosal IgA and systemic IgG Abs against serotype B when administrated mucosally (Figure ). Moreover, it is important to emphasize that in the absence of CT adjuvant, nasal immunization with our recombinant Ad2F fusion vaccine stimulated similar levels of fecal IgA Abs as in mice co-immunized with CT (Figure ). In the absence of the Ad2F targeting moiety, Hcβtre induced only a weak mucosal Ab response even with CT. These findings suggest perhaps Ad2F possesses adjuvant-promoting properties, which, in turn, could limit the use of adjuvants and diminish the number of immunizations required to stimulate protective immunity. Current studies are addressing such possibilities. Interestingly, our study showed that mice given our fusion vaccine via the i.m. route produced elevated levels of both mucosal IgA and systemic IgG Abs (Figure ). Notably, i.m. immunization with Hcβtre-Ad2F in the absence of CT showed elevated IgG Ab titers that remained elevated for at least 35 days post-primary immunization. One possible explanation for our findings could be attributed to the interactions between Ad2F and the coxsackie adenovirus receptor, which is expressed in a wide range of cell types. This interaction may allow more effective delivery of the vaccine into the cells. In addition, similar to our previous study [23
], the Hcβtre's immunogenicity was markedly improved by the addition of Ad2F (Figures , , and ). However, only a low level of mucosal Abs after day 35 was observed (Figure ), suggesting that long term mucosal memory was not induced when the Ad2F-based vaccine was given parenterally. Since very few IgA AFCs were detected in any of the lymphoid tissues of i.m.-immunized mice (Figure ), such evidence suggests that transient IgA Ab responses were induced during immunization.
Few studies have evaluated whether Th1 or Th2 cells are beneficial for optimal Ab production for the Hc-based vaccines. A recent study by Kobayashi et al. [30
] suggests that BoNT/A-specific Abs induced by mucosal BoNToxoid immunization are mediated via Th2-type cytokines. Herein this report, a combination of Th cell types supports the BoNT/B-specific Ab responses (Figures and ). Notable enhancements in Th2-type cells were detected in the spleens and MLNs of i.n. Hcβtre-Ad2F plus CT-immunized mice (Figure ), and likewise for i.m. Hcβtre-Ad2F plus CT-immunized mice (Figure ) suggesting these responses supported the observed, elevated IgG1 Ab responses (Figure ). These findings further corroborate the results of our previous study showing that the Hcβtre/A vaccine for BoNT/A also elicits a mixed Th cell response [23
Nasal administration of Hcβtre-based vaccines induced significant systemic and mucosal Abs. To assess their protective efficacy, a mouse neutralization assay, was performed (Table ). Our data showed that passively administered sera from animals immunized with Hcβtre or with Hcβtre-Ad2F protected 100% mice for 24 hr against 2.0 LD50, suggesting neutralizing Abs are induced.